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Primer plasma membrane repair. This was that upon Ca2+ influx through the
an important conceptual advance, as wound, pre-existing intracellular vesicles
Plasma membrane well as the first step in overturning the
commonly held perception that cells
would fuse with each other to form a
membrane ‘patch’, which would then
repair can spontaneously reseal their plasma merge with the injured plasma membrane
membrane. For example, microinjection restoring its integrity. This is supported
of cells in Ca2+-free media inevitably by electron microscopic observation
Norma W. Andrews*
results in cell death, but the need for of large intracellular vesicles close to
and Matthias Corrotte
extracellular Ca2+ as a trigger for the wound sites and the rapid formation of a
active repair of microneedle wounds is diffusion barrier when Ca2+-rich solutions
Tissue wound repair has been studied rarely mentioned when this technique were injected into oocytes. The second
extensively. It involves the coordinated and other procedures that cause plasma model proposed that reduced plasma
activation of several intracellular membrane injury are discussed. membrane tension resulting from multiple
and intercellular pathways, as well Once it became established that exocytic events close to a wound would
as remodeling from the sequential Ca2+ influx is an absolute requirement facilitate transition from a disordered
recruitment of different cell types to the for plasma membrane repair, attention lipid phase to a continuous planar lipid
wound site. There is, however, an equally turned to the identification of relevant bilayer. This model is supported by
important process that happens at the Ca2+ targets in the cytosol. Several direct measurements of reduced plasma
single cell level, when the integrity of cytosolic Ca2+-binding proteins have membrane tension after injury. Despite
the plasma membrane is compromised. been implicated, such as the Ca2+- limitations in these early models, there is
Individual eukaryotic cells can rapidly dependent cysteine protease calpain, a strong consensus that Ca2+-regulated
repair their plasma membrane after injury, Ca2+-activated transglutaminases that exocytosis of endomembrane vesicles, a
through a process that restores internal have protein cross-linking activity and few seconds after injury, is essential for
homeostasis and prevents cell death. annexins, which respond to Ca2+ by plasma membrane repair.
Despite its importance, investigations of binding to phospholipids and in some The term ‘constitutive exocytosis’
this fascinating mechanism have been cases forming membrane-associated refers to the process by which newly
limited. Only recently have we begun to scaffolds. Transglutaminase-mediated synthesized molecules are delivered
understand that plasma-membrane repair protein cross-linking and annexin- to the plasma membrane of eukaryotic
resembles tissue healing, in the sense mediated two-dimensional membrane cells or secreted into the extracellular
that it also involves sequential, highly scaffolds may form temporary diffusion medium. Molecules secreted through
localized remodeling steps that ultimately barriers at the wound site, preventing constitutive exocytosis are first
eliminate all traces of the injury. cytosol loss while the integrity of the translocated into the endoplasmic
lipid bilayer is restored. Annexins may reticulum and traffic through the Golgi
Ca2+ influx as a trigger also be indirectly involved in facilitating complex, where they are packaged into
By actively pumping Ca2+ ions from the Ca2+-regulated exocytosis, a process vesicles that ultimately fuse with the
cytosol into the extracellular space, cells that plays a key role in restoring plasma plasma membrane. By contrast, the term
maintain cytosolic resting concentrations membrane integrity as discussed below. Ca2+-regulated exocytosis is reserved for
of calcium in the nanomolar range. This Importantly, two classes of membrane- secretory fusion events that only occur in
results in a steep gradient across the associated proteins containing Ca2+- response to elevations in cytosolic Ca2+
plasma membrane, which serves as the binding C2-domains, the ubiquitously concentration. It was initially thought
only separation from an extracellular expressed synaptotagmin 7 and the that only specialized secretory cells
environment containing very high, muscle protein dysferlin, appear to act as were able to undergo Ca2+-regulated
millimolar concentrations of Ca2+. When regulators of Ca2+-regulated membrane exocytosis. However, it is increasingly
the integrity of the plasma membrane is fusion events that are essential for clear that this pathway is present in many
breached, a rapid influx of Ca2+ generates plasma membrane repair. different cell types. An important advance
localized and transient increases in the came when lysosomes, ubiquitous
cytosolic free Ca2+ concentration . Early Ca2+-regulated exocytosis organelles traditionally regarded as
studies in sea urchin eggs showed that A major breakthrough in our terminal degradative compartments of
Ca2+ influx through plasma membrane understanding of plasma membrane the endocytic pathway, were found to
wounds triggered a rapid surface repair came when experiments in sea rapidly fuse with the plasma membrane
reaction followed by full restoration of urchin eggs and mammalian cells in response to elevations in cytosolic
the eggs’ integrity, allowing subsequent revealed extensive and localized Ca2+ concentration. This was initially
fertilization and normal development. fusion of intracellular vesicles with the surprising, but extensive evidence
In contrast, when Ca2+ was removed plasma membrane, a few seconds confirmed that conventional lysosomes
from the seawater surrounding the after injury and Ca2+ influx. Inhibiting in many cell types can behave as
eggs, wounding resulted in progressive vesicle exocytosis by interfering with the Ca2+-regulated exocytic vesicles, and
loss of cytosol and cell death. These formation of SNARE complexes impaired that mammalian cells have a peripheral
initial observations were confirmed plasma membrane resealing, leading population of lysosomes ideally
in mammalian cells, establishing the to the first two models that explain the positioned for rapid fusion with the
concept that influx of extracellular Ca2+ mechanism of plasma membrane repair plasma membrane. Thus, lysosomes
into injured cells is essential for triggering (Figure 1A,B). The first model proposed have emerged as strong candidates for

R392 Current Biology 28, R367–R420, April 23, 2018 © 2017 Elsevier Ltd.
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A Patch B Tension reduction

Vesicle
Wound homotypic fusion Vesicle recruitment Plasma membrane
formation and exocytosis tension reduction
and Ca2+ entry Patch fusion/
Vesicle 2+
Ca Wound
recruitment wound closure
2+ closure
2+ Ca
Ca
2+
Ca

C Exocytosis/Endocytosis D Budding

Lysosomal exocytosis Membrane Vesicle Vesicle and wound

ESCRT Vesicle budding
and enzyme secretion remodeling complex shedding
formation
Endocytosis recruitment
2+ of wounds
Ca
2+
Ca 2+
Ca

Current Biology

Figure 1. Proposed mechanisms of plasma membrane repair.

Ca2+ influx through plasma membrane wounds triggers exocytosis. Initially it was proposed that this would result in formation of a repair ‘patch’ (A)
and/or a reduction in membrane tension facilitating spontaneous resealing (B). More recent studies proposed that lesions are actively removed from
the membrane, through a massive form of endocytosis that follows Ca2+-triggered lysosomal exocytosis (C), or by the shedding of ESCRT complex-
dependent membrane buds in the case of small lesions (D).

the ubiquitous population of exocytic Exocytosis of lysosomes as a defense an arsenal of membrane damaging
vesicles that mediate plasma membrane So why did ‘dangerous’ organelles such agents that include pore-forming toxins
repair. Exocytosis of lysosomes has as lysosomes, which have a very low and specialized secretion systems that
been extensively observed in injured luminal pH and contain a large load of permeabilize eukaryotic membranes
cells, and plasma membrane repair is degradative enzymes, evolve for the and promote Ca2+ influx. Given that
impaired after inhibition of lysosomal fundamental role of plasma membrane eukaryotic cells and bacteria co-evolved
exocytosis. Impaired plasma membrane repair? The use of lysosomes for this for millions of years, it is conceivable that
repair is also seen after inhibition of role is at first glance counter-intuitive, before the appearance of multicellular
synaptotagmin 7, a member of the but this perception changes when one organisms there was no significant barrier
synaptotagmin family of Ca2+ sensors considers that prokaryotes are likely to to prevent frequent contacts between
present on the membrane of lysosomes, have represented a major early threat these two cell types. Thus, a response
or of components of SNARE complexes to the plasma membrane integrity to plasma membrane injury in the form
that mediate lysosome exocytosis. of eukaryotic cells. The single lipid of lysosomal exocytosis might have
Interestingly, inhibition of the lysosomal bilayer that surrounds eukaryotic cells provided a strong evolutionary advantage
Ca2+ channel mucolipin-1 also impairs enabled the development of dynamic to eukaryotic cells. Lysosomes contain
plasma membrane repair, suggesting that membrane fusion/fission events, which numerous molecules and enzymes that
Ca2+ stored in the lumen of lysosomes gave rise to complex organelles and can compromise the viability of bacteria,
may also contribute to the increase robust intracellular membrane traffic. In and a localized release of these agents
in cytosolic Ca2+ concentration that is contrast, bacteria are often protected at the site of the injury might have
critical for plasma membrane resealing. by more rigid cell walls and evolved reduced the microbes’ ability to inflict

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Bacteria secrete
membrane-damaging
agents
Ca2+ influx

Bacterial survival Bacterial killing

Lysosomal Lysosomal
secretion secretion
No lysosomal Endocytosis
Residual
secretion
pores

N N N

Restoration of
Removal of
plasma membrane
bacterial pores
integrity
Death of all the Bacterial killing Lysosomal-mediated
cell population facilitates survival of plasma membrane repair
some cells in the population promotes survival of all
the cell population
Current Biology

Figure 2. Evolutionary advantage of plasma membrane repair mediated by exocytosis of lysosomes.

Membrane-damaging molecules produced by microbes are likely to have represented an early threat to the integrity and survival of eukaryotic cells.
Exocytosis of lysosomes as a rapid and localized response to membrane permeabilization may have provided a survival advantage to eukaryotic
cells, by promoting plasma membrane resealing and at the same time releasing molecules capable of killing the microbes inflicting the damage.

further damage, in addition to promoting allows synchronization of the wounding difficult to envision how the originally
plasma membrane resealing (Figure 2). step. Studies using this system have proposed ‘patch’ or ‘tension reduction’
Consistent with this view of lysosomes as revealed that mammalian cells can fully models of plasma membrane repair
primordial exocytic organelles engaged restore their plasma membrane integrity could promote the resealing of stable,
in cellular defense, it is noteworthy after permeabilization with SLO, and protein-lined transmembrane pores.
that cytotoxic lymphocytes perform concluded that this process has several Instead, it became necessary to identify
their critical role in immune defense by elements in common with the resealing a mechanism capable of physically
secreting the contents of lysosome- of mechanical wounds. First, SLO-pore removing pores from the plasma
related organelles, in a very localized and repair is also strictly dependent on the membrane.
targeted fashion. As discussed below, presence of Ca2+ in the extracellular A different model for plasma
evidence is emerging that extracellularly medium. Second, restoration of plasma membrane repair began to emerge when
released lysosomal enzymes may also membrane integrity is also rapid, being mammalian cells injured mechanically
remodel the outer leaflet of the plasma completed within ~30 seconds after or by SLO pores were found to undergo
membrane to facilitate wound repair. SLO permeabilization. Third, lysosomal massive endocytosis, after Ca2+-triggered
exocytosis is also required for the repair exocytosis of lysosomes (Figure 1C).
Lesion removal by endocytosis of SLO pores. These common properties Rapid endocytosis following elevation
Bacterial pore-forming toxins, such as suggested that a common mechanism of cytosolic Ca2+ concentration was also
Streptolysin O (SLO), are useful tools involving lysosomal exocytosis might detected in patch-clamp experiments
for studying plasma membrane repair, be involved in the resealing of different and called ‘MEND’ (for massive Ca2+-
because pre-binding of toxin monomers types of lesions, as long as they allow activated endocytosis). Importantly,
at low temperatures followed by warming Ca2+ influx. However, it became very these electrophysiology experiments

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Lysosomal enzyme Production of

secretion (ASM) ceramide domains
Membrane Ca2+-dependent
wounding exocytosis
and Ca2+ entry Ca2+ of lysosomes Membrane Endocytosis
Ca 2+ invagination and lesion
removal

Lesion
degradation
in lysosomes

Lesion Vesicle
fusion with
SNARE complex early endosomes
Lysosomal acid sphingomyelinase
Ceramide-enriched domain
Ubiquitin Lesion sorting into
intraluminal vesicles
ESCRT-0 complex Late endosome of late endosomes
ESCRT-III complex maturation

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Figure 3. Plasma membrane injury triggers a repair response that involves exocytosis of lysosomes followed by lesion endocytosis and
intracellular degradation.
Ca2+ influx through plasma membrane wounds triggers localized exocytosis of lysosomes, releasing enzymes that remodel the cell surface facilitat-
ing lesion endocytosis. After internalization in endosomes, membrane lesions traffic to the lumen of lysosomes where they are degraded.

defined the interval between elevation in lesion removal from the plasma internalized toxin travels down the
of intracellular Ca2+ concentration membrane. Providing an important endocytic pathway and is delivered to
and endocytosis as approximately 30 link between lysosomal exocytosis lysosomes for degradation (Figure 3).
seconds, closely mirroring the time scale and endocytosis-mediated plasma Caveolae are plasma membrane
of plasma membrane repair. This injury- membrane repair, the lysosomal enzyme invaginations with a diameter of ~80
dependent massive endocytic process acid sphingomyelinase (ASM) was nm that are associated with membrane
offered an alternative explanation for found to promote endocytosis and microdomains enriched in cholesterol and
the intracellular vesicle accumulation plasma membrane resealing when sphingomyelin (frequently described as
that is seen close to wounds, which added extracellularly to cells injured lipid rafts). Caveolae are present in many
were previously considered primarily mechanically or by SLO permeabilization. cell types, and particularly abundant
exocytic. This unusual form of Collectively, these findings led to a in cells that are under significant
endocytosis observed after Ca2+ multi-step model of plasma membrane mechanical stress in vivo such as muscle
influx is independent of classical repair (Figure 3). According to this model, fibers, cardiomyocytes and endothelial
endocytosis proteins such as clathrin, plasma membrane injury triggers Ca2+ cells. Caveolae were described to
requires the presence of cholesterol influx and exocytosis of lysosomal detach from the plasma membrane
in the plasma membrane, and can be hydrolases, including ASM, which in turn and become internalized after certain
triggered by extracellular exposure to remodel the outer leaflet of the plasma stimuli, but there is still controversy
the enzyme sphingomyelinase. These membrane. This triggers a ceramide- about the frequency of this event and its
findings provided useful opportunities dependent wave of endocytosis that physiological role. Interestingly, electron
for functional inhibition studies, internalizes the lesions. Experiments microscopy studies have revealed a large
which demonstrated a requirement following the intracellular fate of SLO number of internalized vesicles with the
for injury-dependent endocytosis in resealed cells have shown that the size and morphology of caveolae in the

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cytoplasm of injured cells, or in intact away from the cytosol, in the repair type IV, which is characterized by
cells treated with sphingomyelinase. SLO of small plasma membrane wounds progressive neurodegeneration, motor
was directly visualized traveling into cells (Figure 1D). It is important to note that dysfunction and muscle weakness in
inside internalized caveolar vesicles, and the ESCRT-generated vesicles seen so addition to more generalized symptoms
inhibition of caveolae formation blocked far in other systems are much smaller such as retinal degeneration and anemia.
plasma membrane repair, strongly (around 50 nm) than the transient, large The realization that endocytosis plays
suggesting that wounds can be removed plasma membrane blebs commonly a role in plasma membrane repair has
from the plasma membrane through seen in injured cells. Thus, for firmly provided important new insights into the
caveolar endocytosis. Interestingly, implicating ESCRT-mediated vesicle pathology associated with mutations in
intracellular caveolae-like vesicles shedding in plasma membrane repair, it caveolae-specific proteins. Mutations
were reported to accumulate inside the will be important to show that ESCRT- in the muscle-specific caveolin-3
highly fragile skeletal muscle fibers of dependent vesicles in the predicted isoform Cav3 cause an autosomal
Duchenne muscular dystrophy patients, size range are formed in the plasma dominant form of muscular dystrophy
suggesting that a weak sarcolemma membrane and then shed from wounded in humans (LGMD1C). Cavin (PTRF)
may lead to frequent cycles of injury cells. These additional experiments are mutations are linked to cardiomyopathy
and caveolae-mediated repair. These important to rule out potential non- and various forms of skeletal muscle
findings add to the growing evidence canonical roles of ESCRT components pathology in humans. In mice, Cav1
that point to cholesterol-dependent, in plasma membrane repair, or the (the ubiquitously expressed caveolin-1
clathrin-independent endocytosis as a possibility that the ESCRT complex isoform) deficiency is associated with
pathway to remove plasma membrane promotes plasma membrane remodeling impairment in endothelial relaxation/
lesions in mammalian cells. In B at a later step, after resealing. contractility, defective maintenance of
lymphocytes, cells that do not appear myogenic tone and lung fibrosis, while
to contain morphologically-defined Plasma membrane repair and disease animals lacking both Cav1 and Cav3
caveolae, plasma membrane wounding Many tissues are under mechanical develop severe cardiomyopathy. These
triggers an ASM-dependent, clathrin- stress in vivo, which at the cellular level caveolin/cavin-linked pathological
independent form of lipid raft endocytosis is expected to result in frequent cycles of changes are associated with a complete
that is associated with Ca2+-dependent plasma membrane injury and repair. This absence of caveolae on the plasma
lysosomal exocytosis and plasma is particularly true for skeletal muscle, membrane, and have been attributed
membrane repair. These are exciting where frequent injury during exercise is to defects in signaling and/or lipid
developments, because they suggest well documented through the entry of metabolism. However, the demonstration
that the poorly understood pathway of membrane impermeant molecules and that lesions can be directly removed
clathrin-independent endocytosis of extracellularly released cytosolic markers from the plasma membrane through
cholesterol and sphingomyelin-enriched such as creatine kinase and lactate caveolar internalization suggests that
lipid rafts may have a central role in dehydrogenase. In agreement with a defective plasma membrane repair
promoting plasma membrane repair. critical need to restore plasma membrane may represent, at least in part, a
integrity after injury in muscle, genetic component of the muscle pathology
Are lesions also removed by blebbing mutations in several proteins involved in associated with the lack of caveolae.
and shedding? plasma membrane repair are increasingly Reinforcing this view, Cav3 binds to two
A frequent observation in wounded being found to cause muscle pathology. additional muscle proteins important for
cells is the formation of highly dynamic, Autosomal recessive mutations in plasma membrane repair, dysferlin and
large plasma membrane blebs (in the dysferlin, a sarcolemma protein with mitsugumin 53, forming a complex that
µm range). This morphologically striking multiple Ca2+-binding C2 domains, may regulate membrane traffic events
surface reaction has led, understandably, underlie a late onset form of human limb- essential for plasma membrane repair.
to the assumption that shedding of girdle muscular dystrophy (LGMD2B). A As our understanding of the molecular
injured membrane in the form of blebs similar pathology develops in dysferlin- machinery mediating plasma membrane
might represent a mechanism by which null mice, and knockout mice lacking repair increases, more human diseases
cells remove lesions and reseal their the lysosomal Ca2+ sensor Sy7 develop associated with this process are likely to
plasma membrane. However, careful a form of inflammatory myopathy with be identified, paving the way for future
investigations suggest that large foci of muscle fiber death and infiltration therapeutic approaches.
wounding-induced plasma membrane by inflammatory cells. Another form of
blebs do not detach from cells, but human limb girdle muscular dystrophy, Outlook
rather reflect transient, reversible Ca2+- LGMD2A, is associated with autosomal Despite major progress in recent
dependent disassembly of the cortical recessive mutations in the Ca2+-activated years, several aspects of the plasma
actin cytoskeleton. Consistent with this protease calpain 3a. Mouse cells lacking membrane repair mechanism remain
view, drugs that abolish formation of the lysosomal Ca2+ channel mucolipin-1 poorly understood. Understanding
large plasma membrane blebs do not are also defective in plasma membrane how cells reseal very large plasma
inhibit cell resealing. On the other hand, resealing after injury, and knockout membrane wounds represents a
genetic depletion experiments have animals develop early onset progressive particular challenge. Repair of large
implicated components of the ESCRT muscular dystrophy. In humans wounds was proposed to occur by
complex, which promotes membrane mucolipin-1 mutations cause the severe the gradual constriction of plasma
deformation and vesicle budding lysosomal storage disease mucolipidosis membrane openings driven by massive

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FURTHER READING
endocytosis, or through a membranous Primer
patch. Regarding the ‘patch hypothesis’, Andrews, N.W., Almeida, P.E., and Corrotte, M.
it has been hard to envision how a large,
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(2014). Damage control: cellular mechanisms of
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Cheng, X., Zhang, X., Gao, Q., Ali Samie, M., assembly and
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channel MCOLN1 is required for sarcolemma
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Jimenez, A.J., and Perez, F. (2017). Plasma
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plasma membrane continuity after damage in the lung cancer cell line A549. Cell including those of the secretory and
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repair is likely to play an important role recognized by the 2013 Nobel Prize in
in controlling inflammation associated Physiology or Medicine. The SNARE
Department of Cell Biology and Molecular
with tissue injury, and this should be Genetics, University of Maryland, College proteins syntaxin, synaptobrevin (also
an exciting and fertile area for future Park, MD 20742, USA. known as VAMP) and SNAP-25 were
investigation. *E-mail: andrewsn@umd.edu discovered in early biochemical studies

Current Biology 28, R367–R420, April 23, 2018 © 2018 Elsevier Ltd. R397