ICAL2020

International Conference on Autophagy and Lysosomes 2020

Time DAY 1 (16-01-2020) Thursday
07:30-09:00 Breakfast and Tea/Coffee
08:00-09:00 Registration and Poster set up (A1 to A33)
09:00-09:20 Welcome address
Session -1: Autophagy: process and dynamics
Chair: Sascha Martens
09:20-09:55 David Rubinsztein
“Autophagy and neurodegeneration”
09:55-10:30 Juan S. Bonifacino
“Regulation of LC3 levels by ubiquitination and proteasomal degradation”
10:30-10:55 Ravi Manjithaya
“Insights into mechanisms of autophagy flux by genetic and chemical biology
approaches”
10:55-11:10 Coffee/Tea break (poster set up)
Session -2: Autophagy: assembly and disease
Chair: David Rubinsztein
11:10-11:45 Sharon A. Tooze
“Initiation of autophagosome formation”
11:45-12:10 Santosh Chauhan
“Controlling Inflammation and Autoimmunity by Autophagy”
12:10-12:35 Benu Brata Das
“Mitochondrial dysfunction promotes the etiology of neurodegenerative
syndrome SCAN1”
12:35-13:00 Anu Rangarajan
“Tug of war among cellular-kinases in the regulation of autophagy and anoikis”
13:00-14:00 Lunch Break (Networking and poster preview)
Session -3: Autophagy: cargo sorting and turnover
Chair: Fulvio Reggiori
14:00-14:35 Suresh Subramani
“Overview of selective autophagy - lessons learned from studies on pexophagy”
14:35-15:10 Masaaki Komatsu
“Selective turnover of p62/SQSTM1 by autophagy: The molecular mechanism
and physiological significance”
15:10-15:45 Sascha Martens
“Mechanisms of Selective Autophagy - From Phase Separation to Cargo
Degradation”
15:45-15:55 Photo Session
15:55-16:10 Coffee/Tea break
16:10-16:20 Sabuj Bhattacharyya
Innovative funding opportunities for Biomedical & Health Research in India
Session -4: Autophagosome: membrane assembly and formation
Chair: Sharon A. Tooze
16:20-16:55 Fulvio Reggiori
“Supplying the forming autophagosomes with lipids”
16:55-17:20 Lipi Thukral
“How protein shape governs membrane trafficking events during autophagy?”
17:20-17:45 Shailendra Asthana
“Discovery of novel autophagy inducers by exploring the dynamic protein-protein
interaction interfaces through computational approaches”
17:45-18:10 Sunando Datta
“Role of retromer-Snx27 in MT1-MMP trafficking in MDA-MB-231 cells”
18:10-19:40 Poster session 1 (A1 to A33)
19:40-21:00 Dinner
21:00 Departure to accommodation sites

P1
ICAL2020
DAY 2 (17-01-2020) Friday
08:00-09:00 Breakfast and Tea/Coffee; Poster set up (L1 to L31)
Session-5: Autophagy linking viruses/gases
Chair: Suresh Subramani
09:00-09:35 Aleem Siddiqui
“Mitophagy in Viral Hepatitis B and C”
09:35-10:00 Ghulam Hussain Syed
“Mitophagy during Flaviviral infections: Significance in Viral disease
pathogenesis”
10:00-10:25 Manjula Kalia
“Impact of autophagy on Japanese encephalitis virus replication and
pathogenesis”
10:25-10:50 Ashwani Kumar
“Hydrogen Sulfide-Induced GAPDH Sulfhydration Disrupts the DBC1-SIRT1
Interaction to Initiate Autophagy”
10:50-11:05 Coffee/Tea break (poster set up)
Session-6: Lysosome: cargo sorting, biogenesis and disease
Chair: Juan S. Bonifacino
11:05-11:40 Judith Klumperman
“Unravelling lysosome biogenesis by correlative light electron microscopy”
11:40-12:15 Thomas Wollert
“Targeting of protein aggregates to lysosomes and implication for
neurodegenerative diseases”
12:15-12:40 Subba Rao Gangi Setty
“Phosphatome modulation promotes the lysosome function/biogenesis in LSDs”
12:40-13:05 Amit Tuli
“Regulation of osteoclast function by lysosomal GTPase Arl8b”
13:05-14:00 Lunch Break (Networking and poster preview)
Session-7: Endo or auto-lysosomes
Chair: Judith Klumperman
14:00-14:35 Graca Raposo
“From Immunity to Skin: Adaptations of the endo-lysosomal system in
Intercellular communication”
14:35-15:00 Varadharajan Sundaramurthy
“Adaptive endo-lysosomal homeostasis as a defining feature of Mtb infected
macrophages”
15:00-15:25 Nitin Mohan
“Microtubule post-translational modifications spatially constrain lysosomes to
regulate autophagy”
15:25-15:50 C.V. Srikanth
“A SUMO tug-of-war at epithelial-immune cell interface mediate intestinal
homeostasis”
15:50-16:05 Coffee/Tea break
Session-8: Membrane dynamics: PIs and AAs
Chair: Aleem Siddiqui
16:05-16:40 Lois Weisman
“Uncovering novel roles for PIKfyve in cell migration”
16:40-17:05 Raghu Padinjat
“Regulation of membrane dynamics by phosphoinositides”
17:05-17:30 Sunil Laxman
“Methionine as a growth signal and anabolism regulator”
17:30-19:00 Poster session 2 (L1 to L31)
19:00-19:30 Cultural Programme
19:30-22:00 GALA Dinner
22:00 Departure to accommodation sites

P2
ICAL2020
DAY 3 (18-01-2020) Saturday
8:00-09:00 Breakfast and Tea/Coffee
Session-9: Cell organelles: maturation and physiology
Chair: Masaaki Komatsu
09:00-09:25 Sandhya P. Koushika
“A common genetic pathway for cargo retention and polarized cargo distribution
in neurons”
09:25-09:50 Sangeetha Nath
“Tunneling nanotubes: the novel long range intercellular pathway of pathology
spreading exploits PAK1 kinase dependent macropinocytic vesicle recycling”
09:50-10:15 Thomas Pucadyil
“Membrane fission: Insights from reconstituting organelle form and chemistry”
10:15-10:30 Shalini Rawat (Mahak Sharma’s lab)
“Rabip4’ interacts with the small G protein Arl8b and regulates membrane
trafficking in the endolysosomal pathway”
10:30-10:45 Coffee/Tea break
Session-10: Cell surface mechanisms and cellular processes
Chair: Thomas Wollert
10:45-11:10 Deepak Nair
“Nanoscale Organization of amyloidogenic machinery at excitatory synapse”
11:10-11:35 Umasankar PK
“Regulatory Mechanisms of Clathrin-Mediated Endocytosis: Fishing for ON-OFF
Switches”
11:35-12:00 Sivaram V S Mylavarapu
“Endocytic-Exocytic Crosstalk during Cytokinesis”
12:00-12:15 Bharat Bhatt (KN Balaji’s lab)
“BRD4, an epigenetic Reader, orchestrate mycobacterial pathogenesis by co-
modulating host angiogenesis and lipophagy”
Session-11: Short talks selected from Abstracts
Chair: Graca Raposo and Lois Weisman
12:15-12:30 Sarmistha Mahanty
“Differentiated keratinocytes generate unique Golgi-associated lysosomes”
12:30-12:45 Veena Ammanathan
“TFEB-mediated xenophagy: a host defence pathway involved in restricting
intracellular pathogens”
12:45-13:00 Navin Baid
“Antimycobacterial effect of IFNG-induced autophagy depends on HMOX1-
mediated increase in intracellular calcium levels and modulation of
PPP3/calcineurin-TFEB axis”
13:00-14:00 Lunch break
Session-11: Short talks selected from Abstracts
Chair: Graca Raposo and Lois Weisman
14:00-14:15 Krishnendu Roy
“A Redox-dependent Membrane Fission Catalyzed by α-synuclein”
14:15-14:30 Edries Yousaf Hajam
“Adaptive immune cells control lipophagy to maintain dermal white adipose
tissue (DWAT) homeostasis”
14:30-14:45 Viji Vijayan
“Itinerary of Abeta42 whilst under nicotine influence– a lysosome related story”
14:45-15:00 Sravanthi Nadiminti
"LRRK2/LRK-1 and Liprin-α/SYD-2 regulate trafficking of synaptic vesicle and
lysosomal proteins"
15:00-16:00 Panel Discussion:
Data interpretation methods related to lysosome biology and autophagy
16:00-16:30 Coffee/Tea break (High Tea)
16:30-17:30 Quiz, Fun activities and Feedback
17:30-18:00 Poster Awards & Concluding session
18:30 - Dinner (Departure to Hostels @ 20:00)

P3
 Posters: Day-1 (16-01-2020)
Poster Name TITLE
No.
A1 A. Niharika Activation of autophagy by nitazoxanide through mitochondrial
Reddy uncoupling confers protection in cellular and animal models of
Parkinson’s disease

A2 Aitizaz ul The citrus flavonoid (ZO-93) reduces amyloid -β (1-42) load by

Ahsan upregulating autophagy through AMPK/ULK axis in neuronal cells
A3 Ananya Ray Interplay between ER homeostasis, signaling and autophagy in
Plasmodium falciparum.
A4 Anindita Investigating the role of autophagy in the background of monocyte to
Bhattacharya macrophage differentiation
A5 Anirban Autophagy induction exhibits enhanced host immune response via its
Sengupta modulatory influence on splenic macrophages and dendritic cells in
experimental cerebral malaria

A6 Assirbad Behura ESAT-6 regulates Calcimycin-induced autophagy through miR-30a-

3p and 30a-5p
A7 Bharati Singh Mitochondrial injury and quality control in Dengue infection

A8 Edries Yousaf Adaptive immune cells control lipophagy to maintain dermal white
Hajam adipose tissue (DWAT) homeostasis.
A9 Sahar Rafat Therapeutic effect of SMAC mimetic (BV6) targeting IAPs and
autophagic proteins in breast cancer cell lines
A10 Mehboob Ali IIIM-941 a small-molecule induces autophagy by attenuating NLRP3
inflammasome via mTOR pathway.
A11 Mrityunjay WRN, a DNA repair helicase regulates maturation of
Tyagi autophagosomes in response to cisplatin
A12 Mouliganesh Role of PPARγ on Autophagy

A13 Mubashir J IIIM-NPC-290 promotes cell death by inducing autophagy in Mia

Mintoo PaCA-2 pancreatic cancer cells
A14 Nandita Mishra Cytoplasmic vacuolation induced cell death by Piperlongumine in
cancer cells: An interplay between paraptosis and autophagy
A15 Navin Baid Antimycobacterial effect of IFNG (interferon gamma)-induced
autophagy depends on HMOX1 (heme oxygenase 1)-mediated
increase in intracellular calcium levels and modulation of
PPP3/calcineurin-TFEB (transcription factor EB) axis.
A16 Nitin Mohan Microtubule post-translational modifications spatially constrain
lysosomes to regulate autophagy
A17 Rabiya Majeed AMP-activated Protein Kinase induced autophagy is a feed forward
signal for mTORC1 activation.
A18 Rajalakshmi Gcn4 mediates a methionine-dependent anabolic program by
Srinivasan controlling amino acid supply

PL-1
A19 Ram Kumar Arsenic Induces Autophagy in Developmental Mouse Cerebral
Manthari Cortex by Inhibiting Blood Brain Barrier’s Tight Junction Proteins

A20 Rama Gopal Mitochondrial dysfunction affects translational initiation pathways

Reddy Koncha and cellular homeostasis: Importance of Integrated stress response
(eIF2ak-eIF2α phosphorylation-ATF4) pathway
A21 Saiful Anam HBV activates Parkin-mediated mitophagy leading to attenuated
Mir ATP level in human macrophages
A22 Salini K Secondary apoptosis mediated by autophagosomes in hawthorn
extract treated breast cancer cell line in vitro
A23 Sangam Rajak Crinophagy regulates glucagon content via a non-macroautophagic
mechanism
A24 Sheikh Tahir AMP-activated Protein kinase; Map-kinase interacting kinase;
Majeed eIF4E; S6Kinase 1 axis is a rescue to synchronize autophagy with
cell growth.
A25 Shivani Sahu Hydrogen Sulfide-Induced GAPDH Sulfhydration Disrupts the
DBC1-SIRT1 Interaction to Initiate Autophagy
A26 Sushmita Das Activation of cytosolic immune surveillance induces autophagy in
visceral leishmaniasis
A27 Sreedevi Approaches to study the crosstalk of secretory autophagy and
Padmanabhan unconventional protein secretion (UPS)
A28 Swati Swagatika Cantharidin inhibits autophagic flux by impairing cellular
phosphatidylethanolamine pool mediated Atg8 lipidation in
Saccharomyces cerevisiae
A29 Surendra Kumar Pharmacological induction of autophagy as a potential therapeutic
Prajapat target for Japanese encephalitis
A30 Suresh Antiviral interferon response is enhanced upon suppressing foot-and-
Basagoudanavar mouth disease virus induced autophagy
A31 Syed Shadab O2•− and H2O2 activated Pro-autophagic proteins AMBRA1 and
Raza Beclin1 determines the fate of transplanted human stem cells in an
ischemic brain: An in vitro study on human dental and mesenchymal
stem cells
A32 Veena TFEB-mediated xenophagy: a host defence pathway involved in
Ammanathan restricting intracellular pathogens
A33 Yesheswini Investigations on Rab7 in human IBD: An unanticipated role in
Rajendran autophagy mediated disease pathogenesis
A34 Saravana Babu Melatonin improves AKT/mTOR/LC3 II autophagy signalling in
C sleep deprived mice brains

PL-2
 Posters: Day-2 (17-01-2020)
Poster Name TITLE
No.
L1 Anshul Milap STX1A regulates maturation of lysosomes and lysosome–related
Bhatt organelles
L2 Anuradha Neuronal regulation of organ maturation: role of DMon1
Ratnaparkhi
L3 Chandan Understanding the Role of a Novel Dynein Interactor in Mitosis
Kumar
L4 Chandrima Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via
Gain Autophagy-Lysosomal Pathway
L5 Gaurav Kumar Regulation of osteoclast function by the small GTPase Arl8b

L6 Gregor P Jose PLiMAP: A generic, high throughput and sensitive membrane-binding

assay
L7 Gurranna Nucleophagy: Role in nuclear organization
Male
L8 Harini MYCOBACTERIAL-HOST INTERACTION A TRICK OR TREAT:
Laxminarayan Inhibition of autophagy by mycobacteria through STPKL in a nutrition
starvation model.
L9 Himani Deciphering the role of the pleckstrin-homology domain in endocytic
Khurana dynamins
L10 Jahnavi K A study on the effects of Krill oil on autophagy-lysosomal functions
and amyloid-β clearance in scopolamine intoxicated mice brains
L11 Jeganath A Unravelling lysosome mediated nutrient sensing with super-resolution
STORM imaging
L12 Kautilya IRGM is a Master Negative Regulator of Interferon Response by
Kumar Jena Controlling the Activation of cGAS-STING and RIG-I-MAVS
Signaling Pathways
L13 Krishnendu A Redox-dependent Membrane Fission Catalyzed by α-synuclein
Roy

L14 Lakshmi Biochemical characterization of lysosomal enzymes, β-hexosaminidase

Surekha and α L fucosidase from Hydra vulgaris Ind-Pune
Krishnapati
L15 Mahadeva Role of Mint family of Proteins in Localization, Trafficking and
Swamy Processing of Amyloid Precursor Protein
L16 Monika Rawat The Role of RNA binding proteins in Tunneling Nanotube Formation
and Function
L17 Navyasree KV Investigating role of endo-lysosomal cholesterol transport in mTOR
signaling
L18 Prabha M The Specific activities of Acid Phosphatase related to lysosomal
activation and Correlation of Anaplastic Oligodendroglioma G-III to
Meningiomas G-I among all Malignant and Benign Brain Tumors

PL-3
L19 Rohit Sai Purification, biochemical and biophysical characterization of
Reddy Konada lysosomal β-D-glucuronidase from an edible freshwater mussel,
Lamellidens corrianus
L20 Sakshi Gupta ARL6ip5 interacts with α-Synuclein and modulates its neurotoxicity

L21 Saloni Patel Modulation of pseudophosphatase STYXL1 function restores

lysosomal activity in Gaucher’s disease
L22 Sameena Deciphering the role of STX7 in breast cancer cell invasion
Parveen
L23 Sameer Ullah IIIM-3 activates lysosomal mediated cell death via mTOR-TFEB axis
Khan in MIA PaCa-2 cells
L24 Sarmistha Differentiated keratinocytes generate unique Golgi-associated
Mahanty lysosomes
L25 Shaini Rawat Rabip4’ interacts with the small G protein Arl8b and regulates
membrane trafficking in the endolysosomal pathway
L26 Shikha T Activation of AP-2 adaptor complex via FCHO-BMP2K axis promotes
Ramesh clathrin-mediated endocytosis
L27 Soumya Analysis of cellular functions of the ATP-dependent membrane fission
Bhattacharyya catalyst EHD1
L28 Sravanthi Investigating the roles of SYD-2/Liprin-α and LRK-1/LRRK2 as
Nadiminti common genetic regulators of lysosomal and synaptic vesicle proteins
L30 Viji Vijayan Itinerary of Abeta42 whilst under nicotine influence– a lysosome
related story
L31 Vinoth Kumar Fucoidan hydrolyzed by AMG improve Endothelial Progenitor Cells
Rethineswaran functional activities via blocking lysosome localization of TSC2

PL-4
Abstracts of
Speakers
Day-1
(16.01.2020)

A1
Autophagy and Neurodegeneration

David C Rubinsztein
Cambridge Institute for Medical Research and UK Dementia Research
Institute, University of Cambridge, The Keith Peters Building, Cambridge
Biomedical Campus, CB2 0XY, UK

Intracellular protein aggregation is a feature of many late-onset neurodegenerative diseases,

including Parkinson’s disease, tauopathies, and polyglutamine expansion diseases (like
Huntington’s disease (HD)). Many of these mutant proteins, like that causing HD, cause dis-
ease via toxic gain-of-function mechanisms. Therefore, the factors regulating their clearance
are crucial for understanding disease pathogenesis and for developing rational therapeutic
strategies.
We showed that the autophagy inducer, rapamycin, reduced the levels of mutant huntingtin
and attenuated its toxicity in cells, and in Drosophila, zebrafish and mouse HD models. We
have extended the range of intracellular proteinopathy substrates that are cleared by autophagy
to other related neurodegenerative disease targets, like alpha-synuclein in Parkinson’s disease
and tau in various dementias. While autophagy induction is protective in models of various
neurodegenerative diseases, many of these diseases are associated with compromised auto-
phagy, including Huntington’s disease. I will discuss some of our studies describing how
autophagy can be regulated. I will consider how the amino acid leucine regulates autophagy
via mTORC1, and how interferon-b regulates autophagy and neurodegeneration. Finally, I will
describe our drug repurposing efforts to identify compounds already used in humans for other
indications that may be suitable as autophagy inducers in the brain.

Regulation of LC3 levels by ubiquitination and proteasomal

degradation

Juan S. Bonifacino

Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland, 20892, USA

To gain further insights into the regulation of autophagy, we conducted a genome-wide

CRISPR-Cas9 knockout (KO) screen using H4 neuroglioma cells expressing endogenous
LC3B tagged with GFP-mCherry as a reporter. Mutant cells expressing increased
GFP:mCherry ratio were selected by FACS, and the mutated genes were identified by next-
generation sequencing. This screen resulted in the identification of the ubiquitin-activating
enzyme (E1) UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase (E2/E3)
BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate
monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B
available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B,
enhancing the degradation of autophagy receptors and the clearance of intracellular proteins
aggregates in both non-neuronal and neuronal cells. These findings thus identified a novel

A2
mechanism for the regulation of autophagy, which could be exploited for the development of
pharmacological inhibitors to treat protein aggregation disorders.

Insights into mechanisms of autophagy flux by genetic and

chemical biology approaches

Ravi Manjithaya

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced
Scientific Research (JNCASR)

Autophagy is an evolutionary conserved, catabolic process wherein unwanted and excess

cellular contents are degraded inside vacuole or lysosomes. This is brought about by capturing
the cargo in double membranous vesicles called autophagosomes, which then fuse with
lysosomes and deliver the contents for degradation. The rate at which cargo capture, delivery
to lysosomes, and degradation take place is called “autophagy flux.” Autophagy has several
implications in health and disease, and therefore, modulation of autophagy flux is very
important. In my lab, we employ chemical biology and genetics approaches to understand the
mechanism of different stages of autophagy flux. I will highlight the identification of two
protein complexes- septin and exocyst through an unbiased genetic screen. While septins
migrate from bud-neck to PAS and form novel ring-like structures around autophagosomes,
the exocyst subunit form an autophagy-specific sub-complex. Both these complexes were
found to regulate Atg9 trafficking and were involved in autophagosome biogenesis during
autophagy prevalent conditions. Further, I will also discuss about our recent findings where we
identified two compounds by unbiased chemical screening: an inhibitor that inhibits loading of
Syntaxin17 on autophagosomes and the other compound that acts as xenophagy inducer
restricting replication of Salmonella in TFEB dependent but mTOR independent
manner.

Initiation of autophagosome formation

Sharon A. Tooze
The Francis Crick Institute, London UK

Autophagy is a highly conserved cell survival pathway. It is required for cell health, and to
combat infection and disease. Autophagy performs these tasks by removing and degrading
harmful proteins and pathogens. The process is mediated by a double membrane vesicle called
the autophagosome. Autophagosome formation is a complex process that requires a cohort of
ATG (autophagy related) proteins acting in a highly coordinated manner. This core machinery
of ATG proteins drives the formation of the phagophore, the precursor structure for the
autophagosome, and expansion of the nascent autophagosome. Under amino acid starvation,
where autophagy is induced to recycle proteins into amino acids to replenish the pool, this
coordinated response is initiated by ATG9A trafficking and the ULK1/2 complex, a serine-
threonine protein kinase complex. Following initiation, the Class III PI3 kinase complex I, and
its effector WIPI2 mediate recruitment of ATG12-5-16L1, and the ATG8 family of proteins.
ATG8s are required for cargo selection and maturation of the autophagosome. The

A3
autophagosome then undergoes fusion with the endosome and lysosome after which the
contents sequestered by the autophagosome are degraded. In an effort to understand the
formation of autophagosomes we are studying the trafficking and function of ATG9A,
substrates of the ULK1/2 complex, and WIPI proteins. Our recent results will be presented
which provide new insight into these processes.

Controlling Inflammation and Autoimmunity by Autophagy

Santosh Chauhan
Institute of Life Sciences, Bhubaneswar, India

Autophagy play seminal role in controlling the inflammation. Uncontrolled inflammation leads
to several autoimmune and cancer. Activation of type 1 interferon response is extensively
connected with the antiviral immunity and pathogenesis of autoimmune diseases. We found
that IRGM, whose deficiency is linked with the genesis of several autoimmune disorders, is a
master negative regulator of the interferon response. Mechanistically, we show that IRGM
interacts with nucleic acid sensor proteins, including cGAS and RIG-I, and mediates their
autophagic degradation to restrain activation of interferon signaling. Further, IRGM maintains
mitophagy flux, and its deficiency results in the accumulation of defunct leaky mitochondria
that releases cytosolic DAMPs triggering activation of interferon responses via cGAS-STING
and RIG-I-MAVS signaling axis. Due to an enduring type 1 IFN response in IRGM-deficient
cells and mice, they were intrinsically resistant to infection of the Japanese Encephalitis virus,
Herpes Simplex virus, and Chikungunya virus. Altogether, this study defines the molecular
mechanisms by which IRGM maintains interferon homeostasis and protects from autoimmune
diseases. Further, it identifies IRGM as a broad therapeutic target for defense against viruses.

Mitochondrial dysfunction promotes the etiology of neurodegenerative

syndrome SCAN1.

Benu Brata Das

Associate Professor and Wellcome Trust/ IA fellow, Laboratory of Molecular

Biology, School of Biological Sciences, Indian Association for the Cultivation of
Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, INDIA

A homozygous mutation of human tyrosyl-DNA phosphodiesterase 1 (TDP1) causes the

neurodegenerative syndrome, spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1
hydrolyzes the phosphodiester bond between DNA 3′-end and a tyrosyl moiety within trapped
topoisomerase I (Top1)-DNA covalent complexes (Top1cc). TDP1 is critical for mitochondrial
DNA (mtDNA) repair; however, the role of mitochondria remains largely unknown for the
etiology of SCAN1. We demonstrate that mitochondria in cells expressing SCAN1-TDP1
(TDP1H493R) are selectively trapped on mtDNA in the regulatory non-coding region and

A4
promoter sequences. Trapped TDP1H493R-mtDNA complexes were markedly increased in the
presence of the Top1 poison (mito-SN38) when targeted selectively into mitochondria in
nanoparticles. TDP1H493R-trapping accumulates mtDNA damage and triggers Drp1-
mediated mitochondrial fission, which blocks mitobiogenesis. TDP1 H493R prompts PTEN-
induced kinase 1–dependent mitophagy to eliminate dysfunctional mitochondria. SCAN1-
TDP1 in mitochondria creates a pathological state that allows neurons to turn on mitophagy to
rescue fit mitochondria as a mechanism of survival.

Tug of war among cellular-kinases in the regulation of

autophagy and anoikis

Anu Rangarajan

MRDG, Indian Institute of Science, Bangalore, India

Normal epithelial cells grow and proliferate when attached to proper extracellular matrix
(ECM). Detachment of cells from the ECM promotes cell death, which is known as anoikis, a
Greek word meaning ‘homelessness’. Anoikis plays a key role in tissue homeostasis,
preventing growth of cells in inappropriate location. However, cancer cells must develop the
ability to survive in ECM-deprived condition during their journey through the circulation to
initiate a successful metastatic. Therefore, understanding the mechanisms that enable the
survival of ECM-detached cancer cells would help in mitigating metastasis – a major cause of
cancer-related death. Our laboratory has identified a novel role for the energy sensor and
central metabolic regulator AMP-activated protein kinase (AMPK) in the promotion of cell
survival in matrix-deprived condition. Yet, mechanisms by which AMPK regulates survival of
ECM-detached cells is not fully understood. I will duscuss on our recent findings revealing a
role for AMPK in positively regulating autophagy flux by interacting with other cellular
kinases such as Akt and AMPK in overcoming anoikis.

Overview of selective autophagy in yeast

Suresh Subramani

University California, San Diego, USA

This talk will present a historical perspective on selective autophagy in general,

focusing on the contributions from my lab. I will discuss the morphological intermediates
involved in pexophagy, genetic screens for pexophagy components, the discovery of selective
autophagy receptor (SAR)/co-receptor components, the role of
phosphorylation/dephosphorylation of SARs, the signaling pathways that trigger pexophagy
and mitophagy, the molecular players involved and their engagement with the core autophagy
machinery components. I will end with the description of a new selective autophagy pathway
in Pichia pastoris that degrades cytosolic pools of peroxisome targeting signal (PTS) receptors
independently of the pexophagy-specific SAR.

A5
Selective turnover of p62/SQSTM1 by autophagy: The
molecular mechanism and physiological significance

Masaaki Komatsu

Department of Physiology, Juntendo University School of Medicine

p62/SQSTM1(hereafter referred to as p62) is a stress-inducible protein able to change among

binding partners, cellular localizations and form liquid-droplet structures in a context-
dependent manner. This protein is mainly defined as a ubiquitin-binding receptor protein for
selective autophagy. Besides this role, its ability to interact with multiple binding partners
allows p62 to act as a main regulator of the activation of the Nrf2, mTORC1 and NF-κB
signaling pathways, linking p62 to the oxidative defense system, nutrient-sensing and
inflammation, respectively. Recent reports revealed that once p62 binds to ubiquitin chains, it
acquires liquid-like properties. Such phase-separated droplets allow the exchange of their
components, including ubiquitin and LC3, with the surrounding environment. Consequently,
the droplets can also function as nodes from which signaling cascades can be activated in the
context of selective autophagy. Herein, I introduce the physiological role of p62 as well as
molecular mechanism by which the p62 is degraded by autophagy.

Mechanisms of Selective Autophagy - From Phase Separation

to Cargo Degradation
Sascha Martens
Max F. Perutz Laboratories, University of Vienna, Vienna BioCenter, Vienna,
Austria

Autophagy is an intracellular lysosomal bulk degradation pathway that ensures cellular

homeostasis by the removal of damaged and dangerous material from the cytoplasm. This is
achieved by the sequestration of the cytoplasmic cargo material within double membraned
organelles called autophagosomes. The selective sequestration of only specific cargo material
is mediated by cargo receptors that link the cargo to the nascent autophagosomal membrane.
How cargo selection, membrane nucleation and growth are coupled is unclear. I will present
our recent work on the human cargo receptors and the autophagy machinery derived from in
vitro reconstitution systems and cell biology. In particular, I will discuss how the p62 cargo
receptor and the autophagy machinery including FIP200 and ATG8 proteins act sequentially
during cargo condensation, membrane nucleation and elongation to mediate the specific
sequestration and subsequent degradation of cellular material.

A6
Supplying the forming autophagosomes with lipids

Fulvio Reggiori

Department of Biomedical Sciences of Cells & Systems, University of

Groningen,The Netherlands

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the
hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and
transmembrane Atg9 remains largely unknown despite their importance in autophagy. In the
presented study, we provide insights into the molecular role of these proteins by identifying
and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates
to autophagosomal membranes through lipid binding and independently from Atg9. Its
interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore
extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex
is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn,
disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both
Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy.
Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and
highlight the possible functional relevance of the phagophore-ER contact sites in phagophore
expansion. This notion is supported by a series of very recent structural studies that have
revealed that Atg2 has the in vitro capacity of transferring lipids from adjacent membranes.

How protein shape governs membrane trafficking events

during autophagy?

Lipi Thukral

Institute of Genomics and Integrative Biology, New Delhi, India

Multimachine power systems in biology may be described as a combination of robust protein

state space and transiently stable conformations that are capable of producing controlled
functional outcome. The mechanisms underlying these cellular processes, such as Autophagy,
which involves precise intreraction of protein complexes and spatially distributed membrane
lipids are largely unknown. We are interested in addressing autophagic initiation that embarks
by recruitment of LC3 family members to expand and form autophagosome. While LC3B and
related homologs (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and
GATE16/GABARAPL2) are key regulators of autophagy, their distinguishing roles have not
been determined. Because the protein family members share highly congruent topologies,
mapping their unique functional interactions is a long-standing open question in Autophagy
field. In this talk, I will report a novel computational framework to determine factors that lead
to preferential substrate recognition of Atg8 orthologs. We construct a multifaceted approach
by looking at the sequence evolution, structure, type of binding partners, functional impact of
different proteins, and clinical implications of their genomic variations. By comparing
understudied LC3 family members, we provided the molecular basis of understanding their

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properties that may act as a general mechanism in expanding protein machinery across cellular
pathways.

Discovery of novel autophagy inducers by exploring the

dynamic protein-protein interaction interfaces through
computational approaches.

Shailendra Asthana

Translational Health Science and Technology Institute (THSTI), Faridabad, Haryana, India

Protein-protein (PP) complexes orchestrate most of the aspects of cellular biology. Cellular
events encompass obligate and transient PP interactions that accurately define the interactome
in a time and location dependent manner. Mimicry of interfacial protein domain of interactome
offers a promising strategy to rationally design modulators from the PP interaction interfaces.
Here, we are exploring the PP interfaces at dynamic and atomic level to identify the autophagy
inducers. Autophagy has been found to be dysregulated in many diseases. The interfaces of
Beclin-1 (+ve regulator) and GAPR-1 (–ve regulator) were exploited to generate the autophagy
inducers in m-TOR independent manner by using various PP docking algorithms to predict the
most likely binding modes of Beclin-1 GAPR-1 complexes. The microsecond (μs) molecular
dynamics simulations (MD) were performed on complex poses to determine and confirm the
‘hot-spot’ interface regions. The multiple sets of peptides were generated from different ‘hot-
spot’ regions of Beclin-1 and docked on GAPR-1 to generate the protein-peptide complexes.
Based on docking scores, residue wise contacts and binding free energies the top peptides were
picked for in vitro and binding studies through SPR. From peptide’s residue-wise contribution,
alanine-scanning and binding architecture of GAPR-1 were used for small molecule generation
through virtual screening and peptidomemitics approaches. More than 120 molecules were
synthesized to measure the autophagy induction by monitoring the LC3-II and P62 levels, lipid
body content and TG level. The identified lead compound was tested in different therapeutic
areas in which the role of autophagy induction is well documented like infectious and for non-
alcoholic steatohepatitis.

Role of retromer-Snx27 in MT1-MMP trafficking in MDA-

MB-231 cells

Sunando Datta

Indian Institute of Science Education and Research, Bhopal, India

A variety of metastatic cancer cells utilize actin-rich membrane protrusions, invadopodia, for
efficient ECM degradation, which involves trafficking of proteases from intracellular
compartments to these structures. We have found that in metastatic breast cancer cell line,

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MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix
metalloprotease, MT1-MMP. Further MT2-MMP, another abundantly expressed
metalloprotease, showed association with invadopodia. MT1 and MT2-MMP showed
colocalization but were located on the distinct endosomal domains. Retromer and its associated
sorting nexin, SNX27 phenocopied each other in matrix degradation via selectively recycling
MT1-MMP but not MT2-MMP. ITC based studies revealed that both SNX27 and retromer
directly interact with MT1-MMP. Analysis from the publicly available database showed
overexpression or frequent alteration of SNX27 in the patients having invasive breast cancer.
In Xenograft-based studies SNX27 depleted cell line showed prolonged survival of SCID mice,
suggesting a possible implication for over-expression of the sorting nexin in tumor samples.

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Abstracts of Speakers
Day-2
(17.01.2020)

A10
Mitophagy in Viral Hepatitis B and C
Aleem Siddiqui.
Division of Infectious Diseases, School of Medicine, University of
California, San Diego, CA.,

Mitochondrial injury and oxidative stress are prominent features of chronic hepatitis associated
with Hepatitis B (HBV) and C (HCV) viral infections. Hepatitis virus-induced oxidative stress
leads to dysfunctional mitochondria, which are toxic and can cause damage to adjacent
mitochondria and promote cell death. Hence a rapid turnover of the damaged mitochondria is
crucial for survival of HBV/HCV-infected cells and for maintenance of persistent/chronic
infection. Mitochondrial dynamics is tightly regulated in response to alterations in cellular
physiology and its dysfunction that occurs during chronic hepatitis B and C. Mitochondria are
dynamic organelles that constantly undergo fission, fusion, and mitophagy to facilitate
mitochondrial quality control, which is crucial for maintaining cell viability and bioenergetics.
Mitochondrial fission/fragmentation is mediated by recruitment of cytosolic Drp1 to the
mitochondria and mitophagy, which normally follows, is marked by recruitment of Parkin to
mitochondria. Both HBV/HCV induce perinuclear mitochondrial clustering and mitophagy.
Mitochondrial translocation of Parkin, an E3 ubiquitin ligase linked to Parkinson’s disease,
initiates mitophagy leading to the removal of damaged mitochondria. Interestingly, we
observed that HBV/HCV-induced mitochondrial dynamics attenuated apoptosis. Inhibiting of
mitophagy via Parkin silencing caused a robust cytochrome C release and an increase in
apoptotic signaling molecules. These results clearly implicate that modulation of mitochondrial
dynamics and induction of mitophagy by HBV/HCV is required for maintenance of persistent
infection. Our results also revealed that HBV/HCV-induced Parkin translocation to
mitochondria leads to its massive ubiquitination of antiviral mitochondrial signaling protein
(MAVS), further aided by LUBAC complex. Together, this strategy results in the inhibition
of interferon (IFN) synthesis, thus crippling innate immunity, the first line of defense. These
studies provide a unique insight into the underlying mechanisms of mitochondria-mediated
liver injury that contributes to the viral infectious process and liver disease pathogenesis
including progression to hepatocellular carcinoma associated with this infection.

Mitophagy during Flaviviral infections: Significance in

Viral disease pathogenesis

Ghulam Hussain Syed

Institute of Life Sciences, Bhubaneswar, India

Mitochondria are dynamics organelles and undergo cycles of fusion and fission
(fragmentation). The mitochondrial dynamics and mitochondrial-selective autophagy
(mitophagy) in tandem contribute to rapid turnover of damaged mitochondria and maintenance
of mitochondrial & cellular homeostasis. Many viruses target and exploit mitochondrial
functions to facilitate their proliferation and have evolved strategies to impede mitochondria-
associated antiviral signalling. The interactions between the virus and mitochondria regulate
the mitochondrial and cellular homeostasis and govern the outcome of viral infection. Upon
infection with Flaviviruses, such as Hepatitis C virus (HCV), Dengue virus (DV) and Japanese

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Encephalitis virus (JEV), we observe differential regulation of mitochondrial homeostasis in
the host cells. Both HCV and DV cause mitochondrial injury, however HCV promotes
mitochondrial fragmentation and mitophagy to sustain mitochondrial homeostasis; in contrast,
DV inhibits mitophagy and perturbs mitochondrial homeostasis. HCV-induced mitochondrial
quality control is pivotal to sustain cellular homeostasis and promote viral persistence. On the
contrary, defect in mitochondrial quality control in DV-infected cells leads to higher
inflammation and death of infected cells. Our observations suggest that the complex
interactions between viruses and host cell mitochondria govern the outcome of viral infection
and associated diseases.

Impact of autophagy on Japanese encephalitis virus

replication and pathogenesis
Manjula Kalia

Regional Centre for Biotechnology, NCR-Biotech Science Cluster, Faridabad, Haryana

Autophagy is crucial for the modulation of the innate and adaptive immune response against
viral and bacterial pathogens. Several viruses can lead to the induction of autophagy which is
closely linked to their propagation and/or pathogenesis. Flaviviruses that include Japanese
encephalitis Virus (JEV), West nile virus and Dengue virus are important arthropod-borne
viruses that cause human disease globally. The focus of our research group is to understand
how autophagy impacts the replication and pathogenesis of JEV. In recent years we have shown
that autophagy plays an anti-viral role in the context of JEV infection and is closely associated
with the generation of oxidative and ER stress in the virus infected cell. Using TMT based
quantitative proteomic analysis of JEV infected Atg5-deficient mouse embryonic fibroblasts
we have generated a comparative proteomic landscape of JEV infection in the context of
autophagy deficiency. Our recent observations from this study will be discussed in the talk.

Hydrogen Sulfide-Induced GAPDH Sulfhydration Disrupts

the DBC1-SIRT1 Interaction to Initiate Autophagy

Ashwani Kumar

Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh,

India 160036; Academy of Scientific and Innovative Research, Ghaziabad, India 201002.

Hydrogen sulfide (H2S) is a cytoprotective gasotransmitter, known to activate sirtuin 1

(SIRT1) and autophagy; however, the underlying mechanisms for both remain unknown.
Herein, we demonstrate that H2S sulfhydrates the active site cysteine of the glycolytic enzyme
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to induce its redistribution into the
nucleus. Importantly, nuclear localization of GAPDH is critical for H2S mediated activation of
autophagy. Inside the nucleus, GAPDH interacts with deleted in breast cancer 1 (DBC1, also

A12
known as CCRA2). DBC1 is known to interact with SIRT1 to inhibit its activity. Interaction
of GAPDH with DBC1 disrupts the inhibitory effect of DBC1 on SIRT1. Activated SIRT1
then deacetylates microtubule associated protein 1 light chain 3 beta (LC3) to induce its
translocation into cytoplasm and to activate autophagy. Additionally, we demonstrate the
physiological role of this pathway in autophagy-mediated trafficking of Mycobacterium
tuberculosis into lysosomes to restrict growth of intracellular mycobacterial cells. We believe
that this pathway plays an important role in other health benefits associated with H2S.

Unravelling lysosome biogenesis by correlative light electron

microscopy

Judith Klumperman

Section of Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht,
The Netherlands

Understanding membrane trafficking requires an integrated knowledge on the dynamics,

molecular composition and ultrastructural organization of the membranes under study.
Studying these different parameters, however, requires distinct sets of technologies, which is
why they are typically derived from separate samples. Consequently, our understanding of
membrane trafficking is based on an average of dynamic and molecular data and an average of
light or electron microscopy (EM) images. To be able to perform multiparameter analysis at
the level of a single membrane, we developed a novel approach in which we correlate live cell
imaging to 3D EM, using focused ion beam scanning electron microscopy (FIB-SEM) (Fermie
et al., Traffic, 2018). Hence, we visualize membrane trafficking events in live cells together
with molecular and functional probes and then directly link this to 3D ultrastructural
information. All this is done at the level of a single organelle, allowing integrated structure-
function microscopy of single organelles. We use this approach to study the roles of CORVET
and HOPS complexes in membrane trafficking. These multitasking complexes link membrane
fusion to cargo recognition, vesicle formation, signaling, and organelle motility and are
required for endocytosis, lysosome biogenesis, endosomal recycling, autophagy and
phagocytosis (van der Beek et al., J Cell Science, 2019). Mutations in these complexes are
increasingly found as causative genes for human pathologies with overlapping phenotypes.
Interestingly, we found that individual subunits can also function outside complexes, typically
in pathways that regulate transport of selective cargo’s (Pols et al., Nature Comm. 2013; Jonker
et al., Nature Comm., 2018). Currently we investigate the effects of recently discovered
disease-causing mutations in the HOPS complex subunit Vps41 on endocytosis, lysosome
biogenesis, regulated secretion and transport of lysosomal membrane proteins.

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Targeting of protein aggregates to lysosomes and
implication for neurodegenerative diseases
Thomas Wollert

Membrane Biochemistry and Transport, Centre Francois Jacob, 3er etage,

Room 26-03-07b, Institut Pasteur, 75015 Paris, France

The accumulation of protein aggregates is involved in the onset of many neurodegenerative

diseases. Aggrephagy is a selective type of autophagy that counteracts neurodegeneration by
recycling such aggregates. The molecular machinery involved in targeting autophagic
membranes to protein aggregates during autophagosome biogenesis has been well
characterized. However, later steps of autophagy including targeting of autophagosomes to
lysosomes are still not fully understood. Through a combination of classical biochemistry and
cell biology with cutting edge in vitro reconstitutions, we have been able to discover a selective
pathway that targets autophagosomes to lysosomes during aggrephagy. A dedicated set of
autophagy proteins is involved in this pathway and their close cooperation is required to target
autophagosomes to lysosomes for degradation. Importantly, neural cells are particularly
sensitive to perturbations of this pathway, demonstrating that aggrephagy is essential to
maintain neural homeostasis.

Phosphatome modulation promotes the lysosome

function/biogenesis in LSDs

Subba Rao Gangi Setty

Department of Microbiology and Cell Biology, Indian Institute of

Science, Bangalore 560012 India

Lysosomes function as primary site for catabolism and cellular signaling. These
organelles receive cargo through endocytosis and autophagy processes and degrade
them into basic cellular components by a variety of acid hydrolases. These enzymes
are synthesized in the ER and then transported to lysosomes via Golgi. Inability in
the function or defective trafficking of lysosomal hydrolases lead to the
accumulation of intermediate degradative substrates, results in lysosomal storage
disorders (LSDs). In Gaucher’s disease, lysosomes accumulate glucosylceramide
due to mutation/s in GBA1 (encode β-glucocerebrosidase, b-GC) that causes
enlargement, dysfunction and activation of signaling pathways. We hypothesize that
increased lysosome function/biogenesis by altering cellular pathways possibly cure
or delay the disease progression in Gaucher’s and other LSDs. Phosphatases are one
of the key molecules known to regulate different cellular pathways and maintain
organelle homeostasis through post-translational modification. We used genome-
wide RNAi screen against the cellular phosphatases in HeLa cells and measured the
lysosome enzyme activity using biochemical assay. Our screen identified few
phosphatases, whose knockdown improved the lysosome enzyme activity by

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increasing the trafficking of lysosomal hydrolases or altering organelle distribution
and/or biogenesis. Similarly, Gaucher’s patient fibroblasts restored the lysosome
function upon depletion of these phosphatases. Interestingly, our studies showed
individual phosphatases variably modulated the b-GC activity in patient cells
carrying different Gaucher’s mutations suggesting that multiple mechanisms exist to
cure the disease. Overall, these studies provide evidence that altering phosphatome
activity possibly cure LSDs and may form an alternative therapeutic strategy for
these genetic diseases.

Regulation of osteoclast function by lysosomal GTPase Arl8b

Amit Tuli

Institute of Microbial Technology,Sector 39A, Chandigarh, India

Recent studies have established Arl8b-mediated positioning of lysosomes and lysosomes-

related organelles as a crucial factor regulating amino acid sensing, cell migration and
metastasis, NK cell-mediated cytotoxicity and antigen presentation. Arl8b mediates lysosomal
transport to the cell periphery by recruiting its effector, SKIP, which in turn recruits the motor
protein kinesin-1 on lysosomes. Arl8b also binds to the Rab7 effector- PLEKHM1, and this
interaction repositions Arl8b-positive lysosomes to the perinuclear region of the cell and
promotes autophagosomes-lysosome fusion. Interestingly, frameshift mutations in PLEKHM1
result in Osteopetrosis where the bone resorbing functions of osteoclasts is impaired.
Osteoclasts resorb bone by secreting their lysosomal contents within the confines of a sealing
zone between themselves and the bone surface. Here, we have explored the role of Arl8b in
bone remodeling and identified a novel effector of Arl8b that is specifically expressed in
osteoclasts. Arl8b showed a striking localization beneath the actin rings and ruffled borders in
osteoclasts, and lysosome secretion was significantly impaired in Arl8b-deficient osteoclasts.
Further, unlike control osteoclasts, lysosomes in Arl8b-deficient osetoclasts failed to localize
beneath the actin rings or in the ruffled borders. Taken together, our findings establish Arl8b
as a crucial component of osteoclast-mediated bone remodeling.

From Immunity to Skin: Adaptations of the endo-lysosomal

system in Intercellular communication
Graça Raposo

Department of Cell Biology and Cancer, Head Structure and Membrane

Compartments, Institut Curie, CNRS UMR144
Our major goals are to shed light on how the endocytic pathway and how the component
organelles are altered in disease such as cell transformation, neurodegenerative and lysosomal
diseases and pigmentary disorders. We aim to gain a better understanding of the biogenesis and

A15
functions of two such specialized endosomal organelles: exosomes, which are secreted from
multivesicular bodies, and melanosomes, the lysosome related organelles of melanocytes.
Melanosomes are cell type-specific organelles within retinal pigment epithelial cells and
epidermal melanocytes in which melanin pigments are synthesized and stored. Our studies have
started to unravel the structural and molecular mechanisms involved in early and late
melanogenesis but also general features of intercellular communication in the skin between
melanocytes and keratinocytes. We have addressed, the formation of the amyloid-like fibrils
and the trafficking and sorting of melanosomal proteins in normal and diseased states. Our
studies delineate the endo-melanosomal system of melanocytes but also uncover novel
concepts of protein processing, endosomal sorting and motility. Altogether our research opens
new avenues to understand the pathogenesis of the Hermansky-Pudlak Syndrome (HPS) and
Ocular Albinism (OA1) and the formation of amyloid fibrils in neurodegenerative diseases.
Recent studies reveal the cellular and molecular basis of intercellular communication in the
skin with consequences on skin homeostasis and pigmentary disorders.

Adaptive endo-lysosomal homeostasis as a defining feature of

Mtb infected macrophages

Varadharajan Sundaramurthy
NCBS, Bengaluru

Intracellular pathogens commonly manipulate the host trafficking pathways for their survival,
however whether or how this affects the organization and functioning of the concerned
pathways itself is not clear. Here, we show that the endo-lysosomal system is globally altered
in M. tuberculosis infected macrophages relative to uninfected cells, thus defining an altered
homeostasis upon infection. These alterations are robust and indeed distinguish the infected
cell population. Alterations in endosomes and lysosomes, while related, are caused by different
mechanisms. In this talk, I will discuss these mechanisms, the consequences of this alteration,
its influence on pathogenesis outcome and a broader endocytosis-phagocytosis nexus.
Chemical and genetic perturbation of the adaptive homeostasis point to a finely nuanced
negotiation between the host and the pathogen that determines the ultimate balance between
the two. These results define the global alterations in the endo-lysosomal system as an
important homeostatic feature of M. tuberculosis infected macrophages and provide insights
into the cellular determinants of the fine balance operating between host and pathogen.

Microtubule post-translational modifications spatially

constrain lysosomes to regulate autophagy

Nitin Mohan
Biological Sciences and Bioengineering Department, IIT Kanpur, Uttar Pradesh, India

Autophagy is a vital cellular process where the phagosome double membrane invaginates
around the protein aggregates to be degraded, followed by sequestration of membrane
proteins-Atg8/LC3 to form mature autophagosome, which then is actively transported by motor

A16
proteins on microtubules to eventually fuse with lysosome to form autolysosomes, where the
proteins will be degraded by the lysosomal hydrolases. Since, both autophagosome and
lysosome are highly mobile organelles, their transport regulations would play significant role
in effective autophagy. Here we explore the role of microtubule post-translational
modifications (PTMs) in regulating lysosome and autophagosome trafficking and the role of
microtubule PTMs in autophagy. We used super-resolution microscopy method STORM to
quantify different microtubule PTMs and sub-populations in epithelial cells. Our results show
that acetylated and tyrosinated microtubules are predominant subsets of microtubules while
only a small subset (30% of the total microtubule) is detyrosinated. Interestingly, a large
fraction of lysosomes is enriched on the detyrosinated microtubules. Also, motility of lysosome
is hindered on detyrosinated microtubules. Furthermore, autophagosomes and autolysosomes
showed similar enrichment and motility behavior on detyrosinated microtubules. Finally,
correlative live-cell and STORM imaging showed that detyrosinated microtubules facilitate
efficient autophagosomes and lysosome fusion, and depletion of detyrosinated microtubules
led to significant reduction in autophagy. Taken together our study brings forth a new role for
microtubules detyrosination in regulating autophagy.

SUMO tug-of-war at epithelial-immune cell interface mediate

intestinal homeostasis
Chittur V. Srikanth

Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad

Uncontrolled inflammation is a major culprit in a number of gut illnesses including

inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn’s disease (CD) are two
forms of IBD, involving chronic overactivation of inflammatory mechanisms in the gut leading
to severely compromised lifestyle. While a lot of research has gone in, complete understanding
of the mechanisms that mediate the pathophysiology of IBD is not well understood. Here using
a mutli-pronged methodology, we have embarked studies to investigate the role of a
SUMOylation a post-translation modification (PTM) mechanisms in IBD. In a range of
different model systems including human IBD patient biopsies, a dramatic alteration in several
SUMOylation pathway genes was observed. In line with this, a distinct SUMO-modified
proteome (SUMOylome) was observed in inflamed tissue. Regulators of crucial mechanisms
such as vesicular transport pathway (VTS) and immunocyte signaling were part of the altered
SUMOylome in inflamed tissues. These changes appeared to be crucial in tilting epithelial-
immune cell signaling balance and intestinal homeostasis. In vivo perturbations of VTS
pathway regulator Rab7 in mice model of IBD showed an exacerbated inflammation.
Furthermore, Rab7 expression in Paneth cells and Goblet cells of mice intestine was observed
to be altered mice model of IBD, hinting towards a potential connection of Rab7 in
antimicrobial peptide secretions. The precise role of SUMOylation in epithelial-immune cell
crosstalk and its connection to IBD is being investigated. We anticipate that these mechanisms
may potentially offer avenues for novel means of therapeutic interventions.

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Uncovering novel roles for PIKfyve in cell migration

Lois Weisman

Life Sciences Institute and Department of Cellular and Developmental

Biology, University of Michigan, Ann Arbor, MI

PIKfyve is the sole lipid kinase that converts phosphatidylinositol-3-phosphate to

phosphatidylinositol-3,5-bis phosphate (PI(3,5)P2). PIKfyve is also responsible for most of the
cellular pools of PI5P. These low abundance lipids are critical for cell function. Minor
mutations in proteins that regulate PIKfyve have been linked to various neurological diseases,
including Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis, while more severe
mutations impact multiple organs. Inhibition or depletion of PIKfyve results in enlargement of
endosomes and lysosomes and impairs lysosomal function, which led to the view that the main
roles for PIKfyve are at the lysosome. Our recent studies at the neuronal synapse, revealed that
PIKfyve plays a role in the dynamic recycling of AMPA-receptors, and that this role most
likely does not involve lysosomes. However, the mechanistic basis for this role of PIKfyve was
not known. Here we show another situation where PIKfyve plays a role in a recycling pathway,
and in this case, we uncovered molecular insight into how PIKfyve is involved. We and others
found that inhibition of PIKfyve inhibits cell migration. Here we show that conversely,
activation of PIKfyve promotes cell migration. Insight into how PIKfyve regulates cell
migration, came from our finding that the levels of β1-integrin on the cell surface are altered
concomitant with changes in PIKfyve activity. β1-integrin is a transmembrane protein that
binds to both the extracellular matrix, as well as regulators of the actin cytoskeleton, and is a
major regulator of focal adhesion complexes. Thus, β1-integrin plays a key regulatory role in
cell migration. Notably, we found that PIKfyve inhibition causes depletion of β1-integrin at
the cell surface through defects in recycling of β1-integrin from endosomes to the plasma
membrane. Insights into how PIKfyve might regulate β1-integrin trafficking, comes from our
finding that PIKfyve colocalizes with SNX17-Retriever-CCC complex proteins, and that the
intracellular location of this complex is defective during PIKfyve inhibition. The SNX17-
Retriever-CCC complex was previously shown to regulate trafficking of β1-integrin from
endosomes back to the plasma membrane. Together these findings suggest that some lipids
regulated by PIKfyve may be important for the regulation of this retrograde traffic pathway.

Regulation of membrane dynamics by phosphoinositides

Raghu Padinjat

National Centre for biological Sciences-TIFR, GKVK Campus, Bellary

Road, Bangalore 560065. India

Phosphoinositides have been implicated in multiple aspects of membrane dynamics including

autophagy. While the roles of phosphoinositide 3 phosphate are well understood, the function
of other phosphoinositides in these processes remains obscure. I will present our work on the
role of phosphatidylinositol 5 phosphate 4-kinase in the regulation of membrane dynamics
including autophagy.

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Methionine as a growth signal and anabolism regulator
Sunil Laxman

inStem, Bangalore

The amino acid methionine can strongly inhibit autophagy and regulate growth. Even
during amino acid limited conditions, the presence of methionine can strongly trigger growth
and proliferation in cells. Indeed, decades-old studies suggest that many cancers are dependent
on methionine metabolism. Using a simple model system (yeast) we recently discovered that
methionine controls a strong growth program, which is organized hierarchically. Here, cells
activate ribosome biogenesis, as well as key nodes in carbon and nitrogen metabolism, and
induce general amino acid biosynthesis to fuel an anabolic state and rapid cell growth
(Walvekar et al MBoC 2018). Surprisingly, we now discover that this anabolic program
depends on the conserved transcription factor Gcn4/Atf4, which is normally studied in the
context of survival/autophagy and not growth. I will discuss new, unpublished studies that
show how Gcn4/Atf4 directly as well as indirectly enables growth, by producing anabolic
precursor molecules that sustain high rates of translation and nucleotide synthesis. This Gcn4
induction is required for the synthesis of most amino acids, but notably arginine, which helps
cells maintain translation capacity. Collectively, we decipher highly conserved processes
through which methionine can act as a universal growth signal in cells, to drive cell
proliferation.

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Abstracts of Speakers
Day-3
(18.01.2020)

A20
A common genetic pathway for cargo retention and polarized
cargo distribution in neurons
Sandhya P. Koushika
DBS-TIFR, Mumbai, India

Neurons are polarized cells where cargo such as synaptic vesicles are present in the axonal
compartment and excluded from the dendrite. Using the C. elegans model we identified a
genetic pathway that controls biogenesis of synaptic vesicles leading to polarized cargo
distribution. The same genetic pathway also appears critical in retention of lysosomes in the
cell body. We find that UNC-16/JIP3, a MAPK scaffolding molecule, is essential for exclusion
of golgi resident enzymes from the synaptic vesicle protein transport carrier. UNC-16/JIP3
recruits LRK-1/LRRK2, the gene mutated in some forms of familial Parkinson’s disease, to
the golgi. LRK-1 regulates the inclusion of different synaptic vesicle proteins into a single
transport carrier through the adaptor protein complex AP3 and the size of the transport carrier
through the adaptor protein complex AP1. LRK-1 also regulates polarized distribution of
synaptic vesicle proteins through AP1 and helps retain a lysosomal protein in the cell body.
We identified a novel role of SYD-2/Liprin-α, a synapse assembly protein, in the LRK-
1/LRRK2-AP complex genetic pathway. We see that SYD-2 and AP1 act redundantly to
prevent entry of synaptic vesicle proteins into the dendrite. Likewise SYD-2 and AP3 act
redundantly to prevent entry of lysosomal proteins into the axon. These early biogenesis steps
appear essential for regulated exit of pre-SVs into the axon using the synaptic vesicle kinesin
motor UNC-104/KIF1A. The LRK-1-SYD-2-AP complexes dependent biogenesis steps of
inclusion and motor recruitment are critical for polarized distribution of synaptic vesicle
proteins. Further, the same genetic pathway appears to differentially regulate synaptic vesicle
protein transport carriers and lysosomes.

Tunneling nanotubes: the novel long range intercellular

pathway of pathology spreading exploits PAK1 kinase
dependent macropinocytic vesicle recycling

Sangeeta Nath
Manipal Institute of Regenerative Medicine, Manipal Academy of Higher
Education, Bangalore, 560065, India

Alzheimer’s disease (AD) pathology progresses gradually through the anatomically connected
brain regions. The progressive pathology development in AD is due to direct transfer of
amyloid-β1-42 oligomers (oAβ) between connected neurons. However, the mechanism of
transfer is not revealed yet. We observe that tunneling nanotubes (TNTs) propagate oAβ and
organelles directly from one cell-to-another. TNTs are nanoscaled, F-actin containing
membrane nanotube connections between neighboring cells. Interestingly, preceding the
formation of TNTs, we detect oAβ induced plasma membrane damage and repair through
lysosomal-exocytosis and significant membrane surface expansion, followed by rapid
endocytosis to re-establish the plasma membrane. The rapid endocytosis causes spontaneous
internalization of exogenous oAβ and ultimately gradual accumulation in lysosomes. Rapid

A21
internalization/endocytosis of toxic exogenous oAβ occurs via PAK1 (p21 activated kinase)
kinase dependent macropinocytosis. Moreover, formation of TNTs can be inhibited by
inhibiting PAK1 kinase dependent pinocytic vesicles recycling and actin re-modulation.
Phenomena are independent of cell types and differentiation stages of neuronal cells (SH-
SY5Y). Direct transfer of PrPSc, α-synuclein, Aβ, tau and polyQ via tunneling nanotubes
(TNTs) has recently been suggested as means of pathology spreading in neurodegenerative
diseases. However, molecular basis of TNTs formation is unexplored. The present study gives
the insight that sprouting of TNTs might instigate as consequences of oAβ induced membrane
damage and rapid membrane repair process via PAK1 kinase dependent recycling of pinocytic
vesicles and actin remodeling, probably to maintain cell surface expansion and/or membrane-
tension in equilibrium.

Membrane Fission: Insights from Reconstituting Organelle

Form and Chemistry

Thomas J. Pucadyil
Indian Institute of Science Education and Research, Pune

The lipid bilayer is highly resilient to rupture and explains why it was selected over
the course of evolution to serve a barrier function. Yet fission, or the splitting of a membrane
compartment, is a central theme in biology that manifests during cell division, organelle
biogenesis and vesicular transport. Fission involves the local application of forces to bend and
constrict or thin down a membrane tube. Since bending requires the bilayer to deviate from its
preferred planar configuration, fission is energetically unfavorable. Using reconstitution
approaches that involve biochemical screens, we have discovered novel proteins that catalyze
fission and have elucidated their functional basis. My talk will describe these recent
developments.

Short talk: Rabip4’ interacts with the small G protein Arl8b and regulates
membrane trafficking in the endolysosomal pathway
Shalini Rawat

Department of Biological Sciences, Indian Institute of Science Education and Research Mohali
(IISERM), Punjab, India

Lysosomes are primarily known for their catabolic function in degradation of cellular
macromolecules, but recent findings reveal that lysosome is also a site for processes like
nutrient signalling, plasma membrane repair, and in the long-range transport of RNA.
Lysosomal sub-cellular distribution and fusion with other compartments govern its diverse
roles in the cell. Our research group is interested in small GTP binding protein, Arl8b which
localizes on lysosomes and via interaction with its effectors proteins regulates lysosomal
positioning and cargo trafficking to lysosomes. Here we report a new Arl8b interaction partner,
Rabip4' (RUN and FYVE domain-containing protein) that interacts with Arl8b via its N-
terminal RUN domain. Rabip4’ localizes to the early endocytic compartments positive for

A22
Rab14 and early endosomal antigen (EEA1). Our findings suggest that both Arl8b and Rabip4'
are present on a subset of early endocytic compartments that also contain Rab14 and the Cation-
Independent Mannose-6 phosphate receptor (CI-M6PR). Arl8b siRNA treated cells show
redistribution of endogenous Rabip4’ to the cytosol, suggesting that Arl8b is required for stable
membrane localization of Rabip4’.To investigate the role of Rabip4’ in late endocytic
compartments, we visualized the late endosomes/lysosomes in Rabip4’siRNA treated cells.
Depletion of Rabip4' leads to an increase in the size of LAMP1-positive compartment and
enhanced accumulation of lysotracker dye that is retained only in acidic organelles. We are
exploring the mechanism by which Rabip4' is regulating morphology, composition and
function of late endocytic compartments and also want to determine the relevance of its
interaction with Arl8b in this process.

Nanoscale Organization of amyloidogenic machinery at

excitatory synapse
Deepak Nair
Centre for Neuroscience, Indian Institute of Science

Amyloid precursor protein (APP) is a key player in Alzheimer’s disease (AD). Despite the
importance of APP in the generation of Aβ, there exist few attempts to dissect the molecular
localization of APP at excitatory synapses. With the help of microscopy and nanoscopy, we
evaluate the compartmentalization of major components of amyloidogenic machinery at
functional zones of the synapse. We incorporate the spatio-temporal details of amyloidogenic
machinery using insilico experiments in reconstructed physiology of the CA1 dendrites. Our
paradigm of integrating nanoscale localization and real-time trafficking of APP in conjunction
with biophysically realistic 3D models of spines allows us to predict the modulation of APP
processing by amyloidogenic pathway. We predict that the dynamic molecular organization of
APP and secretases at the endocytic zone might serve as a pivotal link in amyloidogenic
processing of APP. Thus, we illustrate fundamental mechanisms where alterations in real-time
nanoscale association of components of amyloidogenic machinery may contribute towards
long term deficits like those seen in AD.

Regulatory Mechanisms of Clathrin-Mediated Endocytosis:

Fishing for ON-OFF Switches.

Perunthottathu K. Umasankar

Intracellular Trafficking Laboratory, Interdisciplinary Biology Research

Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala,
India

Clathrin-mediated endocytosis (CME) is a major vesicular transport pathway pivotal for

lysosome biogenesis and function. Lack of evident GTPases for initiation separates CME from
other membrane trafficking events. This also poses the obvious question of how CME initiates

A23
and progresses in cells. We recently discovered that Fes / CIP4-Homology domain (FCHO)
proteins trigger clathrin-coated pit (CCP) formation by allosterically activating the central
clathrin adaptor protein complex, AP-2 on plasma membrane. We now demonstrate that this
activation promotes AP-2 phosphorylation via recruitment and stabilization of a bona fide AP-
2 kinase- BMP2K, leading to CCP maturation. Accordingly, FCHO knockouts defective in AP-
2 activation or BMP2K mutants that cannot localize to CCPs fail to induce phosphorylation. In
addition, re-expression of FCHO, but not kinase, rescues phosphorylation defects in FCHO
knockout cells implying membrane activation of AP-2 is a prerequisite for kinase function.
Functional inactivation of kinase in the presence of FCHO impairs AP-2 phosphorylation
leading to altered lattice morphology and CME phenotypes reminiscent of CCP maturation
defects. Further, our in vivo studies show that gain- and loss-of function phenotypes of FCHO
and BMP2K are analogous and mirror altered AP-2 functions during zebrafish embryogenesis.
Together, our findings reveal FCHO-AP-2-BMP2K feed-forward axis for operation of CME.

Endocytic-Exocytic Crosstalk during Cytokinesis

Sivaram V S Mylavarapu
Regional Centre for Biotechnology, Faridabad
Cytokinesis is the final step of cell division following chromosome
segregation that generates two daughter cells. The conserved exocyst complex is
required for scission of the intercellular cytokinetic bridge, although the molecular mechanisms
it employs in this process are unclear. We identify and validate the early endocytic GTPase
Rab5 as interacting with the exocyst complex in mammalian cells. Rab5 localizes in the
cytokinetic bridge at the midbody ring in a manner similar to the exocyst complex. Depletion
of Rab5 led to delayed abscission. Caenorhabditis elegans orthologs of both exocyst complex
subunits and Rab5 localize along the cleavage furrow and are required for cytokinesis in early
embryos. Cytokinetic cells depleted of either Rab5 or the exocyst subunits Exoc3 and Exoc4
showed impaired deposition of the endosomal sorting complexes required for transport
(ESCRT) III subunits CHMP2B and/or CHMP4B near the midbody ring. The study reveals an
evolutionarily conserved role for the early endocytic marker Rab5 in cytokinetic abscission. In
addition, it uncovers a key requirement of the exocyst and Rab5 for the delivery of components
of the membrane-severing ESCRT III machinery to complete cytokinesis.

Short talk: BRD4, an epigenetic Reader, orchestrate mycobacterial

pathogenesis by co-modulating host angiogenesis and lipophagy

Bharat Bhatt

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore

Mycobacterium tuberculosis (Mtb), etiological agent of tuberculosis (TB) skews host immune
responses in order to benefit its own survival. Lipid homeostasis and angiogenesis are two
critical cellular processes which are deregulated during progression of tuberculosis and are
coupled with dissemination of Mtb along with delivery of potent anti- mycobacterial drugs to

A24
granuloma. However, the role for epigenetic modifiers in governing TB granuloma- associated
angiogenesis and lipid homeostasis modulation remain poorly explored. Here, we unravel the
role for histone acetylation reader, bromodomain containing protein 4 (BRD4), in Mtb
pathogenesis. We demonstrate that BRD4 modulates the expression of angiogenic genes in in
vitro and in vivo models of Mtb infection. We found that Mtb-activated Epidermal Growth
Factor Receptor (EGFR) signaling induces BRD4, which in conjunction with KLF5
differentially regulates angiogenic markers such as Vegfa and Vegfr2. In addition to this, lipid
accumulation by Mtb was perturbed upon inhibition of BRD4. Using ptf-PLIN2 construct and
ATG5 knockdown approaches, we observed that, inhibition of BRD4 activity during Mtb
infection promote lipophagy (autophagic degradation of lipids) accounting for reduced lipid
accumulation. Altogether, we demonstrate by employing both in vivo TB mouse model and in
vitro studies, that pharmacological inhibition of BRD4 normalizes angiogenesis and augments
lipophagy thereby restricting Mtb growth and subsequent severe TB pathology.

Short Talks
Differentiated keratinocytes generate unique Golgi-associated lysosomes

Sarmistha Mahanty

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore

Epidermis, the outer skin layer provides hydrophobic and antimicrobial barrier to the body and
is critical in maintaining skin health and hygiene. Keratinocytes are the major constituent cell
type of epidermis which maintains skin homeostasis through cellular differentiation. However,
the molecular link between keratinocyte differentiation and skin homeostasis is critically
unknown. In that end, we have used human neonatal primary keratinocytes and differentiated
the cells using high extracellular calcium. We demonstrated that upon differentiation,
keratinocytes produce large number of peripherally distributed globular lysosomes.
Differentiated keratinocytes also exhibit high autophagy turnover which corroborates to the
enhanced lysosomal activity. Further, both these processes are independent of mTOR signaling
and is regulated by ER stress induced UPR pathway. Notably, differentiated cells showed
highly fragmented and dispersed Golgi. Fluorescence microscopic analyses illustrated that
certain Golgi proteins are associated with the lysosomes of differentiated keratinocytes,
suggesting a possible influence of Golgi in the secretion of these lysosomes. These organelles
retain classical lysosomal characteristics despite they are associated with the Golgi proteins.
As expected, disruption of Golgi function or its association led to the malformation/tubulation
of the lysosomes and affected keratinocyte differentiation. However, how this Golgi-
association is important for lysosomal secretion and epidermal homeostasis is currently under
investigation. In future, we would like to identify the skin diseases wherein the Golgi-
associated lysosomal activity is altered.

TFEB-mediated xenophagy: a host defence pathway involved in restricting

intracellular pathogens
Veena Ammanathan

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Bangalore, India.
A25
Xenophagy (Autophagy of pathogens) is considered as one of the host innate immune response
to intracellular pathogen invasion. Modulating xenophagy is proven beneficial to prevent
pathogen replication and thereby ameliorate disease phenotype. However, pathogens have
evolved ways to subvert xenophagy to facilitate their survival. Hence, restoration of xenophagy
block imposed by pathogens through genetic or pharmacological methods would enhance the
clearance of intracellular pathogens and provide insights into host-pathogen mediated
molecular events that control xenophagy. Yeast based high throughput screening performed
previously in our laboratory have identified a number of autophagy inducers and inhibitors. I
further screened the autophagy inducers to check for their ability to clear intracellular
Salmonella typhimurium. Screening yielded a compound, which showed more than two fold
decrease in intracellular pathogen burden. The identified compound has following
characteristics: 1) it induces host xenophagy process and does not directly affect the pathogen
growth, 2) through post transcriptional mechanisms, enhances recruitment of xenophagy
mediators to efficiently capture the pathogen, 3) acts through an autophagy and lysosomal
biogenesis transcriptional master regulator TFEB, 4) activation of TFEB increases the active
lysosomes in cells leading to enhanced fusion with Salmonella containing vacuoles thereby
restricting its replication, and 5) is effective in an in vivo mouse model of bacterial infection.
Additionally, the interplay between xenophagy process and other innate immunity pathways is
probed by microarray analysis. Putative regulators from microarray analysis are accessed for
their role in modulating TFEB nuclear translocation and xenophagy mediated clearance.

Antimycobacterial effect of IFNG (interferon gamma)-induced autophagy

depends on HMOX1 (heme oxygenase 1)-mediated increase in intracellular
calcium levels and modulation of PPP3/calcineurin-TFEB (transcription
factor EB) axis

Navin Baid

CSIR-IMTECH, Chandigarh

IFNG (interferon gamma)-induced autophagy plays an important role in the elimination of

intracellular pathogens, such as Mycobacterium tuberculosis (Mtb). However, the signaling
cascade that leads to the increase in autophagy flux in response to IFNG is poorly defined.
Here, we demonstrate that HMOX1 (heme oxygenase 1)-generated carbon monoxide (CO) is
required for the induction of autophagy and killing of Mtb residing in macrophages in response
to immunomodulation by IFNG. Interestingly, IFNG exposure of macrophages induces an
increase in intracellular calcium levels that is dependent on HMOX1 generated CO. Chelation
of intracellular calcium inhibits IFNG-mediated autophagy and mycobacterial clearance from
macrophages. Moreover, we show that IFNG-mediated increase in intracellular calcium leads
to activation of the phosphatase calcineurin (PPP3), which dephosphorylates the TFEB
(transcription factor EB) to induce autophagy. PPP3-mediated activation and nuclear
translocation of TFEB are critical in IFNG-mediated mycobacterial trafficking and survival
inside the infected macrophages. These findings establish that IFNG utilizes the PPP3-TFEB
signaling axis for inducing autophagy and regulating mycobacterial growth. We believe this
signaling axis could act as a therapeutic target for suppression of growth of intracellular
pathogens.

A26
A Redox-dependent Membrane Fission Catalyzed by α-synuclein
Krishnendu Roy
IISER Pune, India
α-synuclein (AS) is a vertebrate-specific, abundant neuronal protein that binds negatively-
charged membranes. Several lines of evidence causally link its tendency to aggregate into
extracellular plaques to the pathogenesis of Parkinson's Disease (PD). Despite these reports,
very little is known about the native function of AS. AS is intrinsically unstructured in solution
and adopts a partial helical conformation on membranes. Previous reports using very high
protein concentrations have suggested that membrane-bound AS can deform liposomes into
narrow tubules and small vesicles. Using a novel method to assay membrane binding properties
of peripherally-associated proteins, we discover that AS displays preferential affinity for the
mitochondrial lipids cardiolipin (CL) and phosphatidyl glycerol (PG). We then tested AS
functions on CL-containing supported membrane tubes that mimic mitochondria in membrane
topology and lipid composition. Such experiments showed no apparent membrane remodeling
or vesiculation. Remarkably however, similar assays carried out under conditions that mimic
the intracellular reducing environment showed dramatic fission of tubes. Together, our results
point to the possibility that AS functions in a novel redox-dependent mitochondrial fission
pathway.

Adaptive immune cells control lipophagy to maintain dermal white adipose

tissue (DWAT) homeostasis

Edries Yousaf Hajam

Centre for the Regulation of Cell Fate, Institute for Stem Cell Science and Regenerative
Medicine, Bangalore

Homeostasis is not a static process as the tissues in our body continuously undergo various
changes to maintain their structure and function. For instance, the various compartments of our
skin, such as the hair follicle and DWAT (dermal white adipose tissue) undergo continuous
cyclic changes of growth and regression. Understanding the mechanism of cyclic changes in
DWAT is important as these changes are known to play crucial roles in homeostasis and disease
conditions. During homeostasis, various immune cells undergo cyclic changes in numbers as
well as in the activation status and are in sync with cycling of DWAT. Thus, we asked if
inflammatory cells regulate the cycling of DWAT in the skin. To investigate this hypothetical
link, we employed a mouse model with functionally impaired regulatory T cells (T-regs),
(which are known to supress both innate and adaptive immune cells). Strikingly, we observed
a complete loss of DWAT in the mouse model with impaired T regs. Mechanistically we found
that CD4+ and CD8+ T cells create an inflammatory microenvironment prior to the loss of
DWAT. Rescue experiments suggest that CD4+ T cells have a very specific role to play in
DWAT homeostasis. Further, we observed that lipophagy is the main pathway (and not
lipolysis) that causes the loss of DWAT. Thus, we found a novel link of an adaptive immune

A27
cell in controlling DWAT homeostasis. Currently we are investigating how CD4+ T cells
induce autophagy in the DWAT.

Itinerary of Abeta42 whilst under nicotine influence– a lysosome related

story
Viji Vijayan

Molecular Science Lab, National Institute of Immunology, New Delhi

Nicotine, the addictive substance of tobacco is speculated to have a role in the etiology of
Alzheimer’s disease (AD). This alkaloid is fundamentally a hydrophobic weak base that can
cross plasma membrane and organelle membranes. But when such small molecules cross
lysosomal membrane and encounter the acidic lumen of the lysosome, these get protonated
hampering its transport back to the cytoplasm. We hypothesized that this lysomotropic
behavior of nicotine may hamper the Abeta42 degradation machinery of brain cells notably the
astrocytes. Astrocytes are not professionally phagocytic cells, but these do produce several
enzymes that degrade Abeta plaques. In our study we found that nicotine exposure led to higher
uptake of Abeta42 and increased sequestration of Abeta42 in cell without degradation.
Molecular analysis of proteins involved in uptake of Abeta42 demonstrated that the higher
uptake was owing to augmented CD36/TLR2/MyD88 signaling while sequestration occurred
due to defective Rab7 signaling. We found that nicotine augmented CD36/TLR2/MyD88
signaling facilitated greater interaction of MyD88 (a central player in innate immune signaling)
with mTORC1 (a regulator of the subcellular localization of TFEB) causing cytosolic retention
of TFEB (the master regulator of lysosome biogenesis) and defective lysosomal protein
expression and activity. Absence of MyD88 did not evoke cytosolic sequestration of TFEB
despite nicotine treatment demonstrating that innate immune signaling in astrocytes do play
critical role in lysosome biogenesis. Consequences of such altered lysosome biogenesis
induced by nicotine included increased synthesis and release of pathogenic extracellular
vesicles (EV) harboring Abeta42 and cytotoxic cytokines like TRAIL. These Evs potentially
transferred Abeta42 cargo to healthy neurons and induced apoptosis, not seen in Abeta42 alone
treated cells. To conclude, nicotine mediated cross-talks between innate immune signaling and
mTORC1/TFEB signaling in astrocytes alters amyloid degradation machinery of astrocyte and
creates a pathogenic milieu for development of neurodegenerative diseases.

Investigating the roles of SYD-2/Liprin-α and LRK-1/LRRK2 as common

genetic regulators of lysosomal and synaptic vesicle proteins
Sravanthi SP Nadiminti

Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha

Road, Navy Nagar, Colaba, Mumbai

Distinct compartments of the neuron i.e. dendrites, axons, and the synapse have characteristic
protein compositions that allow them to perform different roles in neurotransmission. Polarized
distribution of proteins to axons and dendrites is a result of protein trafficking events that
happen at the neuronal cell body. We show that SYD-2/Liprin-α, a synapse assembly protein,

A28
and LRK-1/LRRK-2, a protein mutated in familial Parkinson’s disease, regulate the polarized
trafficking of lysosomal and synaptic vesicle (SV) proteins. We show that LRK-1, SYD-2 and
the AP3 adaptin complex act to restrict lysosomal proteins to neuronal cell body. Additionally,
these proteins facilitate the inclusion of different SV proteins into one transport carrier, thus
regulating SV membrane composition during SV biogenesis in the cell body. These data
suggest that SV and lysosomal proteins have common genetic regulators for their polarized
distribution and biogenesis. We are testing whether SV and lysosomal proteins undergo similar
trafficking events until they are separately sorted away into distinct membrane compartments.

A29
Abstracts of Poster
presenters
Day-1
(16.01.2020)

A30
Poster: A1

Activation of autophagy by nitazoxanide through mitochondrial

uncoupling confers protection in cellular and animal models of Parkinson’s
disease
A. Niharika Reddy, Srinivas N Puttapaka, TVSS Swaroop and Shasi V Kalivendi

Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad

Parkinson’s disease (PD) is a chronic neurodegenerative disorder, which is characterized by

the loss of dopaminergic neurons in the Substantia nigra pars-compacta region and intra-
cellular a-synuclein positive aggregates called ‘lewy bodies’. The accumulation of these
aggregates disrupts proteostasis machineries such as chaperones, proteasome or autophagy
leading to neuronal degeneration. Amongst the proposed pathways responsible for the
pathogenesis of PD, mitochondria were found to play a precipitating role. Hence, small
molecules that nullify mitochondrial ROS and relieve inhibition of oxidative phosphorylation
by Parkinson’s neurotoxins are being considered as promising therapeutic candidates. In the
present study, we have identified that the FDA approved anti-protozoan drug, nitazoxanide
(NTZ) dose-dependently enhanced mitochondrial respiration by enhancing Oxygen
Consumption Rate (OCR). Also, a dose-dependent decrease in ATP production with a
concomitant increase in the protein leak was observed by NTZ, all of which were found to be
the typical properties of a mild-uncoupler. NTZ mitigated MPP+-induced reduction in the
mitochondrial respiration, depolarization and cellular apoptosis as measured by Seahorse flux
analyzer, JC-1 staining and apoptosis. The mitochondrial uncoupling by NTZ was also found
to enhance cellular autophagy by way of modulation AMP/ATP ratio. Also, NTZ reduced the
expression of a-synuclein in cells. Further, NTZ prevented loss of tyrosine hydroxylase-
positive neurons and locomotor activity in MPTP-induced mouse model of PD. As NTZ
happens to be one of the highly bio-available drug and is known to possess better safety,
efficacy and cerebral permeability, it may serve as a potential therapeutic candidate for drug-
repurposing for the treatment of PD.

Poster: A2

The citrus flavonoid (ZO-93) reduces amyloid -β (1-42) load by

upregulating autophagy through AMPK/ULK axis in neuronal cells
Aitizaz Ul Ahsan1, Ajay Kumar*2, Vijay Lakshmi Sharma1, Mani Chopra*1.

1Cytogenetics Lab, Department of Zoology, Panjab University, Chandigarh, 160014, India;

2Division of PK-PD-Toxicology and Formulation Division, CSIR-Indian Institute of

Integrative Medicine, Canal Road, Jammu-180001, India

Alzheimer’s disease is primarily accompanied by accumulation of amyloid beta and formation

of neurofibrillary tangles in the brain. Increased levels of amyloid beta are attributed for various
pathological manifestations including oxidative stress, neuroinflammation and cognitive
impairment leading ultimately to neurodegeneration. The disease is still an incurable
neurodegenerative disorder with a long 10 – 15 years of presymptomatic period. Autophagy is

A31
one of the important mechanisms for clearance of both intracellular and extracellular amyloid-
beta. We evaluated a novel dietary compound (ZO-93) that potently induced autophagy in
differentiated N2a cells and primary neurons and by upregulating AMPK. A downstream target
of activated AMPK that is ULK1 was also upregulated, while expression of p-mTOR was
reduced. To investigate the underlying mechanism of AMPK activation, we analyzed the
expression of two important kinases LKB1 and CaMKKβ responsible for its activation
however came out to be unaffected, indicating its direct influence on AMPK. The effect of ZO-
93 on downstream proteins such as p-ULK1 and mTOR was also restored in AMPK knocked
down cells. ZO-93 further activated different autophagy regulating proteins such as LC3BII,
ATG5, ATG7 and BECN1 in N2a and primary neuronal cells. ZO-93 induced clearance led to
a decline in the Aβ (1-42) cytotoxicity that was evident by affording protection of cells from
mitochondrial membrane potential loss, reduced the production of ROS and increased cell
viability of primary neurons and differentiated N2a cells. Based on these data, ZO-93 could be
a potential candidate that needs further pharmacological investigation for its development as
an anti-Alzheimer’s bioceutical.

Poster: A3

Interplay between ER homeostasis, signaling and autophagy in Plasmodium

falciparum
Ananya Ray*, Namita Surolia

Molecular parasitology lab; MBGU, JNCASR, Jakkur, Bangalore-560064

A response to endoplasmic reticulum (ER) stress is essential for the homeostasis of cells.
Certain environmental factors cause perturbation of the ER function leading to accumulation
of unfolded proteins that initiate a complex signaling pathway known as the Unfolded Protein
Response (UPR). UPR helps reduce ER stress and restores cellular homeostasis. Studies have
highlighted the involvement of UPR in inducing autophagy in eukaryotic cells, a catabolic
process involving lysosomal hydrolases to clear cellular components and damaged organelles.
During autophagy, the pre-autophagosomal structure (PAS) is assembled, and transport of
autophagosome to the lysosome is induced in an Atg protein-dependent manner. P. falciparum
is a protozoan parasite that has a complex life cycle, alternating between different hosts and
developmental forms. Changes in their environment thus expose them to a variety of stress
stimuli leading to ER stress. We aim to decipher, the involvement of autophagy in ER stress
mediated UPR, and its potential interplay with the PERK regulated pathway. Studies related to
autophagy in the malaria parasite have started to unravel. Bioinformatics analysis of P.
falciparum genome has identified putative autophagy orthologues for some of the autophagy
proteins including Atg1 Atg5, Atg12 and Atg8 to name a few. We find that the expression of
PfAtg8, is upregulated upon induction of ER stress. Autophagy dynamics with respect to ER
stress in the parasite is being characterized by using another autophagy marker PfAtg1.
Interestingly, a specific inhibitor of Atg1 is able to completely inhibit parasites’ invasion of
fresh RBC.

A32
Poster: A4

Investigating the role of autophagy in the background of monocyte to macrophage

differentiation
Anindita Bhattacharya†, Deepak K Sinha, Prosenjit Sen*

Department of Biological Chemistry, Indian Association for the Cultivation of Science,

Jadavpur, Kolkata-700032, India

Blood borne monocytes are generally short-lived species. Once these circulating monocytes
are stimulated by some inflammatory stimulus, they activate pro-survival pathways, migrate to
tissues, and differentiate into macrophages. But the detail molecular mechanism, relating the
survival and differentiation of monocytes are still not clear. Emerging researches have
confirmed the antagonistic relation between autophagy and apoptotic cell death. This primarily
led us to investigate the role of autophagy in the differentiation of monocyte to macrophage.
Moreover, previous studies have confirmed that the protein profile of monocyte is significantly
different from that of macrophages. Our data confirms that autophagy acting as protein
recycling machinery, crucially regulates monocyte differentiation. Our result affirms that
blocking autophagy at the very initial step abrogate the process of differentiation completely
whereas blocking the autophagy at the maturation step has a very surprising impact. Monocytes
can adhere and start to express macrophage marker even in the conditions where the autophagic
flux is inhibited. But these cells are not alike as a completely differentiated macrophage. They
have neither gained enhanced phagocytotic potential nor did they acquire inflammatory status.
Thus, we have concluided that physical isolation of the monocyte specific protein may be
essential for the commencing of differentiation. We also have established that autophagy acts
as an enegy yielding process and thus supports the monocyte differentiation by fulfilling the
energy requirement of a differentiating cell. Thus, we have elucidated the step wise
contribution of autophagy from a mechanistic point of view in the context of differentiation.

Poster: A5

Autophagy induction exhibits enhanced host immune response via its

modulatory influence on splenic macrophages and dendritic cells in
experimental cerebral malaria
Anirban Sengupta*, Saikat Mukherjee, Soubhik Ghosh, Arindam Bhattacharyya

Immunology Lab, Dept. of Zoology, University of Calcutta

Cerebral malaria is a fatal disease, causing millions of deaths worldwide every year. Spleen
being a major lymphoid organ and blood filtration unit is majorly affected during Plasmodium
infection. Splenomegaly is a key symptom of the disease. Macroautophagy plays a crucial role
in the degradation and processing of the engulfed pathogens within the macrophages.
Experimental mice group with Plasmodium bergheei ANKA (mice homolog for Plasmodium
falciparum) infection were treated with either of autophagic inducer or inhibitor and splenic
marginal zone macrophages (MZM) were isolated. The autophagic flux induction in MZM was
confirmed by the gene expression status, vacuole bound LC3B level, autophagolysosome

A33
formation. It leads to better parasite clearance from the MZM, as well as from the blood and
brain, thereby increasing the survivability. Autophagy induction skewed the tissue-damaging
aggressive Th1 proinflammatory to Th2 immune response. Inducer treatment enhances MZM
adhesion and phagocytotic potential, costimulatory and MHC marker expression. More
balanced M1 MZM activation observed post autophagic induction. Interestingly, the co-culture
experiment proved that autophagy-inducer treated MZM facilitates immune activation of
CD8T cells directly or via CD8α+ dendritic cells. Our recently published works in this domain
on the splenic red pulp macrophages and plasmacytoid dendritic cells showed the beneficial
host immune response post-autophagic induction. This study on MZM found a significant
influence of autophagy in shaping splenic immune cells crosstalk. Summing up the inference
of all these studies, we strongly recommend the consideration of autophagy inducer as a
potential therapeutic agent for boosting overall splenic immune response in malaria infection.

Poster: A6

ESAT-6 regulates Calcimycin-induced autophagy through miR-30a-3p and

30a-5p
Assirbad Behura1, Abtar Mishra1, Saurabh Chugh2, Shradha Mawatwal1, Ashish
Kumar1, Debraj Manna1, Amit Mishra3, Ramandeep Singh2, Rohan Dhiman1

1Laboratory of Mycobacterial Immunology, Department of Life Science, National Institute of

Technology, Rourkela, 769008, Odisha, India; 2Tuberculosis Research Laboratory, Vaccine
and Infectious Disease Research Centre, Translational Health Science and Technology
Institute, NCR Biotech Science Cluster, PO Box # 4, Faridabad–121001, Haryana, India;
3Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur,
Rajasthan, 342011, India.

Mycobacterium tuberculosis (M. tb) has a sumptuous repertoire of different antigens to counter
host defenses to enable its survival. Some of these antigens inhibit autophagy, a well
characterized antimycobacterial mechanism orchestrated by the host but the mechanism of this
inhibition is not well understood. Previously we have delineated the mechanism of autophagy
induction by Calcimycin, so the present study was undertaken to deduce the effect of
mycobacterial antigens on the Calcimycin-induced autophagy and mechanism involved thereof
if any. We fractionated purified protein derivative (PPD), a complex mixture of proteins from
pathogenic strains of M. tb using 10 (PPD10) and 3 (PPD3) kDa cutters and their effect on
Calcimycin-induced autophagy was checked. We found significant downregulation of
autophagy by PPD3 pre-treatment in Calcimycin-treated dTHP-1 cells compared to PPD 10.
This decrease in autophagy also corroborated with the increased intracellular survival of M.
smegmatis and M. bovis BCG. Early secreted antigenic target-6 (ESAT-6), an immunodominant
antigen of M. tb was found to be a responsible factor in PPD 3 in executing its autophagy
reduction potential that enhanced intracellular survival of mycobacteria. Mechanistic studies
suggested that ESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-a-vis
downregulates miR-30a-5p expression in Calcimycin-treated THP-1 cells. Transfection
experiments involving either miR-30a-3p inhibitor or miR-30a-5p mimic clearly elucidated the
role of miR-30a-3p in ESAT-6 mediated mycobacterial survival through autophagy inhibition.
Taken together, our result evidently highlights the strategy used by mycobacteria to inhibit host

A34
defensive pathways like autophagy through ESAT-6 mediated upregulation of miR-30a-3p
expression for its growth inside macrophages.

Poster: A7

Mitochondrial injury and quality control in Dengue infection

Bharati S*, Kiran A*,Preethy VK*, Poornima K, Faraz M, Subashish S, Alankrita R,
Shamim.AS and Gulam HS.

Institute of Life Sciences, Bhubhaneswar, India

Dengue is a mosquito-borne human pathogen affecting human health globally. Approximately

400 million infections occur every year with the number steadily increasing each year. Dengue
being an obligate parasite relies on the host cell machinery for its proliferation. In this pursuit
Dengue interacts with the cellular organelles including mitochondria and exploits their function
for its benefit. Mitochondria are either directly targeted by viral proteins or influenced by viral
infection associated alterations to the intracellular environment. In this study we characterized
the effect of Dengue on host cell mitochondrial homeostasis and signaling in cell type and
serotype specific manner. The mitochondrial dynamics (fusion and fission) in concert with
mitophagy sustains mitochondrial homeostasis and constitutes an important arm of
mitochondrial quality control. This dynamic process guards against detrimental stresses that
can thwart mitochondrial and cellular homeostasis. Our data suggest that Dengue promotes
significant mitochondrial damage and injury in hepatic cells leading to their necrotic cell death.
Paradoxically, the mitochondrial quality control is not upregulated in Dengue-infected hepatic
cells. We observed that Dengue enhances global autophagy flux but inhibits mitochondrial-
selective autophagy (mitophagy). Further investigations revealed that Dengue perturbs
mitophagy by affecting the Pink-Parkin signaling pathway.

Poster: A8

Adaptive immune cells control lipophagy to maintain dermal white adipose

tissue (DWAT) homeostasis
Edries Yousaf Hajam, J. Haarshaadri, Patricia Panikulam, Anmol Kulkarni, Abrar
Rizvi, Shaheen Thekkumthala, Tafheem Masudi, Abhik Dutta, Husain Miyajiwala,
Rupali Gund, Apurva Sarin, Colin Jamora

IFOM-inStem Joint Research Laboratory, Centre for Inflammation and Tissue Homeostasis;
Centre for the Regulation of Cell Fate, Institute for Stem Cell Science and Regenerative
Medicine, Bangalore 560065 India; Shanmugha Arts, Science, Technology & Research
Academy, Thanjavur, Tamil Nadu 613401

Homeostasis is not a static process as the tissues in our body continuously undergo various
changes to maintain their structure and function. For instance, the various compartments of our
skin, such as the hair follicle and DWAT (dermal white adipose tissue) undergo continuous

A35
cyclic changes of growth and regression. Understanding the mechanism of cyclic changes in
DWAT is important as these changes are known to play crucial roles in homeostasis and disease
conditions. During homeostasis, various immune cells undergo cyclic changes in numbers as
well as in the activation status and are in sync with cycling of DWAT. Thus, we asked if
inflammatory cells regulate the cycling of DWAT in the skin. To investigate this hypothetical
link, we employed a mouse model with functionally impaired regulatory T cells (T-regs),
(which are known to supress both innate and adaptive immune cells). Strikingly, we observed
a complete loss of DWAT in the mouse model with impaired T regs. Mechanistically we found
that CD4+ and CD8+ T cells create an inflammatory microenvironment prior to the loss of
DWAT. Rescue experiments suggest that CD4+ T cells have a very specific role to play in
DWAT homeostasis. Further, we observed that lipophagy is the main pathway (and not
lipolysis) that causes the loss of DWAT. Thus, we found a novel link of an adaptive immune
cell in controlling DWAT homeostasis. Currently we are investigating how CD4+ T cells
induce autophagy in the DWAT.

POSTER: A9

Therapeutic effect of SMAC mimetic (BV6) targeting IAPs and autophagic

proteins in breast cancer cell lines
Sahar Rafat, Shahbaz Khan, Sowmya Ramachandran, Kapil Dev*

Department of Biotechnology, Jamia Millia Islamia, New Delhi, India

Apoptosis and autophagy are programmed cell death mechanisms. Inhibitors of apoptosis
(IAPs) and autophagy have recently emerged as key mechanisms in resistance to apoptosis in
cancers, that lead to carcinogenesis. A study was conducted on breast cancer cell lines (MCF-
7 and MDA-MB-231) to understand the effect of SMAC mimetic compound (BV6) on cell
death mechanisms. MTT assay was performed on breast cancer lines to determine the IC50
value for BV6. After 48 hours of treatment, the IC50 value of BV6 was 5μM and 7μM for
MCF-7 and MDA-MB-231 cell line, respectively. Following the assay, two doses of BV6
(0.5μM and 0.25μM) for MCF-7 and BV6 (0.7μM and 0.37μM) for MDA-MB-231 were
selected. Both the cell lines were treated with different concentrations of BV6 along with a
control. Total RNA was extracted from the treated and control cells for quantitative real time
PCR (q-RT PCR), to analyze the expression of IAPs (cIAP1, cIAP2, XIAP & Survivin) and
autophagic proteins (Beclin-1 & LC-3). A positive effect of BV6 was observed in reduction of
Autophagic and IAPs mRNA expression. In comparison to control of both the cell lines, BV-6
treated dose-1 group and dose-2 group showed significant change in mRNA expression level
of the targeted apoptotic and autophagic proteins. The present study concludes that these IAPs
and autophagic proteins can be potent target molecules for the development of novel anticancer
drugs. To overcome the problem of resistance to cancer therapy, SMAC mimetic compounds
can also be used in synergy with conventional anti-cancer therapies.

A36
POSTER: A10

IIIM-941 a small-molecule induces autophagy by attenuating NLRP3

inflammasome via mTOR pathway
Mehboob Ali1, Abubakar Wani2, Mehak Gupta1, Ankita Sharma1, Dilpreet Raina1, Sushil
Choudary1, Sameer ullah khan1, Mohd Abdullaha3, Sandip Bharate3 and Ajay Kumar1

1PK-PD and Toxicology Division CSIR-Indian Institute of Integrative Medicine, Jammu and
Kashmir; 2Deparment of Neurology, Washington University in St Louis, MO 63110- USA;
3Medicinal Chemistry Division CSIR-Indian institute of integrative Medicine, Jammu and

Kashmir

NLRP3 inflammasome is activated by the PAMPs and DAMPs leads to the activation of
caspase-1 which cleaves the pro-inflammatory cytokines like IL-1β and IL-18 into its mature
form. Prolong activation of the pro-inflammatory cytokines (IL-1β) leads to various
inflammatory diseases like Alzheimer’s disease, Parkinson’s disease, Diabetes type II, etc.
Autophagy can inhibit the NLRP3 inflammasome activation by the removal of pathogens or
damaged organelles, which protect the neuronal cells from damage. In this study, we have
screened a library of small molecules against ATP and nigericin induced NLRP3
inflammasome in mouse macrophage J774A.1 cells. Among these compounds, IIIM-941
effectively inhibits NLRP3 inflammasome activation by preventing the formation of IL-1β
through caspase-1 activation that was analyzed by ELISA and western blot assay. Moreover,
IIIM-941 potentially inhibits ASC oligomerization and speck formation which was determined
by western blot and confocal microscopy. We further tried to establish the molecular
mechanism of IIIM-941. Interestingly IIIM-941 was able to inhibit mTOR in a concentration-
dependent manner which leads to the induction of autophagy as detectable by the conversion
of LC3-I to LC3-II, increased the expression of Beclin 1 in mouse macrophage J774A.1 cells.
Autophagy induced by IIIM-941 inhibits the NLRP3 inflammasome activation indicates the
link between autophagy and NLRP3 inflammasome. In conclusion, IIIM-941 inhibits NLRP3
inflammasome by inducing autophagy in mouse macrophage J774A.1 cells.

POSTER: A11

WRN, a DNA repair helicase regulates maturation of autophagosomes in

response to cisplatin
Dr. Mrityunjay Tyagi, Pooja Gupta, Bhaskar Saha and Birija S. Patro

Bio-Organic Division, Bhabha Atomic research Centre, Mumbai.

Autophagy is known to associate with cancer resistance and needs definite therapeutic
intervention. Activation of autophagy under nutrient starvation is well studied but DNA
damage induced autophagy in response to chemotherapeutic drug needs is not yet well
established. Here we explore a link between autophagy and Werner RECQ helicase (WRN)
protein. WRN is a DNA helicase protein whose deficiency leads to Werner syndrome. These
patients are predisposed to cancers while sarcoma patients with high WRN show poor
prognosis and low survival. The defects in WRN function are shown to be responsible for

A37
genomic instability via the deregulation of DNA repair, replication, recombination, and
telomere stability but its role in modulating autophagy is not very well known. Our
investigation showed that WRN deficient cells display sensitivity to cisplatin, which was
primarily linked to a defective autophagic process in these cells. Although cell cycle and DNA
damage response proteins were altered similarly in both WRN proficient and deficient cells,
autophagy regulating proteins (LC3 and ATG7) were differentially modulated in WRN-
depleted cells. The LC3-I form accumulated in WRN deficient cells, indicating a key role of
WRN in autophagosome maturation. Besides, WRN deficient cells display high amount of
PARylation contributing to ATP consumption and increased AMPK activation. Further, mTOR
and cellular ER stress mediated UPR (unfolded protein response) were significantly higher in
WRN deficient cells too. Above observations and their plausible therapeutic intervention will
be discussed during the meeting.

POSTER: A12

Role of PPARγ on Autophagy

Mouliganesh Sekar, Kavitha Thirumurugan.

SBST, Vellore Institute of Technology, Vellore

Autophagy is a highly conserved cellular cytoplasmic organelle degradation process that

degrades the cellular components and recycles energy production by nutrient sensing pathway
(mTOR) to maintain the cellular homeostasis. Adipocytes are the cells that store the excess
energy in the form of fats/lipids by the process called adipogenesis. Earlier studies revealed the
dual role of autophagy to regulate adipogenesis in pre-adipocytes, and lipolysis and lipophagy
in post-adipocytes. The process of switching over is not clear and the role of PPARγ on
regulation of autophagy is unknown. PPARγ has the negative feedback loop of self-regulating
mechanism to maintain the SIRT1 through SIRT1- PPARγ pathway. SIRT1 belongs to HDAC
that regulates both autophagy and adipogenesis by activating the central nutrient sensing
mTOR pathway. Inhibition of mTOR by SIRT1 regulates the autophagy and inhibits the
adipogenesis. Our work will investigate the mechanism of SIRT1-PPARγ pathway in
regulating autophagy and adipogenesis through PPARγ.

POSTER: A13

IIIM-NPC-290 promotes cell death by inducing autophagy in Mia PaCA-2

pancreatic cancer cells
Mubashir J. Mintoo1, 2, Sameer U. Khan1,2, Abubakar Wani4, Summera B. Malik1, 2,
Sandip B. Bharate2, 3, D. M. Mondhe1, 2, *.

1Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine-180001,

Jammu; 2Academy of Scientific and Innovative Research, CSIR-Indian Institute of integrative
Medicine-180001, Jammu; 3Medicinal Chemistry Division, CSIR-Indian Institute of

A38
Integrative Medicine-180001, Jammu; 4Department of Neurology, Washington University, St.
Louis, MO 63110- USA.

Autophagy is an evolutionarily conserved intracellular degradative process by which it

degrades the abnormal proteins, damaged organelles and eliminates intracellular pathogens.
Autophagy plays a dual role in tumor suppression as well as tumor survival. Despite the
contrasting role, it is clearly evident that autophagy has a significant role in tumor suppression
through the activation of p53, mTOR and other important pathways. There are munerous
pharmacological agents that induce autophagy and suppress various tumors including
leukemia. We have evaluated IIIM-NPC-290 as a potent inducer of autophagy in Mia PaCa-2
pancreatic cancer cells. IIIM-NPC-290 was able to depolarize the mitochondrial membrane as
detected by flow cytometer and accumulate of autophagosomes as observed by the conversion
of LC3-I into LC3-II through western blot and confocal microscopy in Mia PaCa-2 cells. The
expression of other autophagic proteins like BECN1, ATG7, ATG5, and P62 was
downregulated which in turn activated tumor suppressor protein p53 that activated cell death
and reduced the tumor size. The fluorescence intensity of LAMP1 and accumulation of
lysosomes detected by confocal microscopy and transmission electron microscopy respectively
were increased with the treatment of IIIM-NPC-290 in Mia PaCa-2 cells. Our findings suggest
that IIIM-NPC-290 should be further investigated as a potent anticancer agent acting through
autophagy.

POSTER: A14

Cytoplasmic vacuolation induced cell death by Piperlongumine in cancer

cells: An interplay between paraptosis and autophagy
Divya Nedungadi, Anupama Binoy, Sibi Raj, Chinchu Bose, Bipin G Nair, Nandita
Mishra*

School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Clappana P.O., Kollam

690525, Kerala, India

Piperlongumine (PL), an alkaloid present in long pepper was found to induce cell death in triple
negative breast cancer cells, MDA-MB-231. The cell death was preceded by the presence of
large and extensive cytoplasmic vacuolation of ER origin, a hallmark feature of paraptosis. It
was non-apoptotic in nature with the absence of nuclear condensation and DNA fragmentation.
The cell death was not inhibited in the presence of broad-spectrum caspase inhibitor like QVD-
OPH. There was a G2-M phase arrest with PL treatment. The cell death could not be reversed
by autophagy inhibitors suggesting that the mode of cell death is distinct from autophagy.
Acridine orange did not stain the cytoplasmic vacuoles eliminating the role of lysosomes in PL
induced cell death. Intriguingly, we observed a dose and time dependent upregulation of
microtubule-associated protein 1 light chain 3 (MAP1LC3) I & II and p62 proteins which are
autophagy marker proteins. This suggests that LC3 and p62 might play a crucial yet unknown
role in paraptosis. During a typical autophagic process there occurs an increase in expression
of only the LC3 II form and decrease in p62 levels. Increased p62 levels here indicate that the
autophagy is impaired or inhibited during paraptosis. There was an increase in cell death with
pretreatment of autophagy inhibitor and decrease in cell death with induction of autophagy.

A39
These results shed light on the plausible connection of autophagy alteration to paraptotic cell
death which can be of potential therapeutic interest for targeting recalcitrant cancer cells.

POSTER: A15

Antimycobacterial effect of IFNG (interferon gamma)-induced autophagy

depends on HMOX1 (heme oxygenase 1)-mediated increase in intracellular
calcium levels and modulation of PPP3/calcineurin-TFEB (transcription
factor EB) axis
Nisha Singh, Pallavi Kansal, Zeeshan Ahmad, Navin Baid, Hariom Kushwaha, Neeraj
Khatri and Ashwani Kumar

CSIR-IMTECH, Chandigarh

IFNG (interferon gamma)-induced autophagy plays an important role in the elimination of

intracellular pathogens, such as Mycobacterium tuberculosis (Mtb). However, the signaling
cascade that leads to the increase in autophagy flux in response to IFNG is poorly defined.
Here, we demonstrate that HMOX1 (heme oxygenase 1)-generated carbon monoxide (CO) is
required for the induction of autophagy and killing of Mtb residing in macrophages in response
to immunomodulation by IFNG. Interestingly, IFNG exposure of macrophages induces an
increase in intracellular calcium levels that is dependent on HMOX1 generated CO. Chelation
of intracellular calcium inhibits IFNG-mediated autophagy and mycobacterial clearance from
macrophages. Moreover, we show that IFNG-mediated increase in intracellular calcium leads
to activation of the phosphatase calcineurin (PPP3), which dephosphorylates the TFEB
(transcription factor EB) to induce autophagy. PPP3-mediated activation and nuclear
translocation of TFEB are critical in IFNG-mediated mycobacterial trafficking and survival
inside the infected macrophages. These findings establish that IFNG utilizes the PPP3-TFEB
signaling axis for inducing autophagy and regulating mycobacterial growth. We believe this
signaling axis could act as a therapeutic target for suppression of growth of intracellular
pathogens.

POSTER A16

Microtubule post-translational modifications spatially constrain lysosomes

to regulate autophagy
Nitin Mohan

Biological Sciences and Bioengineering Department, IIT Kanpur, Uttar Pradesh 208016

Autophagy is a vital cellular process where the phagosome double membrane invaginates
around the protein aggregates to be degraded, followed by sequestration of membrane
proteins-Atg8/LC3 to form mature autophagosome, which then is actively transported by motor
proteins on microtubules to eventually fuse with lysosome to form autolysosomes, where the
proteins will be degraded by the lysosomal hydrolases. Since, both autophagosome and

A40
lysosome are highly mobile organelles, their transport regulations would play significant role
in effective autophagy. Here we explore the role of microtubule post-translational
modifications (PTMs) in regulating lysosome and autophagosome trafficking and the role of
microtubule PTMs in autophagy. We used super-resolution microscopy method STORM to
quantify different microtubule PTMs and sub-populations in epithelial cells. Our results show
that acetylated and tyrosinated microtubules are predominant subsets of microtubules while
only a small subset (30% of the total microtubule) is detyrosinated. Interestingly, a large
fraction of lysosomes is enriched on the detyrosinated microtubules. Also, motility of lysosome
is hindered on detyrosinated microtubules. Furthermore, autophagosomes and autolysosomes
showed similar enrichment and motility behavior on detyrosinated microtubules. Finally,
correlative live-cell and STORM imaging showed that detyrosinated microtubules facilitate
efficient autophagosomes and lysosome fusion, and depletion of detyrosinated microtubules
led to significant reduction in autophagy. Taken together our study brings forth a new role for
microtubules detyrosination in regulating autophagy.

POSTER: A17

AMP-activated Protein Kinase induced autophagy is a feed forward signal

for mTORC1 activation
Rabiya Majeed1; Sheikh Tahir Majeed2; Shajrul Amin1; Khurshid Iqbal Andrabi1*

1Department of Biochemistry, University of Kashmir, Srinagar, J&K-India; 2Department of

Biotechnology, Central University of Kashmir, Ganderbal, J&K-India.

Nutrient withdrawal leads to inactivation of mechanistic target of rapamycin complex 1

(mTORC1) that shuts down growth and elicits signals for autophagy induction. Sustained
starvation results in activation of energy sensor AMP activated protein kinase (AMPK) that is
recognized as a stress signal to aggravate autophagy and sustained mTORC1 shutdown. We
present evidence that up-regulation of autophagy coinciding with AMPK activation results in
rapamycin sensitive activation of S6K1 to suggest nutrient salvage as the basis of feed forward
activation of mTORC1. We also show that S6K1 activated by ectopic expression of α-subunit
of AMPK or its constitutive active variant corresponds with enhanced eIF4E phosphorylation
and its resultant interaction with S6K1 in a rapamycin dependent manner. Our data suggests
that AMPK may serve as a rescue signal to use autophagy as a salvage to activate mTORC1
concomitant with its prescribed influence on autophagy.

POSTER: A18

Gcn4 mediates a methionine-dependent anabolic program by controlling amino acid

supply

Rajalakshmi Srinivasan1, Adhish Walvekar1, Aswin Seshasayee2,Sunil Laxman1

A41
1Institute
of Stem Cell Science and Regenerative Medicine, Bangalore; 2National Centre for
Biological Sciences, Bangalore.

Amino acid starvation lead to alteration in several cellular processes like transcription,
translation and also shown to have a role in deciding cellular fates such as proliferation,
autophagy. Methionine – a sulfur containing aminoacid is a strong growth signal for cells.
Supplementation of methionine to the cells under aminoacid starvation has been shown to
inhibit autophagy. In our study we show that the anabolic processes like nucleotide
biosynthesis and amino acid biosynthesis, along with ribosomal biosynthesis are all
upregulated transcriptionally when methionine is abundant, even in otherwise amino acid
limited conditions. In this study, we discover the role of the conserved Gcn4/Atf4
transcriptional regulator, typically studied during starvation responses, in mediating the
methionine induced anabolic response. Gcn4 protein is strongly induced by methionine,
independent of carbon sources. Comparing transcriptomes we find that Gcn4 primarily controls
nitrogen flow in the methionine dependent anabolic program. Using ChIP-seq and RNA seq
analysis we identify direct targets of Gcn4 during methionine-abundance. Gcn4 directly
increases the expression of most of the amino acid biosynthetic genes, and indirectly controls
nucleotide biosynthesis, suggesting that Gcn4 controls nitrogen flow required for anabolism.
Here, we show that Gcn4 is critical for the supply of amino acids for the methionine dependent
activation of ribosomal biogenesis.

POSTER: A19

Arsenic Induces Autophagy in Developmental Mouse Cerebral Cortex by

Inhibiting Blood Brain Barrier’s Tight Junction Proteins
Ram Kumar Manthari, Chiranjeevi Tikka, Ruiyan Niu, Zilong Sun, Jundong Wang

Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary

Medicine, College of Animal Science and Veterinary Medicine, Shanxi Agricultural
University, Taigu, Shanxi-030801, China.

For the past decade, there has been an increased concern about the health risks from arsenic
(As) exposure, because of its neurotoxic effects on the developing brain. In this study, we
highlight the involvement of blood-brain barrier’s (BBB) tight junction (TJ) proteins in As-
induced autophagy in mouse cerebral cortex during developmental periods [postnatal day
(PND) 21, 28, 35 and 42]. Administration of arsenic trioxide (As2O3) at doses of 0.15 mg or
1.5 mg or 15 mg As2O3/L in drinking water from gestational to lactational and continued to
the pups till PND42 resulted in a significant decrease in the mRNA levels of TJ proteins
(Occludin, Claudin, ZO-1 and ZO-2) and Occludin protein expression level. Also, As exposure
significantly decreased the mRNA levels of PI3K, Akt, mTOR, and p62 with a concomitant
increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Moreover, As exposure significantly down-
regulated the protein levels of mTOR with a concomitant up-regulation of Beclin 1, LC3 and
Atg12 in all the developmental age points with no significant alterations in low and medium
dose groups of PND42. TEM ultra-structural analysis revealed the occurrence of
autophagosomes and vacuolated axons in the cerebral cortex of the mice exposed to high dose
As at PND21 and 42. Finally, we conclude that the leaky BBB in cortex may facilitate the As
transfer and induces autophagy by inhibiting PI3K/Akt/mTOR signaling pathway in an age-

A42
dependent manner, i.e., PND21 animals were found to be more vulnerable to the As-induced
neurotoxicity than the other three age points.

POSTER: A20

Mitochondrial dysfunction affects translational initiation pathways and

cellular homeostasis: Importance of Integrated stress response (eIF2ak-
eIF2α phosphorylation-ATF4) pathway
Rama Gopal Reddy Koncha, Naresh B Sepuri, Ramaiah V Kolluru*

Department of Biochemistry, School of Life Sciences, UoH.

Carbonyl cyanide m‐chlorophenylhydrazone (CCCP) that affects mitochondrial function by

dissipating membrane potential promotes phosphorylation of eukaryotic initiation factor eIF2α,
which is independent of PERK or GCN2 kinases but dependent of HRI kinase activation.
CCCP induced eIF2α phosphorylation promotes translation of ATF4 and CHOP and plays a
role in promoting autophagy or cell death. In addition, CCCP reduces expression of BiP, an
ER chaperone, XBP1 splicing, PARP cleavage and caspase activity in Thapsigargin treated
cells suggesting that it reduces ER stress induced UPR. Further, CCCP induced eIF2α
phosphorylation is also correlated in to AMPK activation, reduction in S6 kinase and eIF4EBP
phosphorylation. ISRIB, a small molecule inhibitor of ISR pathway reduces CCCP induced
ATF4 & CHOP expression and autophagy but fails to reducing the activation of AMPK.
Further ISRIB reduces CCCP induced cell death which is independent of caspase activation
and ER stress mediated apoptosis in HepG2 cells.

POSTER: A21

HBV activates Parkin-mediated mitophagy leading to attenuated ATP level

in human macrophages
Saiful Anam Mir1, 2, Mohsin Khan1, Hasan Imam1, Geon-Woo Kim1 and Aleem Siddiqui1

1Divisionof Infectious Diseases, University of California, 9500 Gilman Dr., La Jolla, San
Diego, CA- 92093, USA; 2Dept. of Zoology, City College, University of Calcutta, 102/1, Raja
Rammohan Sarani, Kolkata- 700 009, West Bengal, India.

Hepatitis B Virus (HBV) infection leads to development of liver cirrhosis and hepatocellular
carcinoma over time. Removal or recycling of aged or damaged mitochondria within a cellular
environment is done by the selective autophagic process called mitophagy which contributes
to mitochondrial quality control and maintain cellular homeostasis. Our lab has previously
shown that HBV infected hepatocytes display significant features of Parkin induced
mitophagy, which helps the virus establish chronic infection. Therefore, we investigated if
macrophages exposed to HBV virions exhibit activation of mitophagy and whether these
macrophages also display signatures of inflammation. HBV caused a significant decline in

A43
mitochondrial membrane potential in the macrophages. HBV exposure also resulted in
upregulation of E3 ubiquitin ligase, Parkin and its self-ubiquitination, followed by its
translocation from cytosol to mitochondria. Using a dual mito-RFP-EGFP reporter assay in
HBV exposed macrophages, induction of mitophagic events was again confirmed. There were
no obvious signs of apoptotic induction after HBV exposure in these cells. In addition, we also
examined HBV induced inflammation in cultured macrophage cells, which resulted in
moderately increased synthesis of proinflammatory cytokine IL-1b, IL-6 an IL-18. HBV
exposure resulted in a significant decline in the total ATP level in macrophage cells. These
findings suggest that HBV infection contributes to alteration in the mitochondrial dynamics in
macrophages leading to increased mitophagy and might as well act as a platform for induction
of inflammatory response. In our subsequent work, we are keen to characterize the detailed
pathways for mitophagy induction that can be correlated with HBV induced liver inflammation.

POSTER: A22

Secondary apoptosis mediated by autophagosomes in Hawthorn extract

treated breast cancer cell line in vitro
Salini Kombiyil* and Niranjali Devaraj Sivasithamparam

Department of Biochemistry, University of Madras, Guindy campus, Chennai, Tamilnadu

Apoptotic necrosis or secondary necrosis is a natural outcome of complete apoptotic program,

occurs especially in in vitro conditions, in absence of scavengers like phagocytes. This is
characterized by specific morphological features associated with both apoptosis (pyknosis,
karyorrhexis) and necrosis (cytoplasmic membrane rupture, autolysis of cellular contents). An
interesting observation was made in this regard, while evaluating the anti-carcinogenic
potential of a polyphenol rich extract from hawthorn (Crataegus oxyacantha) berry (M.Co),
using MCF-7 cell line as in vitro breast cancer model. Analysis of transcriptional expression
of apoptotic markers Bcl-2, Bax and translational expression of initiator caspase 9 have proven
the apoptosis inducing property of M.Co. Upon examining the ultra-structural changes induced
by M.Co on MCF-7 cell lines, scanning electron microscope analysis showed cell surface
changes characteristic to apoptosis. Transmission electron microscopic analysis revealed both
early and late apoptotic cells among the cells treated with M.Co. Early apoptotic cells exhibited
characteristic features like loss of microvilli, pyknosis and karyorrhexis, initial stages of
chromatin condensation, dense cytoplasm with condensed mitochondria and tightly packed
organelles. Late apoptotic cells were found to undergo secondary necrosis, since it is in in vitro
culture conditions. Interestingly, double membraned autophagosomes containing cellular
organelles, were found in this process of secondary necrosis, where usually autolysis is
expected instead of autophagy. Cells in the later stages showed extensive cytoplasmic
vacuolation and autophagic degradation of organelles. Our study observed that the cell
elimination by secondary necrosis that occurred in drug treated tumour cells in vitro was
mediated by autophagy. This observation is rare and unreported.

A44
POSTER: A23

Crinophagy regulates glucagon content via a non-macroautophagic

mechanism
Sangam Rajak & Rohit A. Sinha

Dept. of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow-

226014, India

Glucagon is an endocrine hormone secreted by pancreatic alpha-cells to increase glucose

output by the liver under conditions of hypoglycemia. However, deregulated secretion of
glucagon in diabetes is major cause of hyperglycemia in humans. Although, autophagy within
the endocrine cells termed as “crinophagy” has been attributed to control intracellular hormone
levels in mammals, its pharmacological induction and mechanism remains elusive. Using, a
mouse alpha cell line aCT9 we demonstrate that mTORC1 is a major regulator of glucagon
turnover in alpha cells and its inhibition leads to degradation of glucagon containing secretory
vesicles within the lysosomes. Using, both genetic and pharmacological approaches we show
that glucagon crinophagy does not involve the macro-autophagy pathway inspite of having a
common stimulation via mTORC1 inhibition. These results therefore put forth mTORC1
inhibition as a promising pro-crinophagy inducer for regulating hormone content in endocrine
pathology.

POSTER: A24

AMP-activated Protein kinase; Map-kinase interacting kinase; eIF4E;

S6Kinase 1 axis is a rescue to synchronize autophagy with cell growth
Sheikh Tahir Majeed1; Rabiya Majeed2; Mohammad Afzal Zargar1; Khurshid Iqbal
Andrabi2*

1Department of Biotechnology, Central University of Kashmir, Ganderbal, J&K-India;

2Department of Biotechnology and Biochemistry, University of Kashmir, Srinagar, J&K-India.

Autophagy is a survival mechanism that helps eukaryotic cells withstand energy crisis due to
nutrient deficiency. This self-eating process delivers cytoplasmic content for degradation at the
lysosomes and consequent recycling for sustained survival. Not-withstanding its pro-survival
intent, autophagy may switch its function to promote cell proliferation during cancer genesis
through mechanism that largely remains undefined. Although it remains settled that the
interplay between two major signaling complexes, the nutrient sensitive mechanistic target of
rapamycin complex 1 (mTORC1) and energy sensitive AMP activated protein kinase (AMPK),
largely regulate the process of autophagy, the mechanistic insights about the cellular
commitment to switch from pro-survival to death or proliferative mode remaining elusive. We
have recently identified eukaryotic initiation factor 4E (eIF4E) phosphorylation as a necessary
signal to mediate S6 kinase 1 (S6K1) regulation. Herein we show that beyond its prescribed
influence on autophagy, AMPK promotes eIF4E phosphorylation in Map-Kinase interacting
kinase (MNK) dependent manner to circumvent S6K1 dependence on mTORC1. We further
go on to show that AMPK mediated mTORC2 activation and change in PHLPP1 binding

A45
dynamics is sufficient to overcome growth factor dependence of S6K1. Our data contextualizes
cyclic regulation of S6K1 during autophagy and identifies AMPK as a rescue signal to support
growth and proliferation during cellular stress.

POSTER: A25

Hydrogen Sulfide-Induced GAPDH Sulfhydration Disrupts the DBC1-

SIRT1 Interaction to Initiate Autophagy
Iram Khan Iqbal, Sapna Bajeli, Shivani Sahu, Shabir Ahmad Bhat and Ashwani Kumar

Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh,

India 160036

Hydrogen sulfide (H2S) is a cytoprotective gasotransmitter, known to activate sirtuin 1 (SIRT1)

and autophagy; however, the underlying mechanisms for both remain unknown. Herein, we
demonstrate that H2S sulfhydrates the active site cysteine of the glycolytic enzyme
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to induce its redistribution into the
nucleus. Importantly, nuclear localization of GAPDH is critical for H 2S mediated activation of
autophagy. Inside the nucleus, GAPDH interacts with deleted in breast cancer 1 (DBC1, also
known as CCAR2). DBC1 is known to interact with SIRT1 to inhibit its activity. Interaction
of GAPDH with DBC1 disrupts the inhibitory effect of DBC1 on SIRT1. Activated SIRT1
then deacetylates microtubule associated protein 1 light chain 3 beta (LC3) to induce its
translocation into cytoplasm and to activate autophagy. Additionally, we demonstrate the
physiological role of this pathway in autophagy-mediated trafficking of Mycobacterium
tuberculosis into lysosomes to restrict growth of intracellular mycobacterial cells. We believe
that this pathway plays an important role in other health benefits associated with H2S.

POSTER: A26

Activation of cytosolic immune surveillance induces autophagy in visceral

leishmaniasis
Sushmita Das,

Department of Microbiology, All-India Institute of medical Sciences (AIIMS), Patna.

Microbial pattern recognition critically contributes to innate response, both at extracellular and
intracellular cytosolic surveillance pathway (CSP) interface. Here, we have demonstrated that
cytosolic targeting of L.donovani DNA (Ld-DNA) inhibits macrophage responsiveness to
IFNɣ, through decreased MHC-II expression and lowered pSTAT1 (Y701) levels, involving
host three-prime repair exonuclease-1 (TREX-1). The Ld-DNA potently induced type-1 IFNs,
i.e. significant over-production of IFNβ through activation of the IRF pathway. Interestingly,
knockdown of TRIF or MyD88 expression in macrophages had no effect on cytosolic Ld-DNA
transfection-mediated IFN-β production, indicating involvement of a TLR independent
pathway. Contrastingly, Ld-DNA failed to induce IFNβ in both TBK-1 and IRF3KO knockout
macrophages. Although IFNβ was not induced by Ld-DNA in STING- knockout macrophages,

A46
STING alone was not enough for the induction. Evidently, besides STING, Ld-DNA
recognition for induction of IFNβ critically required cytosolic cyclic GMP-AMP synthase
(cGAS). Furthermore, the cGAS dependent targeting of Ld-DNA induced IFNβ over-
production that contributed to autophagy induction in L.donovani infection. The number of
infected macrophages decreases dramatically during treatment with autophagy inhibitor
compared to infection control, suggesting a role of host autophagy in Leishmania infection.
The results suggested that Ld-DNA transfection induced expression of autophagic gene Beclin
1 and LC3b of the macrophages. We provide the first evidence that enhanced cytosolic sensing
of Ld-DNA induces autophagy in L.donovani infected host. The findings may help in future
development of policies for novel anti-leishmanial therapeutics.

POSTER: A27

Approaches to study the crosstalk of secretory autophagy and

unconventional protein secretion (UPS)
Sreedevi Padmanabhan and Ravi Manjithaya

Autophagy Laboratory, Molecular Biology and Genetics Unit, JNCASR, Jakkur, Bangalore
560064, India.

Secretory proteins play a multitude of physiological roles such as stress response, intercellular
communication, signal transduction, nutrient uptake and nutrient degradation. Besides the
canonical secretory pathway where proteins with signal/leader peptide travel via ER, Golgi and
secretory vesicles, many proteins which lack well defined signal peptides are also secreted by
unconventional protein secretion (UPS). The latter mostly occurs under cellular stress such as
inflammation, nutrient stress, ER stress, mechanical stress, and are detected in
pathophysiological conditions with dysfunctional autophagy such as neurodegeneration. The
mechanism underlying UPS has not been universally chalked out with diverse process
suggested for individual cargoes. In this regard, the involvement of autophagy machinery in
UPS of a small subset of proteins have been reported (secretory autophagy). Thus, autophagy
intersects with protein trafficking and secretion and appears to play a broad role in the
constitutive biosynthetic pathway, regulated exocytosis and alternative routing of integral
membrane proteins to the plasma membrane. The main lacunae in this field is to identify potent
UPS cargoes and also in delineating their molecular machinery in the protein trafficking
process. We have employed various interdisciplinary approaches in understanding the
crosstalk of UPS and autophagy using yeast and mammalian model systems.

POSTER: A28

Cantharidin inhibits autophagic flux by impairing cellular

phosphatidylethanolamine pool mediated Atg8 lipidation in Saccharomyces
cerevisiae
Swati Swagatika1, Aroshi Mitra, and Raghuvir Singh Tomar1*

A47
1Department of Biological Sciences, Indian Institute of Science Education and Research
(IISER), Bhopal-462066, MP, India

Cantharidin is a terpenoid compound found in blister beetles. It has been widely studied as an
anticancer molecule because of its cytotoxic effects on several human cancer cell lines. The
anticancer properties of this compound are accredited to its ability to suppress protein
phosphatase 2A (PP2A), to induce cell death through G2/M cell cycle arrest, DNA damage and
apoptosis. Additionally, cantharidin has been shown to be a potent inhibitor of
macroautophagy. However, the precise mechanism of how cantharidin blocks autophagy is yet
to be elucidated. In the present study, we used Saccharomyces cerevisiae as a model organism
to unravel the molecular mechanism(s) behind cantharidin mediated autophagy inhibition. Our
initial findings reveal that yeast cells are sensitive to cantharidin in a dose dependent manner
and external supplementation of ethanolamine, a precursor of phosphatidylethanolamine (PE),
could ameliorate this cytotoxicity. Also, cantharidin inhibits phosphatidylserine decarboxylase
1 (PSD1) expression, a mitochondrial enzyme contributing majorly to the cellular PE pool. In
line with this, we show that cantharidin inhibits autophagy by impairing the Atg8 lipidation, a
key step involved in autophagosome biogenesis. Furthermore, with increase in cantharidin dose
there was concomitant decrease in the ability of the GFP- Atg8 fusion protein to deliver the
cargo to the vacuoles. Strikingly, external administration of ETA remediated autophagy
inhibition by contributing to the total PE pool which in turn stimulated Atg8 lipidation crucial
for autophagy and hence cell viability. Together, these findings suggest that cantharidin can be
employed as an autophagy inhibitor in cancer therapeutics.

POSTER: A29

Pharmacological induction of autophagy as a potential therapeutic target

for Japanese encephalitis
Surendra Kumar Prajapat, Manjula Kalia

Virology lab, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad,
Haryana, India

Japanese encephalitis (JE) is the leading global cause of viral encephalitis with a significant
disease burden in India. One-third of JE infections are fatal, and one-third develop permanent
neurological sequelae. Our research has established that the cellular homeostatic process –
Autophagy, becomes dysfunctional during JE infection. Our studies suggest that autophagy
enhancement can be neuroprotective in JE infection. Recent studies have shown the promise
of using autophagy modulators to treat disease conditions. Several FDA approved drugs have
been shown to enhance autophagy, and have the potential to be repurposed for treatment of
diseases where autophagy upregulation is likely to be beneficial. Here we propose to check
novel compounds and FDA approved drugs for their potential to modulate autophagy. One of
the critical aspects of studying autophagy is the measurement of its degradative capacity, also
known as autophagy flux. The measurement of autophagic flux (rate of degradation) is
important to identify novel autophagy inducers and inhibitors. Here, we have established a
stable mammalian cell line expressing the fluorescent probe GFPLC3-RFP to evaluate
autophagy flux using a high throughput imaging platform. We have utilized this assay to screen
a drug library comprising of 2560 compounds to identify autophagy modulators. In addition to

A48
known autophagy effectors we have identified several novel autophagy inducers and inhibitors.
These are currently being tested for their effect on JEV replication and neuronal cell death.

POSTER: A30

Antiviral interferon response is enhanced upon suppressing foot-and-

mouth disease virus induced autophagy
S. H. Basagoudanavara, H.B. Ranjithaa, A. Veenab, G. Nehaa, M. Hosamania, B.P.
Sreenivasaa, R. Manjithayab and A. Sanyala

aIndianVeterinary Research Institute, Hebbal, Bengaluru– 560024; bMolecular Biology and

Genetics Unit, JNCASR, Jakkur, Bengaluru– 560064

Foot-and-Mouth Disease is a highly infectious viral disease of cloven hoofed animals. It is

caused by Foot-and-Mouth Disease Virus (FMDV), a positive-strand RNA virus. The FMDV
infection induces autophagy possibly through PKR-like ER kinase (PERK)-mediated
unfolded protein response. In order to get a precise insight into the role of autophagy during
the virus infection in porcine cells, a systematic study was carried out. The inhibition of
autophagic flux by pharmacological inhibitor resulted in reduced viral progeny, substantiating
the dependency of FMDV replication on autophagy. Effect of autophagy on antiviral response
was studied by assaying the interferon levels following inhibition of autophagy. The RT-qPCR
for IFN-β and IFN-3 transcript levels showed an upregulation in the range of 4.5–6 fold at 6
and 12 hours post infection (hpi) in the presence of autophagy inhibitors. Further, knockdown
of LC3B, a marker of autophagy, significantly affected the virus replication leading to
decreased viral protein expression and reduction in extracellular progeny-virus titer at 12 and
18 hpi. Besides, cells with knockdown of LC3B showed the enhanced transcript levels of IFN-
β and IFN-3 of 8 and11.4 fold respectively at 12 hpi, together with a concomitant increase in
the levels of IFN-β and IFN-3 proteins in the infected culture supernatant. The data suggests
that suppression of autophagy affected the replication ability of FMDV, by enhancing
interferon levels. These findings could help explore molecular targets for the development of
novel antiviral strategies for alleviating FMD infection in affected animals.

POSTER: A31

O2•− and H2O2 activated Pro-autophagic proteins AMBRA1 and Beclin1

determines the fate of transplanted human stem cells in an ischemic brain:
An in vitro study on human dental and mesenchymal stem cells
Eram Fauziaa#, Ravi Prakasha#, Santosh Kumar Yadava, Mohsin Ali Khan b, Syed Shadab
Raza*a,c

aLaboratoryfor Stem Cell & Restorative Neurology, Department of Biotechnology, Era’s

Lucknow Medical College Hospital, Era University, Sarfarazganj, Lucknow−226003, India;
bMetabolic Unit, Era’s Lucknow Medical College Hospital, Era University, Sarfarazganj,

A49
Lucknow−226003, India; cDepartment of Stem Cell Biology and Regenerative Medicine, Era’s
Lucknow Medical College Hospital, Era University, Sarfarazganj, Lucknow−226003, India

Despite the overwhelming pre-clinical outcomes, the stem cell therapy for ischemic stroke has
largely failed at clinics. One of the preliminary reasons is the fate of the transplanted cells in
ischemic brain. Employing dental pulp stem cells (DPSCs) and human mesenchymal stem cells
(MSCs) we decided to look into this crucial aspect. In the present study we employed Oxygen
Glucose Deprivation (OGD) model to study the cell fate regulation. The DPSCs and MSCs
were challenged with the OGD for different timings in the presence of activators and inhibitors
of ROS and autophagy. Our results suggested that OGD induced the abrupt generation of
generation of O2•− and H2O2 in both cell types, which in turns, induces the autophagy in DPSCs
and MSCs. Since the initial result indicated the dysregulation of Ambra1 and Beclin1, thus, we
studied the effect of nucleation proteins: Ambra1 and Beclin1 in the impairment and survival
of the above mentioned cell types during ischemic stroke. Also, we have been able to establish
a relationship between O2•− and H2O2, ROS mediated MAPKs (especially ERK1/2, c-JNK and
p-38), and the dysregulated autophagy observed in human stem cells. Our results propose that
Ambra1 and Beclin1 regulate the down-signalling and the survival percentage of the DPSCs
and MSCs during their treatment to OGD. Thus, altogether the results suggest that modulation
of autophagy in stem cells would provide a novel strategy to rescue these cells during their
treatment to ischemic brain.

POSTER: A32

TFEB-mediated xenophagy: a host defence pathway involved in restricting

intracellular pathogens
Veena Ammanathan and Ravi Manjithaya

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Bangalore, India.

Xenophagy (Autophagy of pathogens) is considered as one of the host innate immune response
to intracellular pathogen invasion. Modulating xenophagy is proven beneficial to prevent
pathogen replication and thereby ameliorate disease phenotype. However, pathogens have
evolved ways to subvert xenophagy to facilitate their survival. Hence, restoration of xenophagy
block imposed by pathogens through genetic or pharmacological methods would enhance the
clearance of intracellular pathogens and provide insights into host-pathogen mediated
molecular events that control xenophagy. Yeast based high throughput screening performed
previously in our laboratory have identified a number of autophagy inducers and inhibitors. I
further screened the autophagy inducers to check for their ability to clear intracellular
Salmonella typhimurium. Screening yielded a compound, which showed more than two fold
decrease in intracellular pathogen burden. The identified compound has following
characteristics: 1) it induces host xenophagy process and does not directly affect the pathogen
growth, 2) through post transcriptional mechanisms, enhances recruitment of xenophagy
mediators to efficiently capture the pathogen, 3) acts through an autophagy and lysosomal
biogenesis transcriptional master regulator TFEB, 4) activation of TFEB increases the active

A50
lysosomes in cells leading to enhanced fusion with Salmonella containing vacuoles thereby
restricting its replication, and 5) is effective in an in vivo mouse model of bacterial infection.
Additionally, the interplay between xenophagy process and other innate immunity pathways is
probed by microarray analysis. Putative regulators from microarray analysis are accessed for
their role in modulating TFEB nuclear translocation and xenophagy mediated clearance.

POSTER: A33

Investigations on Rab7 in human IBD: An unanticipated role in autophagy

mediated disease pathogenesis
Y. Rajendran1, P. Gaur1, A. Suhail1, V. Ahuja2, C. V. Srikanth1*

1Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India; 2All
India Institute of Medical Sciences, New Delhi, India

Intestinal health depends on complex interactions between gut microbes, intestinal epithelium
and the host immune system. At a cellular level, diverse regulatory mechanisms execute to
maintain the normal intestinal homeostasis. A breakdown in these mechanisms attributes to
chronic inflammatory pathology found in Inflammatory Bowel Disease (IBD). Autophagy is
one such mechanism which plays multiple roles in IBD pathogenesis by altering processes that
include intracellular bacterial killing, antimicrobial peptide secretion by Paneth cells, goblet
cell function and proinflammatory cytokine production by enterocytes. Using murine DSS-
Colitis model, alteration in SUMOylation pathway at mucosal epithelium modulating
inflammation in IBD was investigated in our lab (Salman et al., 2017). With tandem mass-
spectrometry, alteration of SUMO-modification of multiple components of autophagy and
endocytic pathway including Rab GTPases, during intestinal inflammation was observed.
Further experiments indicated Rab7, a late endosome marker and key regulator of autophagy
to undergo significant alteration in inflamed intestinal tissue of mice indicating a direct
relevance to human IBD. Furthermore, Rab7 expression in Paneth cells and Goblet cells of
mice intestine was observed to be altered during DSS mediated colitis. Our quest now is to
decipher the precise role of Rab7 in modulating the functioning of paneth and goblet cells via
dysregulating autophagy. We anticipate that these cellular mechanisms may be pivotal in IBD
pathophysiology and if they can be targeted for possible therapeutic interventions.

POSTER: A34

Melatonin improves AKT/mTOR/LC3 II autophagy signalling in sleep

deprived mice brains
Jagadeeswari P1, Mahalakshm AM1, Abid Bhat1, Bishir M1, Bipul Ray1, Saravana Babu
C1,2*

1Dept.of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education &
Research, Mysuru, India; 2Centre for Experimental Pharmacology and Toxicology, Central
Animal Facility, JSS Academy of Higher Education & Research, Mysuru, India.

A51
Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule having diverse
pharmacological action in central nervous system. Impaired autophagy is reported in various
neurological disorders. The present study is designed to investigate the role of melatonin on
autophagy mechanism in chronic sleep restricted (CSR) animals. Modified platform method is
used to induce paradoxical sleep restriction (21 days; 08.00 am to 06.00 pm) in male Swiss
albino mice. The animals were treated with melatonin at 25 and 50 mg/kg, p.o for 21 days.
Cognitive function in the experimental animals was assessed using Morris water maze during
the third week of treatment period. Animals were euthanized and brains were collected to
investigate the effects of melatonin on the hippocampal protein expression of AKT, mTOR1,
LC3 –II and beclin-1. Melatonin significantly improved spatial and working memory in sleep
restricted mice. Melatonin upregulated AKT, LC3-II and beclin-1 and down-regulated mTOR1
in sleep deprived animals. The present study reveals that CSR impairs autophagy and melatonin
facilitates autophagy in turn cognition in sleep restricted animals.

A52
Abstracts of Poster
presenters
Day-2
(17.01.2020)

A53
POSTER: L1
STX1A regulates maturation of lysosomes and lysosome–related organelles

Anshul Milap Bhatt and Subba Rao Gangi Setty

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012
India

Lysosomes are the primary catabolic organelles of all eukaryotic cells, wherein endocytosed
and intracellular defective macromolecules undergo degradation. SNAREs regulate membrane
fusion involving four set of Qa-, Qb-, Qc- and R- SNAREs. In this study, we have attempted
to evaluate the role of Qa-SNARE STX1A in regulating cargo trafficking to lysosomes.
Immunofluorescence microscopy (IFM) studies revealed that GFP-tagged STX1A localizes to
lysosomes in HeLa cells. STX1A-depletion further altered the distribution of lysosomes
without affecting their organelle content. Additionally, the lysosomes are irresponsive to the
serum starvation in STX1A-depleted HeLa cells. Interestingly, overexpression of dominant
negative form of Rab7 results in mislocalization of STX1A to perinuclear area indicating a role
for Rab7 in lysosomal localization of STX1A. Likewise, regulatory domain deficient mutant
of STX1A was also mislocalized to Golgi and altered the distribution of lysosomes in HeLa.
Altogether, these results suggest that STX1A regulates lysosome function possibly by
modifying the lysosomal distribution or biogenesis. In contrast, GFP-STX1A localizes to
melanosomes, a lysosome-related organelle found in melanocytes. Knockdown of STX1A in
melanocytes mislocalized the cargoes TYRP1 and tyrosinase to lysosomes, suggesting a role
in melanosome biogenesis. Unexpectedly, the transcript levels of lysosomal master regulatory
transcription factors like TFEB and TFE3 were enhanced with a concomitant increase in
lysosomal genes in STX1A-knockdown melanocytes. Currently, we are evaluating the function
of STX1A on melanosome membranes. In future, we would like to identify the cognate
SNARE of STX1A which would form a SNAREpin complex during the delivery of cargo to
lysosomes and melanosomes. Overall, these studies will illustrate the new pathways that are
controlled by STX1A during lysosomes and pigment granule biogenesis.

POSTER: L2
Neuronal regulation of organ maturation: role of DMon1
Neena Dhiman1.2, Kumari Shweta1, Shweta Tendulkar2, Girish Deshpande3, Girish
Ratnaparkhi2, Anuradha Ratnaparkhi1

1Agharkar Research Institute, Pune 411 004; 2 Indian Institute of Science Education and
Research, Pune 411008;3 Princeton University, USA.

Mon1 is an evolutionarily conserved endocytic protein required for the recruitment of Rab7
onto late endosomes. In Drosophila, loss of Dmon1 leads to lethality. The adult escapers are
short-lived, have poor motor abilities and exhibit non-sex specific sterility. An examination of
the female reproductive system reveals highly reduced ovaries lacking mature egg chambers.
Surprisingly, knock-down of Dmon1 in neurons but not ovaries, is able to phenocopy the
mutant. Consistent with this, expression of Dmon1 in neurons is able to rescue the ovary defect.
Furthermore, expression in tyraminergic/octopaminergic neurons alone is sufficient to rescue
lethality and the sterility defect. Interestingly, mon1 mutants show reduced expression of

A54
several insulin-like peptides including dilp5 and dilp3 which are expressed in the median
neurosecretory cells or insulin producing cells (IPCs) in the brain and feeding insulin or
expressing either dilp5 or dilp3 is able to rescue the sterility defect in these mutants. Further,
downregulation of mon1 in the octopaminergic/tyraminergic neurons using RNAi reduces
Dilp5 levels in the IPCs supporting a non-cell autonomous mode of regulation of Dilps by
Mon1. Octopaminergic/tyraminergic neurons are seen to make direct synaptic connections
with the IPCs suggesting a possible direct regulation by Mon1. These results thus identify
Mon1 as an important molecular player in a circuit that regulates ovary maturation.

POSTER: L3
Understanding the Role of a Novel Dynein Interactor in Mitosis

Chandan Kumar, Pergu Rajaiah, Sagar P Mahale, Amrita Kumari and Sivaram V S
Mylavarapu*

Laboratory of Cellular Dynamics, Regional Centre for Biotechnology, Faridabad, Haryana,

India

Mammalian cells divide with high fidelity to generate two euploid daughter cells. The
molecular motor cytoplasmic dynein plays several essential roles in mitosis. C23 has emerged
as a prominent novel interactor of mitotic dynein from earlier work in our group. C23 is a
multifunctional nuclear phosphoprotein that is required for ribosome biogenesis, nucleolus
formation, nucleo-cytoplasmic transport of mRNA, chromatin remodelling and cell
proliferation in interphase. C23 function in mitosis has only been partially explored. My work
aims to elucidate the functional and mechanistic importance of this important protein during
mitosis. We show that C23 interacts with dynein in mammalian cell lysates, and siRNA-
mediated depletion phenocopied dynein depletion, demonstrating the requirement of C23 for
chromosome congression, spindle pole integrity and metaphase to anaphase progression. In
order to understand the underlying molecular mechanisms, we determined the mitotic
interactome of C23. Analysis of the interactome revealed key players of the early stages of
mitosis as well as potential engagement of C23 with the early endocytic pathway. We are
validating these findings biochemically, microscopically and functionally. Our study will
highlight potential molecular mechanisms employed by C23 in ensuring proper mitotic
progression.

POSTER: L4

Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via

Autophagy-Lysosomal Pathway
Chandrima Gain, Samaresh Malik, Shaoni Bhattacharjee, Arijit Ghosh, Benu Brata
Das, and Abhik Saha

Dept. of Life Sciences, Presidency University

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation
and subsequent development of several B-cell lymphomas particularly those generated in an
immune-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for

A55
deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C
is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the
viral protein is surprisingly stable in growing B-lymphocytes. In addition, EBNA3C can bypass
autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-
cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as
a prerequisite for B-cell survival particularly under growth deprivation conditions. We now
demonstrate that proteasomal inhibition using MG132 leads to degradation of EBNA3C both
in EBV transformed and ectopic-expression systems. Interestingly, MG132 treatment promotes
degradation of two EBNA3 family oncoproteins – EBNA3A and -EBNA3C, but not the viral
tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition
is partially blocked when autophagy-lysosomal pathway is inhibited. Using confocal
microscopy, we demonstrate that EBNA3C can colocalize with the autophagy-lsyosomal
fraction in the cytoplasm in response to proteasomal inhibition. Further, immunoprecipitation
assay confirms that EBNA3C can colocalize and interact with one of the key autophagy
components LC3B. Using various truncated domains we demonstrate that the degradation
signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal
inhibition reduces the colony formation ability of this important viral oncoprotein, increases
transcriptional activation of both latent and lytic gene expression and induces viral reactivation
from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors
as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where
EBNA3C is expressed.

POSTER: L5
Regulation of osteoclast function by the small GTPase Arl8b

Gaurav Kumar*#, Subhash B. Arya*¶, Sheetal Sharma*, Harmeet Kaur* and Amit Tuli*

*Division of Cell Biology and Immunology, CSIR-Institute of Microbial Technology

(IMTECH), Chandigarh 160036 India; ¶Life Sciences Institute, University of Michigan, Ann
Arbor, Michigan 48109 USA

Recent studies have established Arl8b-mediated positioning of lysosomes and lysosomes-

related organelles as a crucial factor regulating amino acid sensing, cell migration and
metastasis, NK cell-mediated cytotoxicity and antigen presentation. Arl8b mediates lysosomal
transport to the cell periphery by recruiting its effector, SKIP, which in turn recruits the motor
protein kinesin-1 on lysosomes. Arl8b also binds to the Rab7 effector- PLEKHM1, and this
interaction repositions Arl8b-positive lysosomes to the perinuclear region of the cell and
promotes autophagosomes-lysosome fusion. Interestingly, frameshift mutations in PLEKHM1
result in Osteopetrosis where the bone resorbing functions of osteoclasts is impaired.
Osteoclasts resorb bone by secreting their lysosomal contents within the confines of a sealing
zone between themselves and the bone surface. Here, we have explored the role of Arl8b in
bone remodeling. Arl8b showed a striking localization beneath the actin rings and ruffled
borders in osteoclasts, and lysosome secretion was significantly impaired in Arl8b-deficient
osteoclasts. Further, unlike control osteoclasts, lysosomes in Arl8b-deficient osetoclasts failed
to localize beneath the actin rings or in the ruffled borders. Taken together, our findings
establish Arl8b as a crucial component of osteoclast-mediated bone remodeling.

A56
POSTER: L6
PLiMAP: A generic, high throughput and sensitive membrane-binding
assay

Gregor P. Jose and Thomas J. Pucadyil

Pucadyil lab, IISER Pune

Peripherally bound membrane proteins are an important class of biological molecules yet
facile, sensitive and quantitative methods that complement the widely used liposomes-based
co-sedimentation assays remain unavailable. Here, we describe the utility of a photo-reactive
fluorescent lipid (RFL) as a generic reporter of protein-membrane interactions. When
incorporated into liposomes and exposed to UV, proteins bound to liposomes become
crosslinked with the fluorescent lipid and can be readily detected and quantitated by in-gel
fluorescence analysis. This modification obviates the requirement for high-speed
centrifugation common to most assays that detect liposome-bound proteins. We refer to this
assay as Proximity-based Labeling of Membrane-Associated Proteins (PLiMAP). We have
validated the lipid binding specificities and binding affinities of SidM (P4M domain) and PLCδ
(PH domain) proteins using this method.

POSTER: L7
Nucleophagy: Role in nuclear organization

Gurranna Male, Naresh Kumar M, and Krishnaveni Mishra

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad,

India - 500046

The nucleus is the most prominent organelle of the eukaryotic cell. It is organized into various
sub-compartments, with the nucleolus being the most prominent. In a focused genetic screen
for the nuclear organization in S. cerevisiae to ask if the nucleolus influences the nuclear shape,
we find that increasing nucleolar volume triggers non-isometric nuclear envelope expansion
resulting in an abnormal nuclear envelope shape. Autophagy is a physiologic catabolic process
that occurs in all eukaryotic cells to recycle nutrients during starvation or remove defective
organelles or protein aggregates to maintain cellular homeostasis. Selective autophagy or
microautophagy degrades the selective part of the organelles. Piecemeal Nucleophagy (PMN)
is a specialized process that removes abnormal structures of the nucleus through autophagy.
Non-isometric expansion of the nucleus produces abnormal nuclear extensions that are the
substrate for nucleophagy. The results of our study implicating nuclear autophagy in nuclear
shape regulation will be discussed.

POSTER: L8
Mycobacterial-host Interaction a trick or treat: Inhibition of autophagy by
Mycobacteria through stpkl in a nutrition starvation model.
Harini Rajeev Lakshminarayan

1National Institute for Research in Tuberculosis, India; 2University of Cambridge, United

A57
PknL plays an important role in sensing the host environment and adapting itself in slowing
down the growth of the pathogen and persisting within the host. Internal signals used to activate
PknL are most likely the host-associated internal signals and the ability of PknL to respond to
such stresses is relevant to their survival in vivo. Scanning Electron Microscopy indicates that
PknL is also responsible for cell wall homeostasis. Proteome profiling studies have also shown
the involvement of PknL in stress and starvation response of M. tuberculosis. It is proposed
that PknL can sense the environmental cues and act accordingly thereby helping the bacteria in
adaptation to stress conditions inside the host by slowing down the growth of the bacteria
leading to dormant state and thereby persisting within the host. In host phagocytic cells, M.
tuberculosis interferes with the phagosome maturation pathway. The autophagy trafficking
pathway has been originally described as a cellular adaptation to starvation, whereby bulk
cytoplasmic ingredients are engulfed by a membrane of still-unknown origin and degraded for
reprocessing to sustain cellular anabolic needs. Autophagy is a bulk degradation system that
delivers cytoplasmic constituents into lysosomes. This process enables the reuse of intracellular
constituents and supplies an amino acid pool during periods of starvation. The delicate balance
between external energy and nutrient supply and internal production and consumption is a
demanding task. The complex protein network that senses and precisely reacts to
environmental changes is thus mainly regulated by rapid and reversible posttranslational
modifications such as phosphorylation.

POSTER: L9
Deciphering the role of the pleckstrin-homology domain in endocytic
dynamins

Himani Khurana1, Kirtika Jha2, Krishnakanth Baratam2, Anand Shrivastava2 and

Thomas Pucadyil1

1IndianInstitute of Science Education and Research, Pune ; 2Indian Institute of Science,

Bangalore

Dynamin superfamily proteins are multidomain GTPases that bind negatively-charged

membranes and self-assemble into helical scaffolds. Self-assembly stimulates their GTPase
activity, which in turn facilitates membrane fission. Endocytic dynamins act on the necks of
clathrin-coated buds and manage their fission and release into vesicles during clathrin-mediated
endocytosis and synaptic vesicle biogenesis. Interestingly, endocytic dynamins have evolved a
specialized membrane-binding pleckstrin-homology domain (PHD). Using a facile assay
system of supported membrane nanotubes to monitor membrane fission in real-time, previous
work from the lab showed that the absence of PHD renders the dynamin-mediated membrane
fission reaction to proceed slowly with long-lived pre-constricted membrane intermediates.
Together, these results point to a catalytic role of the PHD in dynamin-mediated membrane
fission. To decipher the molecular basis of such catalytic function, we performed atomistic
molecular dynamics simulations of the PHD on a membrane composed of physiologically-
relevant concentrations of negatively-charged lipids. We find the PHD to become stabilized on
the membrane by partial insertion of a highly conserved variable loop 1 (VL1), as reported
earlier, but remarkably, also by the insertion of a novel variable loop (VL4). VL4 is
characterized by the presence of a phenylalanine residue at its tip. Mutation of this residue to
an alanine manifests a dramatic defect in the fission activity, which can be attributed to its

A58
severely compromised membrane binding as revealed by analysis of fluorescently-labelled
protein. Together, these results emphasize the essential role for VL4 in dynamin function and
point to a possible function of VL4 in the PHD to facilitate the high capacity capture,
constriction and fission of the curved necks of transport vesicles.

POSTER: L10

A study on the effects of Krill oil on autophagy-lysosomal functions and

amyloid-β clearance in scopolamine intoxicated mice brains

Jahnavi, K1, Saravanan Bhojaraj2, Muhammed Bishir2, Saravana Babu

Chidambaram2,3, Malathi Srinivasan1

1Department of Lipid Science, CSIR-CFTRI, Mysore -570020 & Academy of Scientific and
Industrial Research; 2Department of Pharmacology, JSS College of Pharmacy, JSS Academy
of Higher Education & Research, Mysuru – 570015; 3Centre for Experimental Pharmacology
and Toxicology, Central Animal Facility, JSS Academy of Higher Education & Research,
Mysuru - 570015

The present study was undertaken to investigate the effects of Krill oil on the autophagy-
lysosomal functions and amyloid- clearance in scopolamine intoxicated mice brains. In
addition, the ability of Krill oil on the cognitive enhancing potential was studied using Morris
water maze and novel object recognition test. Identification of compounds that restore
autophagy-lysosomal functions and that modulate amyloid- depositions is clinically
important in the treatment of Alzheimer’s disease. In the present study, male C57BL/6 mice
were divided in to seven groups: Negative control (received saline); Positive control (received
saline and scopolamine); Treatment groups (received Krill oil and scopolamine and Krill oil
low and high dose + scopolamine), drug control (received donepezil and donepezil and Krill
oil + scopolamine). Administration of Krill oil altered autophagy-lysosomal markers (such as
LC3 and Beclin) expression. Krill oil treated mice also showed changes in amyloid-
expression in the hippocampal region of brain. The improved autophagy-lysosomal functions
exerted by Krill oil was found to be mediated through the up-regulation of Transcription factor
EB (TFEB), a master regulator of autophagy and lysosomal genes. Taken together, these results
indicate the neuroprotective potential of Krill oil against scopolamine induced
neurodegeneration and cognitive impairment. In summary, Krill oil may provide a therapeutic
strategy towards improving autophagy–lysosomal functions through TFEB and in turn, the
cognition, in scopolamine intoxicated mice.

POSTER: L11
Unravelling lysosome mediated nutrient sensing with super-resolution
STORM imaging

Jeganath A, and Nitin Mohan

Biological Sciences and Bioengineering Department, IIT Kanpur, Uttar Pradesh 208016

A59
Lysosome, long been known as the catabolic compartment for intracellular as well as the
exogenous molecules. However, recent studies on nutrient sensing kinase-mTOR (Mechanistic
Target of Rapamycin) and its localization on the lysosome surface suggests that lysosome is a
key player in metabolic signal transduction. mTOR, a serine/threonine kinase of the PI3K
related kinase family, is the master regulator of cell growth and metabolism in response to
energy, nutrients and oxygen level. It nucleates two complexes called mTORC1 and mTORC2,
which regulates the growth and plays a vital role in protein synthesis and autophagy. The
mTORC1 localizes to endomembrane systems such as a late endosome or lysosome and forms
an active complex. Though studies have revealed the mTORC1 components and the structural
basis of the interaction, the active stoichiometry of the mTORC1 in the lysosome and their
effect on the lysosome localization and function are not well understood. In our study, we will
be using super-resolution microscopy techniques to precisely locate the mTORC1 component
proteins and lysosomes spatiotemporally. Our preliminary results showed that the lysosomes
are enriched in the detyrosinated microtubules, a post-translationally modified (PTM) form of
microtubules. We will be probing the lysosomal distribution on different PTM microtubules
when it carries the active mTORC1 as well as modulated mTORC1. We are also interested to
understand the implication of the mTORC1 activation and lysosomal distribution both in
normal and diseased conditions. A successful understanding of this gap will give insights in
controlling various diseases such as diabetes, cancer.

POSTER: L12
IRGM is a Master Negative Regulator of Interferon Response by
Controlling the Activation of cGAS-STING and RIG-I-MAVS Signaling
Pathways

Kautilya Kumar Jena, Subhash Mehto, Parej Nath, NishantRanjan Chauhan,Punit

Prasad, Soma Chattopadhyay, Swati Chauhan, Santosh Chauhan#

Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, 751023,
India

Activation of type 1 interferon response is extensively connected with the antiviral response
and pathogenesis of autoimmune diseases. Here, we found that IRGM whose deficiency is
linked with genesis of several autoimmune disorders is a master negative regulator of interferon
response. Mechanistically, we show that IRGM interacts with nucleic acid sensor proteins
including cGAS and RIG-I and mediates their autophagic degradation to restrain aberrant
activation of interferon signaling. Conversely, IRGM deficiency results in defective mitophagy
flux, accumulation of defunct leaky mitochondria, which releases cytosolic DAMPs that
triggers activation of interferon response via cGAS-STING and RIG-I-MAVS signaling axis.
Due to an enduring type 1 IFN response in IRGM-deficient cells and mice, they were
intrinsically resistant to infection of Japanese Encephalitis virus, Herpes Simplex virus and
Chikungunya viruses. Altogether, this study defines the molecular mechanisms by which
IRGM maintains interferon homeostasis and protects from autoimmune diseases. Further, it
identifies IRGM as a broad therapeutic target for defense against viruses.

A60
POSTER: L13
A Redox-dependent Membrane Fission Catalyzed by α-synuclein

Krishnendu Roy and Thomas Pucadyil

IISER Pune, India

α-synuclein (AS) is a vertebrate-specific, abundant neuronal protein that binds negatively-

charged membranes. Several lines of evidence causally link its tendency to aggregate into
extracellular plaques to the pathogenesis of Parkinson's Disease (PD). Despite these reports,
very little is known about the native function of AS. AS is intrinsically unstructured in solution
and adopts a partial helical conformation on membranes. Previous reports using very high
protein concentrations have suggested that membrane-bound AS can deform liposomes into
narrow tubules and small vesicles. Using a novel method to assay membrane binding properties
of peripherally-associated proteins, we discover that AS displays preferential affinity for the
mitochondrial lipids cardiolipin (CL) and phosphatidyl glycerol (PG). We then tested AS
functions on CL-containing supported membrane tubes that mimic mitochondria in membrane
topology and lipid composition. Such experiments showed no apparent membrane remodeling
or vesiculation. Remarkably however, similar assays carried out under conditions that mimic
the intracellular reducing environment showed dramatic fission of tubes. Together, our results
point to the possibility that AS functions in a novel redox-dependent mitochondrial fission
pathway.

POSTER: L14
Biochemical characterization of lysosomal enzymes, β-hexosaminidase and
α-L-fucosidase from Hydra vulgaris Ind-Pune

Lakshmi Surekha Krishnapati1*, Poorna Manasa Bhamidimarri1*, Rohit Sai Reddy

Konada1*, Surendra Ghaskadbi2, Siva Kumar Nadimpalli1#

1Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, School of

Life Sciences, University of Hyderabad, Hyderabad-500046, India; 2Developmental Biology
Group, MACS-Agharkar Research Institute, G. G. Agarkar Road, Pune-411004, India

Hydra, a classical model to study regeneration and development is becoming a popular and
alternative model for the assessment of toxic pollutants of aquatic origins in recent years. At
the cellular level, lysosomes are the major organelles that undergo substantial changes due to
the toxic effects of many contaminants. It would be interesting to use this information to
decipher the dynamic changes occurring in the lysosomal hydrolases of Hydra to assess aquatic
pollution. Our recently published results have established the conservation of several lysosomal
enzymes and mannose-6 phosphate receptor-dependent lysosomal targeting system in Hydra.
Here, we present the identification and biochemical characterization of β-hexosaminidase
using the soluble protein extracts of Hydra. Purification of β-hexosaminidase was carried out
using size exclusion chromatography and Con-A affinity chromatography. Tissue localization
by whole-mount in situ hybridization revealed predominant expression of β-hexosaminidase in

A61
the endoderm. An attempt was also made to express Hydra β-hexosaminidase in the
mammalian cells. Similarly, identification and in silico characterization of α-L-fucosidase was
carried out. Tissue localization studies showed predominant expression in specific cells of the
ectoderm and the endodermal epithelial cells, while its expression is absent in the cells
undergoing oogenesis. Lysosomal machinery of Hydra can serve as specific molecular marker
for risk assessment of environmental toxins and can be used as biological indicators of
freshwater contamination.

POSTER: L15
Role of Mint family of Proteins in Localization, Trafficking and Processing
of Amyloid Precursor Protein

Mahadeva Swamy.H.S, Vivek Belapurkar, Shekhar Kedia, Deepak Nair

Centre for Neuroscience, Indian Institute of Science Bengaluru-560012

Amyloid beta, a key determinant in the pathology of Alzheimer’s disease is formed by the
cleavage of Amyloid Precursor Protein (APP) by secretases. The recent studies demonstrate
the differential localization of APP in functional domains of synapse. Mint family of proteins,
consisting of Mint1, Mint2 and Mint3 are the adaptor proteins which is shown to rescue
memory deficits in APP transgenic mouse. Here we have addressed the role of Mint isoforms
in regulating the localization and trafficking of APP. We looked at the relative mRNA
expression levels of Mint isoforms in hippocampus and cortex of mice at different
developmental stages namely Post-natal-1, 1 month, 3 month and 6 months. The relative
mRNA expression of Mint1 showed the highest, followed by Mint2 and the lowest by Mint3.
The localization study using super resolution microscopy revealed colocalization of Mint1 and
Mint2 with pre synapse, excitatory post synapse, inhibitory post synapse and endocytic zones.
However, Mint1 and Mint2 showed more increased association with pre synapse and endocytic
zones of the excitatory synapse. Fluorescence Recovery after Photobleaching and single
molecule localization microscopy study showed discrete mobility pattern for Mint isoforms
and regulation of lateral mobility of APP molecules by Mint isoforms. Association of Mint
with APP is important for understanding surface dynamics, endocytosis and proteolysis of
APP. As Mint showed more association with endocytic zone, it can trap APP molecules in the
endocytic zone and effect the processing of APP.

POSTER: L16
The Role of RNA binding proteins in Tunneling Nanotube Formation and
Function

Monika Rawat, Sunayana Dagar and Sivaram V S Mylavarapu*

Laboratory of Cellular Dynamics, Regional Centre for Biotechnology, Faridabad, India

Tunnelling nanotubes (TNTs) are a novel and important, but poorly understood mode of
intercellular communication across various in vitro and in vivo systems. TNTs constitute a
diverse variety of long, membrane-enclosed, tubular cytoplasmic conduits that connect

A62
eukaryotic cells several hundred micrometers apart. TNTs mediate a wide spectrum of critical
cellular functions, including ion signaling, antigen presentation, intercellular organelle transfer,
morphogen transfer during development, glia-neuron communication, metastatic cell homing,
HIV and Chikungunya intercellular viral transmission, prion protein transfer and bacterial
surfing between cells. Despite their demonstrated importance in health and disease, a thorough
molecular mechanistic understanding of the biogenesis, growth, maintenance and function of
TNTs is missing in any system. Analysis of the cellular interactome of MSec (a protein
essential for TNT formation in many systems) determined in our group revealed the presence
of RNA binding proteins (RBPs) that we show are required for TNT formation. Depletion of
the novel MSec interacting RBPs from cells does not appear to affect the cortical enrichment
of MSec, but drastically reduces TNT numbers. These observations suggest that the RBPs bring
some additional activity that is required for TNT biogenesis. Current efforts are focussed on
understanding the role of these RBPs in TNT formation and function. Our study will generate
important molecular mechanistic knowledge regarding the importance of RBPs in TNT
biogenesis and could reveal novel targets for therapeutics against TNT-based diseases.

POSTER: L17
Investigating role of endo-lysosomal cholesterol transport in mTOR
signaling

Kolaparamba V. Navyasree, Shikha T. Ramesh and Perunthottathu K. Umasankar

Rajiv Gandhi Centre for Biotechnology (RGCB), Trivandrum, Kerala

Lysosomes are end compartments of endocytosis and have long been recognized as organelles
to degrade and recycle cell waste. Recent studies radically transformed this idea by identifying
lysosomes as signaling hubs for metabolic sensing. Nutrient-regulated switching of mTORC1
(mechanistic target of rapamycin) kinase, the master regulator of cell growth and metabolism,
between cytosol and limiting membrane of lysosomes strengthened this view. Lysosomes are
also major sorting stations for dietary cholesterol. However, whether lysosomal cholesterol
influences mTOR signaling and the cellular consequences associated with this process remain
poorly understood. Here, we demonstrate that lysosomal cholesterol regulates mTOR signaling
in cells. Depletion of total cholesterol by methyl-β-cyclodextrin (MCD) in the presence of other
nutrients and growth factors inactivates mTORC1 by displacing the kinase to cytosol. We also
show that depletion of cholesterol hampers insulin-dependent mTOR activation. Consistently,
adverse effects on mTOR signaling can be reversed by repleting cholesterol in the form of low-
density lipoprotein (LDL) or free cholesterol but not derivatives of cholesterol. In addition, we
demonstrate that blocking lysosomal cholesterol transport by functionally inhibiting lysosomal
sterol-transporter NPC1 alters lysosome positioning and nutrient-regulated switching of
mTOR. Together, our findings imply novel mechanisms of metabolic sensing and integration
by lysosomes. We anticipate that our investigations on cholesterol-mTOR-lysosome axis
would help identify innovative therapeutic strategies to combat lysosomal storage diseases
such as Niemann-Pick disease type-C (NP-C) and certain cancers.

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POSTER: L18
The Specific activities of Acid Phosphatase related to lysosomal activation
and Correlation of Anaplastic Oligodendroglioma G-III to Meningiomas
G-I among all Malignant and Benign Brain Tumors

Prabha M*1, Ravi V2, Chethan C T3, Chadrasekhar Sagar4, N Ramachandra Swamy5

1Corresponding Author, Associate Professor, Dept. of Biotechnology, Ramaiah Institute of

Technology, Bangalore-54; 2Professor, Department of Neurovirology, NIMHANS, Bangalore
560029; 3Assistant Professor, Dept. of Computer science and Engineering, Ramaiah Institute
of Technology, Bangalore-560054; 4Additional Professor, Departments of Neuro Pathology,
NIMHANS, Bangalore -560029; 5Professor (Retd), Department of Biochemistry, Central
College, Bangalore-560001

The specific activities of acid phosphatase (ACP) was studied in different grades of
Meningiomas (n=75) and gliomas (n=81) compared among brain tumors and normal brain
tissues. Average ACP showed 9.32617 ± 4.1144 for meningiomas (n=55) and 5.91± 5.8305
for gliomas (n=60) respectively as compared to normal brain 7.104 ± 1.33 (n=120) nm/min/mg
of protein. ACP showed higher activity for meningiomas but not in gliomas as compared to
normal brain. The spectrophotometric results were similar to Electrophoresis native gel band
pattern in which similar average specific activities for ACP obtained for gliomas. However,
GBM IV showed higher ACP activity (marker for lysosomes). Electron microscope showed
many number of lysosomes for GBM as compared to meningiomas which were similar to
spectrophotmetric results. ACP showed higher specific activities for Anaplastic
Oligodendroglioma G-III which is the first report in which ACP levels similar to Meningiomas
Grade-I similarly anaplastic meningioma G-III features are similar to some higher grade
gliomas. The ACP expression in brain tumor cell culture constantly lower in meningiomas but
not for gliomas which varies with expression may due to migration of ACP with cell especially
in protoplasmic astrocytoma G-II progressing to G-III. The activation of Acid Phosphatase
confirms lysosomal activation in higher grade gliomas of GBM G-IV for complimentary
diagnosis for brain tumors that demonstrates to know the status of defence function of the cell
against invasion of tumor cell for use in the investigation of the pathophysiology of the
associated disease towards the brain tumors treatment.

POSTER: L19
Purification, biochemical and biophysical characterization of lysosomal β-
D-glucuronidase from an edible freshwater mussel, Lamellidens corrianus

Rohit Sai Reddy Konada, Venugopal A, Siva Kumar Nadimpalli#

Laboratory for Protein Biochemistry and Glycobiology, Department of Biochemistry, School

of Life Sciences, University of Hyderabad, Prof CR Rao road, Gachibowli, Hyderabad-
500046, India

A64
The cation-independent mannose 6-phosphate receptor (MPR 300) and cation-dependent
mannose 6-phosphate receptors (MPR 46) play important role in the biogenesis of lysosomes
by targeting newly synthesized lysosomal enzymes. This function of MPRs is accomplished
by their binding to the mannose 6-phosphate (M6P) residues present on the lysosomal
hydrolases. The M6P binding properties of MPRs have been studied extensively in mammals
and other vertebrates. In invertebrates the binding properties of these receptors is under
explored. Our laboratory has been studying the M6P dependent targeting of lysosomal enzymes
in invertebrates. As a part of the study, we could identify and characterize the lysosomal
enzymes from Lamellidens corrianus, a commonly available mollusc in the freshwater bodies.
The lysosomal hydrolases like α-fucosidase and β-N-acetyl hexosaminidase were reported
earlier from the same species. Although occurrence of β-glucuronidase among invertebrates
was reported, clear biochemical analysis and structural studies were not done. In this study, a
comprehensive biochemical analysis of β-glucuronidase, purified using conventional
chromatography steps from L. corrianus was carried out. In SDS-PAGE, the enzyme was
resolved into four sub units with approximate molecular weights of 90, 75, 65, and 50 kDa
respectively, and subunits 90, 50 kDa cross-reacted with human β-glucuronidase antiserum.
The optimum pH and temperature of the purified glycosidase was 5.0 and 70°C respectively
and the kinetic parameters, KM and Vmax of the enzyme were 0.457 mM and 0.11867 µmol-
1min-1mL-1 respectively. The secondary structure was also determined using CD spectroscopy.
This is the first report to ascertain the biochemical and biophysical features of β-glucuronidase
from a fresh water bivalve.

POSTER: L20
ARL6ip5 interacts with α-Synuclein and modulates its neurotoxicity

Sakshi Gupta, Kajal Kamble, Rajiv Ranjan Singh & Sarika Gupta

National Institute of Immunology, New Delhi, India

α-Synuclein is a presynaptic neuronal protein, neuropathologically linked to Parkinson's

disease (PD). The secreted form of neurotoxic α-synuclein exerts deleterious effects on
neuronal cells via seeding of aggregation contributing to disease propagation. Hence
attenuation of neurotoxicity of alpha-synuclein is proposed as an alternative to current PD
therapy. We have observed that expression of Arl6ip5 goes down with increased α-synuclein
aggregation. Herein we report the role of Arl6ip5, a regulator of glutamate toxicity in
modulating the neurotoxicity of alpha-synuclein. In SH-SY5Y neuronal culture, stably over-
expressing mutant alpha-synuclein, a transient overexpression of ARL6ip5 causes decrease in
cytosolic aggregates and neurotoxic exosome release (evidenced by reduced intracellular
expression of CD63 and flottilin, markers of exosome synthesis). Interestingly, co-
immunoprecipitation experiments, in situ and co-localization experiments in both alpha-
synuclein and Arl6ip5 overexpressing neurons showed that both colocalize and alpha-
synuclein interacts with Arl6ip5. Thus, Arl6ip5 interacts with alpha-synuclein under
neurotoxic conditions and might be critical in modulating neurotoxic effects of alpha-synuclein
under conditions like PD.

A65
POSTER: L21
Modulation of pseudophosphatase STYXL1 function restores lysosomal
activity in Gaucher’s disease

Saloni Patel and Subba Rao Gangi Setty

Dept. of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Pseudophosphatases are catalytically inactive counterpart of classical phosphatases, which

share sequence and structural similarity and form 8% of total phosphatome. They act as
modulators of cellular signalling pathways possibly through inhibiting the function/activity of
phosphatases either directly or indirectly. Genome-wide RNAi screen of phosphatases
identified a pseudophosphatase STYXL1 [also known as MK-STYX; MAPK (mitogen-
activated protein kinase) phospho- serine/threonine/tyrosine-binding protein], whose
knockdown enhanced the lysosome enzyme activity in HeLa cells. STYXL1 belongs to dual
specificity phosphatases known to be involved in apoptosis, stress granule formation and
neurite formation. However, STYXL1 role in cellular trafficking is not yet elucidated. Our
studies show that STYXL1-knockdown enhances -glucocerebrosidase (-GC) trafficking and
its activity in HeLa cells. Further, depletion of STYXL1 causes translocation of lysosomal
biogenesis transcription factor TFEB to the nucleus and enhances ER stress in HeLa cells.
Interestingly, the increase in -GC activity in STYXL1-knockdown cells is independent of
TFEB nuclear localization. In addition, activation of TFEB through starvation response,
inhibition of calcineurin or mTOR activity showed an additive effect to -GC activity in
STYXL1 knockdown cells. In line, elevation of ER stress by thapsigargin also illustrated an
additive effect on enhancing the -GC activity in STYXL1 knockdown cells, suggesting an
indirect role in lysosome function. Consistently, depletion of STYXL1 in human primary
fibroblast cells derived from Gaucher’s patients showed enhanced lysosomal enzyme activity.
Overall, these studies illustrated the novel cellular function of a catalytically inactive
phosphatase in modulating the trafficking of wild type and mutant -GC. In future, designing
of small molecules against STYXL1 possibly restore the lysosome activity in Gaucher’s
patients and may act as possible alternate therapy to lysosome-storage disorders (LSDs).

POSTER: L22
Deciphering the role of STX7 in breast cancer cell invasion

Parveen S.and Datta S

Department of Biological Sciences, Indian Institute of Science Education and Research,

Bhopal, Madhya Pradesh, India

Cancer cell invasion is a complex process, involves breaching of the basement membrane,
degradation of the complex extracellular-matrix and metastasis to distant places.This series of
events require a complex but coordinated delivery of nutrient cargoes, signalling molecules
and the proteases to the matrix degrading devices called invadopodia. SNAREs belong to a
protein superfamily, comprising of more than 60 members reported in mammals and yeast.
They have an evolutionarily conserved coiled-coil stretch containing 60–70 amino acids termed
as SNARE motif. In this study, we have used siRNA based knockdown approach coupled with

A66
assays such as Gelatin degradation assay, Matrigel invasion assay which led us to narrow down
from 14 candidates involved in endocytic and recycling pathway to 4 candidates namely STX2,
STX7, VAMP3 and VAMP7. Our data suggest that STX7 resides in early endosome(EEA1)
as well as in recycling compartments(Rab4 & Rab11). Because of its localization in early and
recycling compartment, we hypothesized that STX7 may have a role in trafficking of certain
cargoes towards the protrusive structure of the plasma membrane which helps in breast cancer
cell invasion. It is found that a significant population of integrin, MT1MMP, MT2MMP, EGF
and transferrin are co-localizing with STX7. STX7 depletion leads to cargo accumulation in
recycling compartments which were unable to fuse with the plasma membrane. The recycling
defect was tracked via TIRFM and biochemical assays. Further to establish STX7 is specific
or acts in general in the recycling route, search for interacting partners by proteomics study is
currently under progress.

POSTER: L23
IIIM-3 activates lysosomal mediated cell death via mTOR-TFEB axis in
MIA PaCa-2 cells.

Sameer Ullah Khan1, Anup Singh Pathania2, Abubakar Wani3, Masroor Ahmad1,
Sameer A. Mir1, Mehboob Ali1, Mubashir Javed Mintoo1, SD Sawant1and Fayaz Ahmad
Malik1

1Division of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu-

180001, India; 2Department of Cancer and Blood disease, University of Southern California
University USA; 3Department of Neurology, Washington University in St. Louis, MO 63110-
USA

Autophagy is a cellular lysosomal degradative process by which it sequesters the damaged

organelles, aggregated proteins into a double membrane structure known as autophagosome.
The lysosome is a membrane-bounded organelle that contains various catabolic enzymes
known as acid hydrolases which play an important role in the progression of cancer at various
stages but the excessive release of these enzymes into the cells induces cell death. Autophagy
in cancer has a dual role, it can promote or suppress cancer depending upon the type and the
stage of cancer. In current study, IIIM-3 was identified as an anti-cancer agent that inhibit the
mammalian target of rapamycin (mTOR) that leads to the activation of autophagy and
lysosomal biogenesis via activation of TFEB in MIA PaCa-2 cells. Our results showed IIIM-3
inhibited the fusion of autophagosome with the lysosome thus resulting in the accumulation of
autophagosomes and ultimately cell death. It was also demonstrated that IIIM-3 promotes the
lysosomal membrane permeabilization which promotes the leakage of hydrolases into cytosol
thus inducing cell death. Knockdown of transcriptional factor TFEB abrogates the effect of
IIIM-3 in MIA Paca-2 cells. Furthermore, bafilomycin A1 which prevents the acidification of
lysosomes rescued IIIM-3 mediated cell death while 3-Methyl adenine an early autophagy
inhibitor, has no impact on their survival indicating independent autophagic death induction by
IIIM-3 in pancreatic cancer cells. Results were further validated by in vivo studies. Based on
our findings, IIIM-3 can be further investigated as a potential candidate for developing
clinically active agent against pancreatic cancer.

A67
POSTER: L24
Differentiated keratinocytes generate unique Golgi-associated lysosomes

Sarmistha Mahanty1, Litralson Eluvathingal2, Vivek Belapurkar2, Deepak Kumaran

Nair2, Amitabha Majumdar3 and Subba Rao Gangi Setty1

1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012
India; 2Centre for Neuroscience, Indian Institute of Science, Bangalore 560 012 India;
3Unilever Industries Pvt. Ltd., Bangalore 560066, India

Epidermis, the outer skin layer provides hydrophobic and antimicrobial barrier to the body and
is critical in maintaining skin health and hygiene. Keratinocytes are the major constituent cell
type of epidermis which maintains skin homeostasis through cellular differentiation. However,
the molecular link between keratinocyte differentiation and skin homeostasis is critically
unknown. In that end, we have used human neonatal primary keratinocytes and differentiated
the cells using high extracellular calcium. We demonstrated that upon differentiation,
keratinocytes produce large number of peripherally distributed globular lysosomes.
Differentiated keratinocytes also exhibit high autophagy turnover which corroborates to the
enhanced lysosomal activity. Further, both these processes are independent of mTOR signaling
and is regulated by ER stress induced UPR pathway. Notably, differentiated cells showed
highly fragmented and dispersed Golgi. Fluorescence microscopic analyses illustrated that
certain Golgi proteins are associated with the lysosomes of differentiated keratinocytes,
suggesting a possible influence of Golgi in the secretion of these lysosomes. These organelles
retain classical lysosomal characteristics despite they are associated with the Golgi proteins.
As expected, disruption of Golgi function or its association led to the malformation/tubulation
of the lysosomes and affected keratinocyte differentiation. However, how this Golgi-
association is important for lysosomal secretion and epidermal homeostasis is currently under
investigation. In future, we would like to identify the skin diseases wherein the Golgi-
associated lysosomal activity is altered.

POSTER: L25
Rabip4’ interacts with the small G protein Arl8b and regulates membrane
trafficking in the endolysosomal pathway

Shalini Rawat1, Rituraj Marwaha1, 2, and Mahak Sharma1 *

1Department of Biological Sciences, Indian Institute of Science Education and Research

Mohali (IISERM), Punjab, India; 2 Tata Institute of Fundamental Research (TIFR), Hyderabad

Lysosomes are primarily known for their catabolic function in degradation of cellular
macromolecules, but recent findings reveal that lysosome is also a site for processes like
nutrient signalling, plasma membrane repair, and in the long-range transport of RNA.
Lysosomal sub-cellular distribution and fusion with other compartments govern its diverse
roles in the cell. Our research group is interested in small GTP binding protein, Arl8b which
localizes on lysosomes and via interaction with its effectors proteins regulates lysosomal

A68
positioning and cargo trafficking to lysosomes. Here we report a new Arl8b interaction partner,
Rabip4' (RUN and FYVE domain-containing protein) that interacts with Arl8b via its N-
terminal RUN domain. Rabip4’ localizes to the early endocytic compartments positive for
Rab14 and early endosomal antigen (EEA1). Our findings suggest that both Arl8b and Rabip4'
are present on a subset of early endocytic compartments that also contain Rab14 and the Cation-
Independent Mannose-6 phosphate receptor (CI-M6PR). Arl8b siRNA treated cells show
redistribution of endogenous Rabip4’ to the cytosol, suggesting that Arl8b is required for stable
membrane localization of Rabip4’.To investigate the role of Rabip4’ in late endocytic
compartments, we visualized the late endosomes/lysosomes in Rabip4’siRNA treated cells.
Depletion of Rabip4' leads to an increase in the size of LAMP1-positive compartment and
enhanced accumulation of lysotracker dye that is retained only in acidic organelles. We are
exploring the mechanism by which Rabip4' is regulating morphology, composition and
function of late endocytic compartments and also want to determine the relevance of its
interaction with Arl8b in this process.

POSTER: L26
Activation of AP-2 adaptor complex via FCHO-BMP2K axis promotes
clathrin-mediated endocytosis

Shikha T. Ramesh1, Kolaparamba V. Navyasree1, Anjitha B. Ashok1, Nishada Qathoon1,

Suryasikha Mohanty2, Rajeeb K. Swain2, Perunthottathu K. Umasankar1

1Intracellular Trafficking Laboratory, Interdisciplinary Biology Research Program, Rajiv

Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India; 2 Institute of Life
Sciences, Bhubaneswar, Orissa, India

Clathrin-mediated endocytosis (CME) is a crucial mechanism that generates endo-lysosomes.

Yet, how CME is orchestrated in cells remains enigmatic. Spatio-temporal regulation of central
adaptor complex, AP-2 is pivotal for CME. Here, we identify a novel endocytic kinase- BMP-
2 inducible kinase (BMP2K) that phosphorylates AP-2 on plasma membrane for the
progression of CME. We show that BMP2K interacts with AP-2 via intrinsically disordered C-
terminus and that this interaction is important for recruitment and function of kinase at clathrin-
coated pits (CCPs). Accordingly, functionally inactive BMP2K or BMP2K mutants that cannot
localize to CCPs fail to induce AP-2 phosphorylation leading to altered lattice morphology and
endocytosis phenotypes reminiscent of CCP maturation defects. Further, we demonstrate that
BMP2K functions in an allosteric feed-forward axis triggered by AP-2 activating protein
FCHO. Consistent with this model, FCHO knockout cells exhibit AP-2 phosphorylation
defects due to destabilization and degradation of BMP2K. Moreover, activating AP-2 by
reexpressing FCHO restores functional kinase and AP-2 phosphorylation defects in FCHO
knockout cells. We also demonstrate that FCHO-BMP2K axis functions in vivo; gain- and loss-
of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions
during zebrafish embryogenesis. Together, our findings reveal a novel mode of AP-2 regulation
for the in vivo operation of CME in vertebrates.

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POSTER: L27
Analysis of cellular functions of the ATP-dependent membrane fission
catalyst EHD1

Soumya Bhattacharyya, Prasanna Iyer, Thomas J. Pucadyil

Indian Institute of Science Education and Research, Pune

The Eps15-Homology Domain (EHD) containing proteins are an evolutionarily conserved

group of ATPases that belong to the dynamin superfamily of proteins. Mammals have four
paralogs (EHD1-4) that share ~70% amino acid identity and have been ascribed membrane
remodelling functions. EHD1 has been the most studied among them and its functions have
been linked to endocytic recycling of receptors. Recent work from the lab shows that EHD1
can catalyse membrane fission in an ATP-dependent manner. We now address the specific
cellular context wherein such fission activity becomes necessary. Using CRISPR-mediated
EHD1 knockout (KO) cells, we find that the absence of EHD1 results in a dramatic elaboration
of endosomal compartments. This observation is consistent with the premise that EHD1
functions in membrane fission of endosomes. Using complementation assays, where we trigger
the expression of GFP-tagged EHD1 in EHD1 KO cells, we plan on addressing the specific
requirements for fission of endosomes. Together, these constitute the first molecular-level
insights into membrane fission mechanisms that functions in endocytic recycling.

POSTER: L28

Investigating the roles of SYD-2/Liprin-α and LRK-1/LRRK2 as common

genetic regulators of lysosomal and synaptic vesicle proteins

Sravanthi SP Nadiminti, Shirley Dixit, Neena Ratnakaran, Sandhya Koushika

Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha

Road, Navy Nagar, Colaba, Mumbai – 400005

Distinct compartments of the neuron i.e. dendrites, axons, and the synapse have characteristic
protein compositions that allow them to perform different roles in neurotransmission. Polarized
distribution of proteins to axons and dendrites is a result of protein trafficking events that
happen at the neuronal cell body. We show that SYD-2/Liprin-α, a synapse assembly protein,
and LRK-1/LRRK-2, a protein mutated in familial Parkinson’s disease, regulate the polarized
trafficking of lysosomal and synaptic vesicle (SV) proteins. We show that LRK-1, SYD-2 and
the AP3 adaptin complex act to restrict lysosomal proteins to neuronal cell body. Additionally,
these proteins facilitate the inclusion of different SV proteins into one transport carrier, thus
regulating SV membrane composition during SV biogenesis in the cell body. These data
suggest that SV and lysosomal proteins have common genetic regulators for their polarized
distribution and biogenesis. We are testing whether SV and lysosomal proteins undergo similar
trafficking events until they are separately sorted away into distinct membrane compartments.

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POSTER: L29
Regulatory Mechanisms of Clathrin-Mediated Endocytosis: Fishing for
ON-OFF Switches

P.K. Umasankar, Ph.D.

Rajiv Gandhi Centre for Biotechnology (RGCB), Trivandrum, Kerala

Clathrin-mediated endocytosis (CME) is a major vesicular transport pathway pivotal for

lysosome biogenesis and function. Lack of evident GTPases for initiation separates CME from
other membrane trafficking events. This also poses the obvious question of how CME initiates
and progresses in cells. We recently discovered that Fes / CIP4-Homology domain (FCHO)
proteins trigger clathrin-coated pit (CCP) formation by allosterically activating the central
clathrin adaptor protein complex, AP-2 on plasma membrane. We now demonstrate that this
activation promotes AP-2 phosphorylation via recruitment and stabilization of a bona fide AP-
2 kinase- BMP2K, leading to CCP maturation. Accordingly, FCHO knockouts defective in AP-
2 activation or BMP2K mutants that cannot localize to CCPs fail to induce phosphorylation. In
addition, re-expression of FCHO, but not kinase, rescues phosphorylation defects in FCHO
knockout cells implying membrane activation of AP-2 is a prerequisite for kinase function.
Functional inactivation of kinase in the presence of FCHO impairs AP-2 phosphorylation
leading to altered lattice morphology and CME phenotypes reminiscent of CCP maturation
defects. Further, our in vivo studies show that gain- and loss-of function phenotypes of FCHO
and BMP2K are analogous and mirror altered AP-2 functions during zebrafish embryogenesis.
Together, our findings reveal FCHO-AP-2-BMP2K feed-forward axis for operation of CME.

POSTER: L30

Itinerary of Abeta42 whilst under nicotine influence– a lysosome related

story

Viji Vijayan and Sarika Gupta

Molecular Science Lab, National Institute of Immunology, New Delhi

Nicotine, the addictive substance of tobacco is speculated to have a role in the etiology of
Alzheimer’s disease (AD). This alkaloid is fundamentally a hydrophobic weak base that can
cross plasma membrane and organelle membranes. But when such small molecules cross
lysosomal membrane and encounter the acidic lumen of the lysosome, these get protonated
hampering its transport back to the cytoplasm. We hypothesized that this lysomotropic
behavior of nicotine may hamper the Abeta42 degradation machinery of brain cells notably the
astrocytes. Astrocytes are not professionally phagocytic cells, but these do produce several
enzymes that degrade Abeta plaques. In our study we found that nicotine exposure led to higher
uptake of Abeta42 and increased sequestration of Abeta42 in cell without degradation.
Molecular analysis of proteins involved in uptake of Abeta42 demonstrated that the higher
uptake was owing to augmented CD36/TLR2/MyD88 signaling while sequestration occurred
due to defective Rab7 signaling. We found that nicotine augmented CD36/TLR2/MyD88

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signaling facilitated greater interaction of MyD88 (a central player in innate immune signaling)
with mTORC1 (a regulator of the subcellular localization of TFEB) causing cytosolic retention
of TFEB (the master regulator of lysosome biogenesis) and defective lysosomal protein
expression and activity. Absence of MyD88 did not evoke cytosolic sequestration of TFEB
despite nicotine treatment demonstrating that innate immune signaling in astrocytes do play
critical role in lysosome biogenesis. Consequences of such altered lysosome biogenesis
induced by nicotine included increased synthesis and release of pathogenic extracellular
vesicles (EV) harboring Abeta42 and cytotoxic cytokines like TRAIL. These Evs potentially
transferred Abeta42 cargo to healthy neurons and induced apoptosis, not seen in Abeta42 alone
treated cells. To conclude, nicotine mediated cross-talks between innate immune signaling and
mTORC1/TFEB signaling in astrocytes alters amyloid degradation machinery of astrocyte and
creates a pathogenic milieu for development of neurodegenerative diseases.

POSTER: L31
Enzyme aided extraction of fucoidan by AMG augments the functionality
of EPCs through regulation of the AKT/Rheb signaling pathway.

Vinoth Kumar R , Yeon Ju Kim, Woong Bi Jang, Seung Taek Ji, Songhwa Kang, Da
Yeon Kim, Ji Hye Park, Le Thi Hong Van, Ly Thanh Truong Giang, Jong Seong Ha ,
Jisoo Yun , Dong Hyung Lee , Sun-Nyoung Yu, Sul-Gi Park, Soon-Cheol Ahn and Sang-
Mo Kwon*

1Convergence stem cell research center, Pusan National University, Yangsan, Republic of
Korea; 2Laboratory for Vascular Medicine and Stem Cell Biology, Department of Physiology,
School of Medicine,Pusan National University, Yangsan 50612, Republic of Korea

The purpose of the present study is to improve the Endothelial Progenitor cells (EPC)
activation, proliferation, and angiogenesis using enzyme aided extraction of fucoidan by AMG
(EAEF-AMG). Enzyme aided extraction of fucoidan by amyloglucosidase (EAEF-AMG)
significantly increased EPC proliferation by reducing the ROS and decreasing apoptosis.
Notably, EAEF-AMG treated EPCs repressed the colocalization of TSC2/LAMP1 and
promoted perinuclear localization of mTOR/LAMP1 and mTOR/Rheb. Moreover, EAEF-
AMG enhanced EPC functionalities, including tube formation, cell migration, and wound
healing via regulation of AKT/Rheb signaling. Our data provided cell priming protocols to
enhance therapeutic applications of EPCs using bioactive compounds for the treatment of
Cardiovascular Disease.

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 SPEAKERS

Aleem Siddiqui Lipi Thukral

University of California, San Diego Institute of Genomics and Integrative Biology, New Delhi
[email protected] [email protected]

Amit Tuli Manjula Kalia

Institute of Microbial Technology, Chandigarh Regional Centre for Biotechnology,
[email protected] [email protected]

Amitabha Majumdar Nitin Mohan

Unilever Industries Pvt Ltd. Bangalore IIT Kanpur, Kanpur
[email protected] [email protected]

Anu Rangarajan Raghu Padinjat

Indian Institute of Science, Bangalore National Centre for Biological Sciences, Bangalore
[email protected] [email protected]

Ashwani Kumar Ravi Manjithaya

Institute of Microbial Technology, Chandigarh Jawaharlal Nehru Centre for Advanced Scientific Research,
[email protected] Bangalore
[email protected]
Benu Brata Das
Indian Association for the Cultivation of Science, Kolkata Sabuj Bhattacharyya
[email protected] India Alliance, Hyderabad
[email protected]
CV Srikanth
Regional Centre for Biotechnology, Faridabad Sandhya P. Koushika
[email protected] Tata Institute of Fundamental Research, Mumbai
spkoushika @ tifr.res.in
David Rubinsztein
Cambridge Institute for Medical Research, Camridge Sangeetha Nath
[email protected] Manipal Institute of Regenerative Medicine, Bangalore
[email protected]
Deepak Nair
CNS, Indian Institute of Science, Bangalore Santosh Chauhan
[email protected] Institute of Life Sciences, Bhubaneswar
[email protected]
Thomas Wollert
Centre Francois Jacob, France Sascha Martens
[email protected] University of Vienna, Austria
[email protected]
Masaaki Komatsu
Juntendo University Graduate School of Medicine, Japan Shailendra Asthana
[email protected] Translational Health Science and Technology Institute,
Faridabad
Fulvio Reggiori [email protected]
University of Groningen, University Medical Center Groningen,
The Netherlands Sharon Tooze
[email protected] The Francis Crick Institute, UK
[email protected]
Ghulam Hussain Syed
Institute of Life Sciences, Bhubaneswar Sivaram VS M
[email protected] Regional Centre for Biotechnology, Faridabad
[email protected]
Graca Raposo
Institut Curie, France Subba Rao Gangi Setty
[email protected] Indian Institute of Science, Bangalore
[email protected]
Juan S. Bonifacino
National Institutes of Health (NIH), Bethesda MD Sunando Datta
[email protected] Indian Institute of Science Education and Research, Bhopal
[email protected]
Judith Klumperman
University Medical Centre Utrecht, The Netherlands Sunil Laxman
[email protected] Institute for Stem Cell Biology and Regenerative Medicine,
Bangalore
Lois Weisman [email protected]
University of Michigan Medical School, USA
[email protected]

R1
Suresh Subramani Umasankar P.K.
University of California San Diego (UCSD), USA Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram
[email protected] [email protected]

Thomas Pucadyil Varadharajan Sundaramurthy

Indian Institute of Science Education and Research, Pune National Centre for Biological Sciences,
[email protected] [email protected]

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