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David Owald: Early Active Zone Assembly in Drosophila

This study examines the role of DSyd-1, a RhoGAP protein, in the early assembly of active zones (AZs) at the Drosophila neuromuscular junction. The study finds that DSyd-1 physically interacts with and regulates the localization of BRP and DLiprin-α, two key AZ proteins. DSyd-1 and DLiprin-α accumulate early in synapse development preceding the accumulation of postsynaptic glutamate receptors and BRP. DSyd-1 is shown to regulate both presynaptic AZ assembly and composition of postsynaptic glutamate receptors. It also regulates the levels of postsynaptic Neuroligin1, indicating it may regulate the Neurexin-Neu

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56 views1 page

David Owald: Early Active Zone Assembly in Drosophila

This study examines the role of DSyd-1, a RhoGAP protein, in the early assembly of active zones (AZs) at the Drosophila neuromuscular junction. The study finds that DSyd-1 physically interacts with and regulates the localization of BRP and DLiprin-α, two key AZ proteins. DSyd-1 and DLiprin-α accumulate early in synapse development preceding the accumulation of postsynaptic glutamate receptors and BRP. DSyd-1 is shown to regulate both presynaptic AZ assembly and composition of postsynaptic glutamate receptors. It also regulates the levels of postsynaptic Neuroligin1, indicating it may regulate the Neurexin-Neu

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neeraj12121
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© © All Rights Reserved
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David Owald

Early Active Zone Assembly in Drosophila


The dissection of presynaptic assembly processes has proven difficult in the past. Apart from
genetic redundancies, this likely reflects a highly cooperative and regulated nature of synapse
assembly, complicating the straightforward deduction of linear molecular models. This study
genetically dissects defined event hierarchies and assembly intermediates and complements
these with biochemical, electrophysiological, ultrastructural and in vivo protein trafficking data.
Analysis of chemically-induced alleles of the active zone (AZ) protein BRP, positions it as a
direct building block of the electron dense cytomatrix at the AZ (CAZ). An unbiased proteomics
screen reveals that the RhoGAP DSyd-1 physically interacts with BRP and localizes to AZs.
Moreover, DSyd-1 closely co-localizes with a further AZ protein, DLiprin-α.
Using transposon-based genetics two dsyd-1 deficient alleles were synthesized. dsyd-1 mutant
animals elicit impaired locomotive behavior and a reduced life span, while NMJ synapse
numbers and evoked synaptic currents are reduced. Furthermore, AZ morphology appears
abnormal in dsyd-1 deficient animals with the CAZ often appearing misshapen. Floating electron
dense material is, moreover, found at ectopic positions.
In vivo imaging reveals that DSyd-1 and DLiprin-α accumulate early during synapse
development, preceding postsynaptic glutamate receptor accumulation, as well as
presynaptically localized BRP. Analysis of dliprin-α; dsyd-1 double mutants indicates that
overgrown BRP accumulations found in dsyd-1 mutants are dependent on the presence of
dliprin-α. In fact, defining a hierarchy of the two proteins, DLiprin-α localization is largely
impaired in dsyd-1 mutant animals, while DSyd-1 localizes normally in mutants for dliprin-α.
Unlike BRP, DLiprin-α and DSyd-1 clusters appear to be able to decompose again, indicating
that early AZ assembly is still reversible. Thus, AZ assembly can be divided into an early,
reversible step at nascent site and a later, largely irreversible maturation phase. DLiprin-
mobility is largely elevated in dsyd-1 mutants, specifically indicating a clamping function of
DSyd-1, which possibly shapes the transition between nascent and maturing synapses.
Presynaptic DSyd-1 is further shown to regulate postsynaptic receptor field composition,
increasing the amount of slow desensitizing IIA subunit-containing glutamate receptor
complexes. This process is independent of DLiprin-α. Following phenotypic similarities,
Drosophila Neurexin is proposed as a further DSyd-1 substrate. Indeed, postsynaptic
Neuroligin1, a potential DNrx interactor, is identified as localizing towards the edge of
postsynaptic densities here. Mutants in dnlg1 exhibit aberrant NMJ morphology with increased
sizes of postsynaptic densities, impaired neurotransmission and boutons lacking postsynaptic
receptor fields. Such “orphan” boutons are also occasionally found in dsyd-1 mutants. Moreover,
DNlg1 immunoreactivity is drastically reduced in the absence of DSyd-1. It thus appears likely
that presynaptic DSyd-1 regulates the levels of postsynaptic DNlg1, potentially via presynaptic
DNrx.
In a proteomics approach, the GTPase Dynamin is uncovered as a potential interactor of BRP.
This physical interaction is confirmed in vitro. The interaction platform is fine-mapped to an N-
terminal 30 aa of BRP and the GTPase effector domain along with a domain towards the very C-
term of Dynamin. This interaction might link the BRP-based CAZ to the SV exo/endo-cycle.

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