Seminars in Cell & Developmental Biology 45 (2015) 1823

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Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

Cell healing: Calcium, repair and regeneration

Alison M. Moe a , Adriana E. Golding a , William M. Bement a,b,
a
Cell and Molecular Biology Graduate Program, Laboratory of Cell and Molecular Biology, 1525 Linden Drive, Madison, WI, USA
b
Department of Zoology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, USA

a r t i c l e i n f o a b s t r a c t

Article history: Cell repair is attracting increasing attention due to its conservation, its importance to health, and its utility
Received 20 August 2015 as a model for cell signaling and cell polarization. However, some of the most fundamental questions
Accepted 24 September 2015 concerning cell repair have yet to be answered. Here we consider three such questions: (1) How are
Available online 26 October 2015
wound holes stopped? (2) How is cell regeneration achieved after wounding? (3) How is calcium inrush
linked to wound stoppage and cell regeneration?
Keywords:
2015 Elsevier Ltd. All rights reserved.
Calcium
Regeneration
Plasma membrane repair
Rho GTPase
Patching

Contents

1. How are wound holes stopped? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1.1. Contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.2. Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.3. Patching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4. Internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.5. Externalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.6. Plugging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2. How is the cell regenerated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3. What is the cellular and molecular basis of the calcium dependence of cell repair? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Until recently, cell repair was a topic ignored by all but a handful membrane repair and rapid reorganization of the cytoskeleton in
of researchers. However, a series of overlapping ndings has moved a variety of cell types and organisms [511]; cell repair represents
this subject, if not into the mainstream, at least out of the back- a powerful model for studying cell polarization signaling mecha-
waters of scientic research. In particular, several general types of nisms [1216]); failed cell repair is linked to diseasepathologies
discoveries have attracted attention to cell healing: cell damage and ranging from muscular dystrophies to diabetes may result from,
healing is surprisingly commonmany cell types are damaged and result in, or be exacerbated by decits in cell repair [1720]. These
heal as a simple consequence of existence [13], this apparently ndings have drawn to cell repair researchers from otherwise dis-
includes metastatic cells, which can survive locomotion-induced parate elds, and as a consequence, the list of potential molecular
damage via upregulation of proteins involved in cell repair [4]; participants in cell repair is growing rapidly.
cell repair mechanisms are conserveddamage consistently elicits However, in spite of the recent progress concerning the details
of cell repair, answers to several big picture questions remain
elusive. Here we consider three of these questions: (1) How are
Corresponding author at: Laboratory of Cell and Molecular Biology, 1525 Linden wound holes stopped? (2) How is cell regeneration achieved after
Drive, Madison, WI, USA. wounding? (3) How is calcium inrush linked to wound stoppage
E-mail address: [email protected] (W.M. Bement). and cell regeneration? We do not pretend to have answers to these

http://dx.doi.org/10.1016/j.semcdb.2015.09.026
1084-9521/ 2015 Elsevier Ltd. All rights reserved.
 A.M. Moe et al. / Seminars in Cell & Developmental Biology 45 (2015) 1823 19

questions, rather, it is our hope to clarify what can be a confusing [8,9,37] and Drosophila embryos [11] and may occur in mam-
literature and to prompt new ways of considering cell repair. malian muscle based on the reported recruitment of myosin-2
[22] and the observation that F-actin disruption suppresses lat-
1. How are wound holes stopped? eral recruitment of dysferlin to wounds [23]. Similarly, EM studies
have localized F-actin and/or myosin-2 to sites of cell damage
It is generally assumed that, unless repaired, plasma membrane in amoeba and plant cells [5,24]. Further, transected invertebrate
breaches will result in rapid cell death as a consequence of inrush axons pinch inward near the cut site [25,26], a behavior consistent
of calcium. That is, the calcium concentration outside the cell is with contraction. Moreover, one of the essential signaling events in
several orders of magnitude higher than it is in the cytoplasm and the wound-induced contraction pathwayRho activationoccurs
prolonged elevation of intracellular free calcium triggers cell death. in frog oocytes and embryos [12], Drosophila syncytial embryos
Independent of calcium, loss of cytoplasm to the extracellular space [16] and in budding yeast [15], arguing that it is a fundamen-
would eventually kill the cell. Thus, stopping holes in the plasma tal feature of wound repair. The problem with the contraction
membrane is widely viewed as being the most imperative event in model is that it seems far too slow to close the hole within the
the cell repair process. few seconds generally thought to be required for healing [6] or
to prevent cell death via calcium inrush, with actin and myosin
1.1. Contraction accumulation beginning within 30 s or more and closure taking
three or more minutes, depending on the wound size and model
One of the rst explicit mechanisms proposed for hole stop- system. Further, in some studies, disruption of F-actin has been
ping was actomyosin-based contraction [9,21]. Here, F-actin and reported to facilitate, rather than inhibit wound resealing (e.g.
myosin-2 accumulate in the cortex around the wound perime- [27]).
ter, forming a continuous network that closes inward, bringing
undamaged plasma membrane and underlying cortical cytoskele- 1.2. Exocytosis
ton with it (Fig. 1). This response occurs in frog oocytes and embryos
Because healing requires a high concentration of extracellular
calcium (see below) and because it is inhibited by toxins that impair
the function of SNARES, proteins essential for membrane fusion,
exocytosis has also been proposed to heal cell wounds [6]. Consis-
tent with this mechanism, exocytosis has been detected in response
to wounding [7,28]. However, by denition exocytosisfusion of an
internal compartment with the plasma membranecannot occur
in a region that, like a hole, lacks plasma membrane. Thus, exocy-
tosis would likely be effective only for healing very small wounds,
and that assumes that membrane inserted into the regions anking
the wound can ow inward (Fig. 1). Since cells can heal wounds that
are tens or even hundreds of micrometers in diameter (e.g. [21,29]),
it follows that exocytosis cannot be the only mechanism involved
in cell repair. Additionally, wounding is thought to be associated
with endocytosis and removal of local membrane protrusions by
scission (see below) which seems odd if the primary hole stopping
mechanism works by inserting new membrane into the plasma
membrane.

1.3. Patching

A third mechanism proposed for stopping holes is membrane

patching [29,30]. Here the idea is that internal compartments fuse
with each other at the site of the wound, creating a patch where the
plasma membrane is missing (Fig. 1). This fusion is accompanied by
fusion of the forming patch with the plasma membrane at the edges
of the wound, thereby creating a continuous membrane over the
wound site. The patching model can explain hole stoppage in both
small and large wounds, and is consistent with several experimen-
tal ndings, most compellingly the demonstration that sea water
microinjected into sea urchin eggs is rapidly walled off from the
cytoplasm [29]. However, patching has yet to be directly observed
in any system and to date there is no consensus on which intracellu-
lar compartment or compartments participate in patching. Further,
it is not clear how the double-membraned structure predicted by
patching would resolve into a single membrane.
Fig. 1. Schematic diagram depicting the various mechanisms proposed to stop holes
made in cells. In each case, the black line with gap represents the plasma membrane
with a wound-induced hole, black circles represent membranous compartments in 1.4. Internalization
the cell and healing progresses from top to bottom. Green lines in Contraction
represent cortical cytoskeleton; yellow dots in Internalization represent machin- A fourth mechanism associated with wound repair and pro-
ery powering endocytotic invagination and pinching; blue dots in Externalization
posed for stopping holes is internalization via endocytosis [31].
represent ESCRT machinery powering scission; red dots in Plugging represent
proteins crosslinking membranous compartments. See text for description of mech-
Here, plasma membrane-containing holes are simply removed
anisms. from the cell surface via invagination of the region containing
20 A.M. Moe et al. / Seminars in Cell & Developmental Biology 45 (2015) 1823

the hole; the invagination would then be pinched off the plasma within a few seconds or less. This attitude is based on the anal-
membrane, similar to the processes of receptor mediated endocy- ogy to regulated exocytosis, on the feeling that calcium inrush
tosis or caveolae internalization (Fig. 1). This model is consistent must be immediately and completely shut off or the cell will die,
with the observation that mechanical wounding or exposure to and the observation that wound-induced increases in intracellular
plasma membrane pore forming agents triggers endocytosis and free calcium cease within 510 s in wounded sea urchin eggs even
that manipulations which inhibit endocytosis inhibit repair [31,74]. when the wounds are extremely large [6,29]. However, more recent
However, as with exocytosis, such a mechanism would be limited studies suggest resealing may often be much slower. For exam-
to small wounds as the hole would have to be accommodated by ple, broblasts require 1590 s to reseal depending on the study
the nascent endocytic compartment. (e.g. [35]). Other cell types require longer: 100300 s for syncytial
Drosophila embryos [11], 60 to 120 s for myoblasts [36], 60300 s
1.5. Externalization for muscle bers [4,17], 90 s for cancer cells [4], 120240 s for HeLa
cells [75], 20120 s for Xenopus blastomeres [37] and 60 s15 m for
A fth mechanism proposed for stopping holes is topologi- frog oocytes ([38]; our unpublished results).
cally the opposite of internalization, namely, externalization via Clearly, not cells can tolerate a hole in the plasma membrane
ESCRT-mediated budding and pinching off of plasma membrane- for longer than a few seconds. Indeed, the results from transected
containing holes ([75]; Fig. 1). This model is consistent with axons suggest that complete hole stopping may take hours (e.g.
localization of ESCRT proteins to wound sites and inhibition of [34]). What explains the differences in hole stopping speeds and
healing following ESCRT inhibition [4,75]. Further, the involvement why do cells not die if the holes remain open for minutes or more?
of ESCRTs could explain the results of older EM studies revealing One simple explanation for the differences between resealing rates
wound-associated compartments comprising vesicles enclosed by in sea urchin eggs and other cells is that the former are normally
other vesicles (e.g. Fig. 1 in Ref. [7]), since ESCRTs are well known wounded in media containing 10 mM CaCl2 (which corresponds to
to promote inward budding of vesicles into endosomes. However, the normal concentration of calcium in sea water) but media for
like exocytosis and internalization, ESCRT-mediated budding and other cells typically have 12 mM calcium. Since healing is calcium
scission is also likely to have an upward limit of utility. Indeed, it dependent (see also below) higher external calcium concentration
was reported that wounds above 100 nm do not require ESCRTs may promote more rapid healing. Consistent with this possibility,
for hole stoppage [75] although this point was challenged [4]. sea urchin eggs wounded in 2 mM external calcium were observed
to take much longer to reseal90120 s [6]. With respect to cells
1.6. Plugging surviving relatively lengthy times to hole stoppage, it may be that
calcium simply does not diffuse very quickly from the site of dam-
All of the above mechanisms are assumed to stop holes by age to the rest of the cell, a possibility suggested by assessment of
restoring membrane continuity at the wound site. However, there calcium mobility in cytoplasm [39]. Testing this notion will require
is a sixth mechanism with the potential to stop holes without estab- that studies on healing in different cell types with longer hole stop-
lishment of a continuous membrane: plugging [32]. In this model, ping kinetics include direct assessment of intracellular free calcium
membranous compartments in the cytoplasm become tightly cross concentration.
linked to each other and the plasma membrane (possibly by pro- Another vexing problem is the identity of the membranous
teins recruited to the wound site), forming a plug that stops or compartments involved in the healing process in general and hole
limits movement of ions or other materials into or out of the cell. stoppage in particular [40]. A variety of candidates have been pro-
Plugging has been visualized in transected axons where a variety posed including lysosomes [41], enlargosomes [42], and reserve
of membranous compartments accumulate at the cut site over the granules [30], but consensus has remained out of reach. And, with-
course of many minutes [32,33] and membranous compartments out knowing the identity of the compartment(s), development of
have been found to form a plug in Drosophila syncytial embryo the proper probes for visualization of healing becomes quite dif-
wounds [11]. Plugging would not have an intrinsic size limit but is cult. Our suspicion is that we are missing something, perhaps an
necessarily a temporary solution. It is also not clear how efciently as-yet unidentied compartment that has evolved specically for
ion inrush could be limited by plugging, although measurements membrane repair. We base this suggestion on the fact that high
in transected axons indicate that calcium inrush is progressively resolution studies of membranes at wound sites typically reveal a
reduced as plugging occurs [34]. And, as discussed below, it riot of external folds, blebs, and tubes extending into the extracel-
may be that cell holes can remain open longer than is generally lular space around the wound (e.g. [7,43,75]; Fig. 2). Further, such
thought. elaborations are apparent even when the wound is comparatively
Why has it been so difcult to determine which of these mech- small. For example, SLO toxin, which produces a pore of 30 nm
anisms are responsible for stopping the hole? It is likely that diameter [44], results in protrusions that may extend six or more
both experimental and conceptual difculties are to blame. From microns into the extracellular space [45].
the experimental standpoint, the healing response is rapid and It is also the case that we naturally tend to seek single, simple
complex, making it difcult to draw rm conclusions from xed explanations where possible. But perhaps this is a mistake. As a
samples. Further, it is currently impossible to directly determine primordial cell polarization event, the mechanistic similarity of cell
wound size in living cells unless the wounds are larger than the repair to cytokinesis has been pointed out previously [8,21,46] and
limit of resolution of the light microscope. A potential exception is the comparison appears increasingly apt as more is learned about
wounds induced by pore-forming toxins, which (presumably) have each process.
a minimum diameter of the pore size. Animal cell cytokinesis results from several complementary
Our assays for hole stopping are also inherently limited. Hole processes: actomyosin-powered ingression of the plasma mem-
stopping is usually assessed by cessation of dye uptake. Dye uptake- brane brings plasma membrane and cortex together across a large
based assays work well for showing differences in rates of hole distance, thereby generating a narrow tube of cytoplasm sur-
stoppage, but they are not so good at providing a measure of hole rounded by plasma membrane (the midbody) that separates
stoppage, as plots typically show a slowing, rather than a cessation the nascent daughter cells. After the midbody has formed, it is
of dye incorporation over time. associated with elaborate membrane extensions (e.g. [47]), and it
From the conceptual standpoint, we have been biased by the undergoes scission in a manner that is thought to involve exo-
notion that hole stopping is or must be extremely fast, occurring cytosis, endocytosis, fusion of the membraneous compartments
 A.M. Moe et al. / Seminars in Cell & Developmental Biology 45 (2015) 1823 21

minutes, given that they may take hours to days to develop in

differentiating tissue.
With the exception of patching and plugging, all of the mech-
anisms described for stopping the hole also have the potential
to aid regeneration: exocytosis of vesicles containing appropriate
plasma membrane proteins and lipids could replace those lost dur-
ing wounding and hole stopping. Conversely, endocytosis could
remove inappropriate proteins and lipids inserted into the plasma
membrane during hole stopping. Scission during externalization
could excise protrusions or plasma membrane containing high con-
centrations of inappropriate proteins and lipids. And contraction
could restore the cell to the undamaged state simply by pulling
unperturbed plasma membrane and cortical cytoskeleton over the
original wound site.
An additional potential contributor to regeneration is wound-
induced microtubule assembly and reorganization. Microtubules
rst disappear from and then accumulate around wounds within
1 to 3 min as a consequence of both de novo assembly and
actomyosin-powered transport [55,56]. The end result is a radial
array of microtubules around the wound that has the potential to
Fig. 2. Confocal micrograph showing membrane extensions extending into the transport material to and from the wound site [55]. Consistent with
extracellular space around a wound made in a Xenopus egg. The wound (which microtubules playing a role in regeneration, their loss does not pre-
is obscured by the extensions) is 5 m in diameter; the region occupied by the
vent healing of an initial wound, but does slow repair of subsequent
extensions is 60 m in diameter.
wounds [56].
Why should we worry about regeneration of damaged cells?
First, failure to recognize regeneration as a part of the cell repair
with each other and the plasma membrane, and ESCRT-mediated
process may lead to the belief that a cell is completely healed when
pinching [4850]. By analogy, one might imagine that wounds
in fact complete healing is hours or even days away, particularly
are likewise stopped via collaboration of many processes, with
if the cell type in question is highly differentiated. Second, distin-
plugging and/or patching limiting calcium inrush, contraction
guishing between regeneration and hole stopping may help clarify
bringing edges of wounds close to each other and, once the hole is
variations in the repair process in wounds of different sizes and in
small enough, ESCRTs, endocytosis and exocytosis nishing things
wounds occurring in different cell types. For example, while ESCRTs
off.
may not be required for healing of larger wounds [75], they could
nonetheless make important contributions to the regeneration pro-
2. How is the cell regenerated? cess by excising membrane protrusions. Third, it seems probable
that pathologies resulting from compromised regeneration rather
Just as tissue healing is not nished when a clot forms, it is than hole stopping exist, but have not been discovered because no
likely that cell repair involves far more than stopping the hole one is looking for them.
[46]. We use the term cell regeneration to refer to the col-
lection of processes hypothesized to return the wound site to
its original state, replete with the normal repertoire of plasma 3. What is the cellular and molecular basis of the calcium
membrane lipids and proteins, as well as a fully restored cor- dependence of cell repair?
tical cytoskeleton. Why posit that the repair process continues
after the hole is stopped, and perhaps even long after the hole is If the process of cell repair seems complicated by the potential
stopped? First, cell damage triggers exocytosis of lysosomes [41]. for multiple hole stopping and regeneration mechanisms, and con-
While the role of this event remains unclear, one consequence is fusion surrounding the identity of potential repair compartments,
undeniable: fusion of the lysosome with the plasma membrane at least one feature of cell repair is not in questionthe requirement
necessarily changes the latter, as lysosome membranes differ sig- for extracellular calcium. In the absence of extracellular calcium,
nicantly from the plasma membrane in terms of lipid and protein neither contraction [8,21], exocytosis [6,7], patching [29], internal-
composition (e.g. [51]). Likewise, patching would be expected to ization [31,74], externalization [4,75], nor plugging [32,33] occur
signicantly perturb plasma membrane composition as a conse- after wounding. Indeed, so important is calcium inrush for repair,
quence of fusion with internal compartment(s). Second, wounding wounding in the absence of external calcium is often used as a
results in loss of the cortical cytoskeleton immediately beneath the negative control for the healing process. But the general accep-
wound, which presumably facilitates membrane fusion [52]. The tance of calcium as a key upstream signal for cell repair masks a
cortical cytoskeleton thus has to be repaired. Third, like the clot, major mystery: Why does the external calcium concentration have
the extravagant membrane protrusions and other material that to be so high? For example, repair of sea urchin eggs requires a
accumulate at wounds likely do not represent functional plasma minimum of 1.2 mM and broblast repair a minimum of 0.8 mM
membrane and these can linger long for 20 min or more after external calcium in the presence of physiological concentrations
wounding (e.g. [32,43,53]). Fourth, proteins recruited to wounds of magnesium [6] and similar results have been obtained in other
can remain there for many minutes to even hours after wound- systems (e.g. [57]; our unpublished results). Neither the cellu-
ing (e.g. [53,54,75]; our unpublished results). Finally, many of the lar process nor the protein targets of the high calcium are clear.
cells routinely subject to wounds (e.g. muscle, enterocytes, skin; see For example, the calcium requirement was originally suggested
[13]) are highly differentiated and contain elaborate cortical spe- to support the idea that plasma membrane resealing resulted
cializations such as microvilli, cellcell junctions, and costameres. from exocytosis, based on the well known calcium dependence
Such structures, if destroyed during wounding, have to be reassem- of regulated exocytosis [6]. However, uncaging studies show that
bled, a process that is unlikely to be nished within just a few exocytosis of synaptic vesicles is efciently triggered at 310 M
22 A.M. Moe et al. / Seminars in Cell & Developmental Biology 45 (2015) 1823

intracellular free calcium [58], more than an order of magnitude [9] Mandato CA, Bement WM. Contraction and polymerization cooperate to
less than the requirement for repair. Similarly, the calcium afnities assemble and close actomyosin rings around Xenopus oocyte wounds. J. Cell
Biol 2001;154(August (4)):78597.
for the synaptotagmins (syts), which confer calcium sensitivity on [10] Schapire AL, Voigt B, Jasik J, Rosado A, Lopez-Cobollo R, Menzel D, Salinas J,
SNARE-dependent membrane fusion events [76], are in the range Mancuso S, Valpuesta V, Baluska F, Botella MA. Arabidopsis synaptotagmin 1
of 120 M depending on the syt and the presence of lipid binding is required for the maintenance of plasma membrane integrity and cell
viability. Plant Cell 2008;20(December (12)):337488.
partners (e.g. [5961]). While it could be argued that exocytosis [11] Abreu-Blanco MT, Verboon JM, Parkhurst SM. Cell wound repair in Drosophila
of other compartments requires far more calcium, exocytosis of occurs through three distinct phases of membrane and cytoskeletal
lysosomes occurs at 1 M intracellular free calcium [62]. Nor does remodeling. J. Cell Biol 2011;193(May (3)):45564.
[12] Benink HA, Bement WM. Concentric zones of active RhoA and Cdc42 around
internalization seem to require high calcium in that it is thought to
single cell wounds. J. Cell Biol 2005;168(January (3)):42939.
be triggered by lysosome exocytosis [74]. Similarly, during exter- [13] Vaughan EM, Miller AL, Yu HY, Bement WM. Control of local Rho GTPase
nalization, calcium sensitivity is thought be conferred by the EF crosstalk by Abr. Curr. Biol 2011;21(February (4)):2707.
[14] Burkel BM, Benink HA, Vaughan EM, von Dassow G, Bement WM. A Rho
hand-containing Alg-2 [4], but its afnity for calcium is in the
GTPase signal treadmill backs a contractile array. Dev. Cell 2012;23(August
13 M range [63]. With respect to contraction, the only known (2)):38496.
calcium-dependent link to Rho GTPase activation is protein kinase [15] Kono K, Saeki Y, Yoshida S, Tanaka K, Pellman D. Proteasomal degradation
C beta [77], but its afnity for calcium is 5 M [64]. resolves competition between cell polarization and cellular wound healing.
Cell 2012;150(July (1)):15164.
Thus, what cellular or molecular targets during cell repair [16] Abreu-Blanco MT, Verboon JM, Parkhurst SM. Coordination of Rho family
account for its extremely high calcium requirement are unknown GTPase activities to orchestrate cytoskeleton responses during cell wound
and it could be argued that until they are identied, issues concern- repair. Curr. Biol 2014;24(January (2)):14455.
[17] Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL,
ing the relative importance of different hole stopping mechanisms Campbell KP. Defective membrane repair in dysferlin-decient muscular
cannot be settled. One general possibility worth considering is that dystrophy. Nature 2003;423(May (6936)):16872.
potential targets with more than one calcium binding site may [18] Han R, Bansal D, Miyake K, Muniz VP, Weiss RM, McNeil PL, Campbell KP.
Dysferlin-mediated membrane repair protects the heart from stress-induced
acquire different functions at extremely high calcium levels (e.g. left ventricular injury. J. Clin. Invest 2007;117(July (7)):180513.
[65,66]). Alternatively, focussing on proteins that have been impli- [19] Mller MW, McNeil PL, Bchler P, Ceyhan GO, Wolf-Hieber E, Adler G, Beger
cated in wound repair and which are known to bind calcium with HG, Bchler MW, Friess H. Acinar cell membrane disruption is an early event
in experimental acute pancreatitis in rats. Pancreas 2007;35(November
relatively low afnity may be a promising approach. For exam-
(4)):e3040.
ple, S100A11, an annexin binding protein, has a calcium afnity [20] Howard AC, McNeil AK, Xiong F, Xiong WC, McNeil PL. A novel cellular defect
of 500 M [67]. Since both S100 proteins [4,68] and annexins in diabetes: membrane repair failure. Diabetes 2011;60(November
(11)):303443, http://dx.doi.org/10.2337/db11-0851 (Epub 2011 Sep 22).
[6971] have been implicated in cell repair, they are attractive
[21] Bement WM, Capco DG. Analysis of inducible contractile rings suggests a role
candidates as targets. for protein kinase C in embryonic cytokinesis and wound healing. Cell Motil.
In summary, while the number of molecular participants impli- Cytoskeleton 1991;20(2):14557.
cated in cell repair grows at an increasing pace [72,73], several [22] Lin P, Zhu H, Cai C, Wang X, Cao C, Xiao R, Pan Z, Weisleder N, Takeshima H,
Ma J. Nonmuscle myosin IIA facilitates vesicle trafcking for MG53-mediated
of the fundamental questions surrounding this important process cell membrane repair. FASEB J 2012;26(May (5)):187583.
remain unresolved. We suspect that the answers will require a [23] McDade JR, Archambeau A, Michele DE. Rapid actin-cytoskeleton-dependent
broadening of outlook, rather than a focus on a particular molecule recruitment of plasma membrane-derived dysferlin at wounds is critical for
muscle membrane repair. FASEB J 2014;28(August (8)):366070.
or process. Regardless, we look forward with excitement to the
[24] Jeon KW, Jeon MS. Cytoplasmic laments and cellular wound healing in
answers which, one assumes, will be forthcoming as more attention Amoeba proteus. J. Cell Biol 1975;67(October (1)):2439.
is directed toward them. [25] Gallant PE. Effects of the external ions and metabolic poisoning on the
constriction of the squid giant axon after axotomy. J. Neurosci 1988;8(May
(5)):147984.
Acknowledgements [26] Krause TL, Fishman HM, Ballinger ML, Bittner GD. Extent and mechanism of
sealing in transected giant axons of squid and earthworms. J. Neurosci
1994;14(November (11 Pt 1)):663851.
We are grateful to Nick Davenport and Kevin Sonnemann (Uni- [27] Togo T, Krasieva TB, Steinhardt RA. A decrease in membrane tension precedes
versity of Wisconsin-Madison) to their ideas about cell repair, and successful cell-membrane repair. Mol. Biol. Cell 2000;11(December
(12)):433946.
to George Bittner (University of Texas-Austin) for discussing ideas [28] Bi GQ, Alderton JM, Steinhardt RA. Calcium-regulated exocytosis is required
and publications concerning healing of transected axons. The work for cell membrane resealing. J. Cell Biol 1995;131(December (6 Pt
in our lab is supported by a grant from the National Institutes of 2)):174758.
[29] Terasaki M, Miyake K, McNeil PL. Large plasma membrane disruptions are
Health (GM052932).
rapidly resealed by Ca2+ -dependent vesiclevesicle fusion events. J. Cell Biol
1997;139(October (1)):6374.
[30] McNeil PL, Vogel SS, Miyake K, Terasaki M. Patching plasma membrane
References disruptions with cytoplasmic membrane. J. Cell Sci 2000;113(June (Pt
11)):1891902.
[1] McNeil PL, Ito S. Gastrointestinal cell plasma membrane wounding and [31] Idone V, Tam C, Goss JW, Toomre D, Pypaert M, Andrews NW. Repair of
resealing in vivo. Gastroenterology 1989;96(May (5 Pt 1)):123848. injured plasma membrane by rapid Ca2+ -dependent endocytosis. J. Cell Biol
[2] McNeil PL, Ito S. Molecular trafc through plasma membrane disruptions of 2008;180(March (5)):90514.
cells in vivo. J. Cell Sci 1990;96(July (Pt 3)):54956. [32] Eddleman CS, Ballinger ML, Smyers ME, Godell CM, Fishman HM, Bittner GD.
[3] McNeil PL, Khakee R. Disruptions of muscle ber plasma membranes. Role in Repair of plasmalemmal lesions by vesicles. Proc. Natl. Acad. Sci. U. S. A
exercise-induced damage. Am. J. Pathol 1992;140(May (5)):1097109. 1997;94(April (9)):474550.
[4] Jaiswal JK, Lauritzen SP, Scheffer L, Sakaguchi M, Bunkenborg J, Simon SM, [33] Eddleman CS, Ballinger ML, Smyers ME, Fishman HM, Bittner GD. Endocytotic
Kallunki T, Jttel M, Nylandsted J. S100A11 is required for efcient plasma formation of vesicles and other membranous structures induced by Ca2+ and
membrane repair and survival of invasive cancer cells. Nat. Commun axolemmal injury. J. Neurosci 1998;18(June (11)):402941.
2014;5(May):3795. [34] Eddleman CS, Bittner GD, Fishman HM. Barrier permeability at cut axonal
[5] La Claire 2nd JW. Actin cytoskeleton in intact and wounded coenocytic green ends progressively decreases until an ionic seal is formed. Biophys. J
algae. Planta 1989;177(January (1)):4757. 2000;79(October (4)):188390.
[6] Steinhardt RA, Bi G, Alderton JM. Cell membrane resealing by a vesicular [35] Togo T, Alderton JM, Bi GQ, Steinhardt RA. The mechanism of facilitated cell
mechanism similar to neurotransmitter release. Science 1994;263(January membrane resealing. J. Cell Sci 1999;112(March (Pt 5)):71931.
(5145)):3903. [36] Azakir BA, Di Fulvio S, Kinter J, Sinnreich M. Proteasomal inhibition restores
[7] Miyake K, McNeil PL. Vesicle accumulation and exocytosis at sites of plasma biological function of mis-sense mutated dysferlin in patient-derived muscle
membrane disruption. J. Cell Biol 1995;131(December (6 Pt 2)):173745. cells. J. Biol. Chem 2012;287(March (13)):1034454.
[8] Bement WM, Mandato CA, Kirsch MN. Wound-induced assembly and closure [37] Clark AG, Miller AL, Vaughan E, Yu HY, Penkert R, Bement WM. Integration of
of an actomyosin purse string in Xenopus oocytes. Curr. Biol 1999;9(June single and multicellular wound responses. Curr. Biol 2009;19(August
(11)):57987. (16)):138995.
 A.M. Moe et al. / Seminars in Cell & Developmental Biology 45 (2015) 1823 23

[38] Luxardi G, Reid B, Maillard P, Zhao M. Single cell wound generates electric [60] Radhakrishnan A, Stein A, Jahn R, Fasshauer D. The Ca2+ afnity of
current circuit and cell membrane potential variations that requires calcium synaptotagmin 1 is markedly increased by a specic interaction of its C2B
inux. Integr. Biol. (Camb.) 2014;6(July (7)):66272. domain with phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem
[39] Allbritton NL, Meyer T, Stryer L. Range of messenger action of calcium ion and 2009;284(September (38)):2574960.
inositol 1,4,5-trisphosphate. Science 1992;258(December (5089)):18125. [61] van den Bogaart G, Meyenberg K, Diederichsen U, Jahn R. Phosphatidylinositol
[40] Bement WM, Yu HY, Burkel BM, Vaughan EM, Clark AG. Rehabilitation and the 4,5-bisphosphate increases Ca2+ afnity of synaptotagmin-1 by 40-fold. J.
single cell. Curr. Opin. Cell Biol 2007;19(February (1)):95100. Biol. Chem 2012;287(May (20)):1644753.
[41] Reddy A, Caler EV, Andrews NW. Plasma membrane repair is mediated [62] Andrews NW. Regulated secretion of conventional lysosomes. Trends Cell Biol
by Ca(2+ )-regulated exocytosis of lysosomes. Cell 2001;106(July (2)): 2000;(August (8)):31621.
15769. [63] Tarabykina S, Mller AL, Durussel I, Cox J, Berchtold MW. Two forms of the
[42] Borgonovo B, Cocucci E, Racchetti G, Podini P, Bachi A, Meldolesi J. Regulated apoptosis-linked protein Alg-2 with different Ca(2+ ) afnities and target
exocytosis: a novel, widely expressed system. Nat. Cell Biol 2002;4(December recognition. J. Biol. Chem 2000;275(April (14)):105148.
(12)):95562. [64] Nalefski EA, Wisner MA, Chen JZ, Sprang SR, Fukuda M, Mikoshiba K, Falke JJ.
[43] Gingell D. Contractile responses at the surface of an amphibian egg. J. C2 domains from different Ca2+ signaling pathways display functional and
Embryol. Exp. Morphol 1970;23(June (3)):583609. mechanistic diversity. Biochemistry 2001;40(March (10)):3089100.
[44] Bhakdi S, Tranum-Jensen J, Sziegoleit A. Mechanism of membrane damage by [65] Lek A, Evesson FJ, Lemckert FA, Redpath GM, Lueders AK, Turnbull L,
streptolysin-O. Infect. Immun 1985;47(January (1)):5260. Whitchurch CB, North KN, Cooper ST. Calpains, cleaved mini-dysferlinC72,
[45] Atanassoff AP, Wolfmeier H, Schoenauer R, Hostettler A, Ring A, Draeger A, and L-type channels underpin calcium-dependent muscle membrane repair. J.
Babiychuk EB. Microvesicle shedding and lysosomal repair fulll divergent Neurosci 2013;33(March (12)):508594.
cellular needs during the repair of streptolysin O-induced plasmalemmal [66] Redpath GM, Woolger N, Piper AK, Lemckert FA, Lek A, Greer PA, North KN,
damage. PLOS ONE 2014;9(February (2)):e89743. Cooper ST. Calpain cleavage within dysferlin exon 40a releases a
[46] Sonnemann KJ, Bement WM. Wound repair: toward understanding and synaptotagmin-like module for membrane repair. Mol. Biol. Cell
integration of single-cell and multicellular wound responses. Annu. Rev. Cell 2014;25(October (19)):303748.
Dev. Biol 2011;27:23763. [67] Zimmer DB, Weber DJ. The calcium-dependent interaction of S100B with its
[47] Crowell EF, Gaffuri AL, Gayraud-Morel B, Tajbakhsh S, Echard A. Engulfment protein targets. Cardiovasc. Psychiatry Neurol 2010;2010, pii: 728052.
of the midbody remnant after cytokinesis in mammalian cells. J. Cell Sci [68] Rezvanpour A, Santamaria-Kisiel L, Shaw GS. The S100A10-annexin A2
2014;127(September (Pt 17)):384051. complex provides a novel asymmetric platform for membrane repair. J. Biol.
[48] Neto H, Collins LL, Gould GW. Vesicle trafcking and membrane remodelling Chem 2011;286(November (46)):4017483.
in cytokinesis. Biochem. J 2011;437(July (1)):1324. [69] McNeil AK, Rescher U, Gerke V, McNeil PL. Requirement for annexin A1 in
[49] Schiel JA, Childs C, Prekeris R. Endocytic transport and cytokinesis: from plasma membrane repair. J. Biol. Chem 2006;281(November (46)):352027
regulation of the cytoskeleton to midbody inheritance. Trends Cell Biol (Epub 2006 Sep 19). PMID: 16984915.
2013;23(July (7)):31927. [70] Potez S, Luginbhl M, Monastyrskaya K, Hostettler A, Draeger A, Babiychuk
[50] Mierzwa B, Gerlich DW. Cytokinetic abscission: molecular mechanisms and EB. Tailored protection against plasmalemmal injury by annexins with
temporal control. Dev. Cell 2014;31(December (5)):52538. different Ca2+ sensitivities. J. Biol. Chem 2011;286(May (20)):1798291.
[51] Schroder BA, Wrocklage C, Hasilik A, Saftig P. The proteome of lysosomes. [71] Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP,
Proteomics 2010;10:405360. Holley-Cuthrell J, Eskin A, Chen Z, Squire K, Heydemann A, Palmer AA, Nelson
[52] Miyake K, McNeil PL, Suzuki K, Tsunoda R, Sugai N. An actin barrier to SF, McNally EM. Annexin A6 modies muscular dystrophy by mediating
resealing. J. Cell Sci 2001;114(October (Pt 19)):348794. sarcolemmal repair. Proc. Natl. Acad. Sci. U. S. A 2014;111(April (16)):60049.
[53] Marg A, Schoewel V, Timmel T, Schulze A, Shah C, Daumke O, Spuler S. [72] Jaiswal JK, Nylandsted J. S100 and annexin proteins identify cell membrane
Sarcolemmal repair is a slow process and includes EHD2. Trafc damage as the Achilles heel of metastatic cancer cells. Cell Cycle
2012;13(September (9)):128694. 2015;14(4):5029.
[54] Mellgren RL. A plasma membrane wound proteome: reversible [73] Boucher E, Mandato CA. Plasma membrane and cytoskeleton dynamics during
externalization of intracellular proteins following reparable mechanical single-cell wound healing. Biochim. Biophys. Acta 2015;1853(July (10 Pt
damage. J. Biol. Chem 2010;285(November (47)):36597607. A)):264961, http://dx.doi.org/10.1016/j.bbamcr.2015.07.012 (Epub ahead of
[55] Mandato CA, Bement WM. Actomyosin transports microtubules and print). Review.
microtubules control actomyosin recruitment during Xenopus oocyte wound [74] Corrotte M, Almeida PE, Tam C, Castro-Gomes T, Fernandes MC, Millis BA,
healing. Curr. Biol 2003;13(July (13)):1096105. Cortez M, Miller H, Song W, Maugel TK, Andrews NW. Caveolae
[56] Togo T. Disruption of the plasma membrane stimulates rearrangement of internalization repairs wounded cells and muscle bers. eLife 2013;2:e00926.
microtubules and lipid trafc toward the wound site. J. Cell Sci 2006;119(July [75] Jimenez AJ, Maiuri P, Lafaurie-Janvore J, Divoux S, Piel M, Perez F. ESCRT
(Pt 13)):27806. machinery is required for plasma membrane repair. Science
[57] Yumura S, Hashima S, Muranaka S. Myosin II does not contribute to wound 2014;343(February (6174)):986, http://dx.doi.org/10.1126/science.1247136
repair in Dictyostelium cells. Biol. Open 2014;3(September (10)):96673. (Epub 2014 Jan 30).
[58] Schneggenburger R1, Neher E. Intracellular calcium dependence of [76] Chapman ER. How does synaptotagmin trigger neurotransmitter release?
transmitter release rates at a fast central synapse. Nature 2000;406(August Annu. Rev. Biochem 2008;77:61541 (review).
(6798)):88993. [77] Vaughan EM, You JS, Elsie Yu HY, Lasek A, Vitale N, Hornberger TA, Bement
[59] Sugita S, Shin OH, Han W, Lao Y, Sdhof TC. Synaptotagmins form a hierarchy WM. Lipid domain-dependent regulation of single-cell wound repair. Mol.
of exocytotic Ca(2+ ) sensors with distinct Ca(2+ ) afnities. EMBO J Biol. Cell 2014;25(June (15)):186776.
2002;21(February (3)):27080.