Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30904, USA
e-mail: pmcneil{at}mcg.edu
![]() |
Summary |
---|
Key words: Lysosome, Fusion, Vesicle, Plasma membrane
![]() |
Introduction |
---|
In addition to suffering from a kind of `guilt by association', resealing
languished for a second reason. It was thought to have a simple and trivial
explanation. Thus, once it was established that the principal cell surface
barrier torn by the microneedle was a fluid lipid bilayer, resealing became
explicable as simply the thermodynamically determined outcome of the well
established principle that `Membranes hate edges'
(Parsegian et al., 1984).
According to this view, still current in textbooks, resealing is an ability
inherent in all membranes, a response requiring nothing more than that the
torn membrane to be held at a temperature above its liquid-crystalline
transition point. This view is no longer tenable, at least in the case of
large (>1 µm) disruptions occurring in nucleated animal cells. Rather,
as outlined here, resealing is now viewed as the outcome of a dynamic and
complex mechanism, one that relies heavily on the participation of numerous
cytoplasmic constituents. Recent work, discussed below, strongly implicates
lysosomes and the actin-based cytoskeleton as two key cytoplasmic players in
the resealing response and actin/myosin-based contraction in the subsequent
repair of wound-associated damage to the cell cortex.
![]() |
The significance of resealing |
---|
For long-lived, multicellular organisms, too, resealing may often be a beneficial and therefore an evolutionarily favored cell response. For example, physical insults (accidents or attacks) that disrupt tissue integrity are an obvious cause of plasma membrane disruption. However, for a single, isolated tissue injury, it could be argued that resealing is of little consequence: `once only' cell replacement costs might be of little overall importance, at least in long-lived organisms. It is only in cases in which the cells experiencing a disruption during an injury are both irreplaceable and essential to the continued functioning of the organism that resealing has an indisputable value. Importantly, this condition is fulfilled in the case of, for example, a severed nerve. The individual neurons suffering disruptions during the injurious (severing) event cannot be replaced. Therefore they must reseal if they are to survive and subsequently `grow' back to their targets in a successful re-innervation effort.
If under normal, physiological circumstances plasma membrane disruption
affects a large proportion of a tissue's cell population (e.g. is a frequent
event) and/or if it affects large cell types, and/or affects cells that are
irreplaceable, then resealing might be important under normal as well as
pathological conditions. Either it reduces the cost to the organism of
complete cell replacement or it prevents an accumulating loss of cells
essential for the functioning of the organism. `Cell wounding', defined as a
survivable plasma membrane disruption event marked by the uptake into the
cytosol of a normally membrane impermeant tracer, is observable under
physiological conditions in the endothelium lining the aorta, the epithelium
lining the gastrointestinal tract, the epithelium of skin, and the myocytes of
cardiac and skeletal muscle (McNeil,
1993). The frequent occurrence of disruptions in cardiac
(irreplaceable) and skeletal (large) myoctes
(Clarke et al., 1995
;
McNeil and Khakee, 1992
),
under physiological conditions, argues that resealing is a fundamental
biological response in mammals. It is possible that resealing is also a
cost-effective cell adaptation in the several other tissues mentioned.
The proportion of cells classifiable as wounded typically increases as a
function of mechanical load. For example, it rises from 4% of the
myocytes of the triceps muscle of the mouse or rat kept in its cage to
20% after these rodents are exercised by running downhill
(Clarke et al., 1993
;
McNeil and Khakee, 1992
),
which results in eccentric (high-force-producing) contractions of this muscle.
Resealing is therefore an essential, if widely overlooked, function of the
many and diverse cells that reside in mechanically challenging tissue
environments of the normally functioning mammalian body.
![]() |
Rapid resealign involves a Ca2+-dependent, exocytotic response in nucleated animal cells |
---|
The first clue to how exactly Ca2+ promotes resealing or
restoration of disrupted lipid bilayer continuity came from work by Steinhardt
et al. (Steinhardt et al.,
1994). They found that resealing is inhibited if fibroblasts or
sea urchin eggs are first injected with botulinum or tetanus toxins and then
60 minutes later wounded by a second microneedle impalement. These toxins
are proteases that are thought to specifically target and thereby inactivate
members of the SNARE family of proteins required for certain exocytotic
events, such as those occurring at the synapse during neurotransmitter release
(Schiavo et al., 1992
).
Therefore the toxin microinjection experiment suggested that exocytosis is
required for resealing. Subsequent work, using both endothelial cells
(Miyake and McNeil, 1995
) and
sea urchin eggs (Bi et al.,
1995
), confirmed that an exocytotic reaction is rapidly evoked in
a Ca2+-dependent fashion by plasma membrane disruption, that this
response is localized to the disruption site and that it is quantitatively
related to disruption magnitude.
What is the function in resealing of this exocytotic delivery of internal
membrane to the surface of the wounded cell? The plasma membrane adheres to
the underlying cortical cytoskeleton (principally filamentous actin). This
generates a `membrane tension' (Raucher
and Sheetz, 1999) that opposes the `line tension' generated by
lipid disordering at the free edge of a disruption (reviewed in
Chernomordik et al., 1987
). It
is this line tension that, theoretically, promotes lipid flow over a
disruption site, as well as the bilayer fusion event required in completing
resealing. If therefore the exocytotic events induced by a disruption could
somehow reduce membrane tension, the predicted result would be the promotion
of resealing through enhanced, line-tension-driven lipid flow. Consistent with
this hypothesis is the observation that a rapid (second timescale),
Ca2+-dependent reduction in membrane tension is induced by membrane
disruption (Togo et al., 1999
;
Togo et al., 2000
). Moreover,
treatment of cells with surface active agents, which might reduce membrane or
`surface' tension, enhances resealing and survival
(Clarke and McNeil, 1994
;
Togo et al., 1999
).
![]() |
Vesicle-vesicle fusion |
---|
Electron micrographs of the cortical cytoplasm bordering on endothelial
cell disruption sites suggest that, in addition to vesicle-plasma membrane
fusion (exocytotic fusion), vesicle-vesicle fusion is induced locally by a
disruption. The cortical cytoplasm surrounding a disruption displays within
seconds after its formation a remarkable abundance of abnormally enlarged
vesicles (Miyake and McNeil,
1995). What is the role of these enlarged vesicles? An answer to
this question was suggested by experiments in which sea water was injected
along with fluorescent tracers into the cytoplasm of starfish and sea urchin
eggs (Terasaki et al., 1997
).
Fluorescent seawater containing a normal level of Ca2+, but not
seawater without Ca2+, was immediately sequestered as it left the
microneedle orifice behind an impermeant barrier. That this barrier was a
membrane was confirmed by electron microscopy, staining with lipidic dyes and
its measured impermeability not only to small fluorescent dies such as
fluorescein stachyose (
1000 MW) but also to Ca2+ and even
H+ ions.
The sea water injection experiment revealed a key concept: cytoplasm by itself, in the absence of plasma membrane and therefore exocytotic events, can form a membrane barrier to prevent further incursion of the toxic extracellular environment. Moreover, it provided a mechanism consistent with the earlier electron microscope observations: given the scale (vesicles >10 µm in diameter form at pipette tips) and rapidity (second or sub second time scale) of the formation of membrane barrier, the process had to be based on a vesicle-vesicle fusion reaction.
![]() |
The patch hypothesis |
---|
|
Four key predictions of this `patch' hypothesis have recently been verified
in the sea urchin egg (McNeil et al.,
2000). First, native, pre-disruption surface membrane is not
present initially over large, resealed disruptions. The membrane covering the
disruption site immediately after resealing must therefore be derived from an
internal source, as predicted. Second, stratification of organelles induced by
egg centrifugation results in a polarization of resealing function. The
distribution or availability of internal membrane is therefore a crucial
determinant of resealing capacity, as predicted. Third, abnormally large
vesicles are readily detected in the cytoplasm underlying a disruption site,
both by light and scanning electron microscopy, and the appearance of these is
rapid and Ca2+ dependent
(McNeil and Baker, 2001
).
Vesicle-vesicle fusion is therefore induced locally by Ca2+ influx
through a disruption, as predicted. Fourth, an egg organelle (the yolk
granule) displays cytosol-independent homotypic fusion in vitro that is
initiated Ca2+ (
10 µM threshold) with a T1/2 of
seconds and results in the production of very large (>50 µm diameter)
vesicles. The egg therefore possesses a vesicle population capable of
homotypic fusion that can occur rapidly in the absence of the time consuming,
cytosol-dependent priming steps of other homotypic fusion reactions and that
can erect large membrane boundaries, as predicted.
The patch hypothesis can explain how extremely large disruptions, requiring
substantial membrane replacement, are resealed; how, in fact, some cells are
able rapidly to replace their entire surface membrane
(Rappaport, 1976). Recent
work, discussed below, now allows us to name the vesicle population used for
patch formation by mammalian cells lysosomes and adds, for
these cells, an additional early step in the mechanism actin
depolymerization. Moreover, it is becoming clear that a wounded cell's repair
work continues after it has patched the surface bilayer discontinuity.
![]() |
What vesicles are used for resealing? |
---|
What is the organelle used in cells that lack yolk granules? Considerable
indirect evidence had accumulated that pointed to the lysosome. Yolk granules
of sea urchin eggs are an acidic compartment
(McNeil and Terasaki, 2001)
that contains hydrolytic enzymes (Armant et
al., 1986
), and yolk granules are known to have, in the species
studied (Raikhel, 1987
;
Wallace et al., 1983
), an
endocytotic origin. Lysosomes of cultured mammalian cells can be induced by
elevated Ca2+ levels, both in vitro
(Mayorga et al., 1994
) and in
situ (Bakker et al., 1997
), to
fuse with one another (homotypically), as is required for patch formation.
Moreover, Ca2+ induces lysosomes to fuse exocytotically with the
plasma membrane (Rodriguez et al.,
1997
), which is another fusion event induced by disruption. This
Ca2+-regulated lysosomal exocytosis depends on synaptotagmin
(Syt)VII, a lysosomal membrane protein and a member of a
Ca2+-binding family of proteins long thought to play a role in
fusion possibly as Ca2+ sensors
(Geppert and Sudhof, 1998
).
Antibodies against the C2A domain of this protein and recombinant Syt VII C2A
domain peptide both inhibit Ca2+-induced (streptolysin-O
permebilized) lysosomal exocytosis
(Martinez et al., 2000
).
Antibodies raised against the C2A domain of a squid synaptogamin inhibited
resealing in the giant squid axon and in cultured PC12 cells
(Detrait et al., 2000a
;
Detrait et al., 2000b
).
However, these axon-resealing studies did not reveal what organelle this
antibody targeted. Lastly, earlier studies employing fluorescent dyes taken up
endocytotically revealed the involvement of the various compartments,
including lysosomes, thus labeled in a disruption-induced exocytotic response
(Miyake and McNeil, 1995
).
Again the organelle involved could not be defined, since this method did not
discriminate between the several compartments labeled, for example, endosomes
(early, late) and lysosomes.
Thus it was important to ask more specifically whether exocytosis of
lysosomes is triggered by a plasma membrane disruption. The approach Reddy et
al. (Reddy et al., 2001) took
in answering this question to look for the disruption-induced
appearance on the cell surface of a lysosmal membrane protein yielded
a striking result. Antibodies against the luminal domain of the
lysosome-specific protein, Lamp-1 (Granger
et al., 1990
), do not stain the surface of undisturbed cells, but
strongly stain the surface of wounded cells
(Fig. 2). This surface exposure
of Lamp-1 is Ca2+-dependent and localized to disruption sites made
with a microneedle. To test the functional importance in resealing of the
disruption-induced lysosomal exocytosis, Reddy et al. introduced into living
cells antibodies to the C2A (calcium-binding) domain of Syt VII, as well as
recombinant peptide fragments of the whole protein. Both of these reagents
inhibited the surface appearance of the Lamp-1 luminal domain and cell
resealing. These inhibitory effects were observed when the disruption event
being monitored was also the route of access of reagent to cytoplasm.
Inactivation must therefore have been extremely rapid, since resealing is
generally complete in <90 sec in these cells. In an independent test of the
role of lysosomes, antibodies against the cytosolic domain of Lamp-1, which
have a lysosome-aggregating activity, also inhibited fusion.
|
These studies, it must be pointed out, do not rule out the participation of
other organelles in resealing. When the cortical granules of the sea urchin
are `undocked' by treatments with stachyose, resealing is reversibly inhibited
(Bi et al., 1995). This is
indirect evidence for a cortical granule contribution to resealing, although
it remains unclear how specific the stachyose treatment is for cortical
granules. Moreover, two separate pools of vesicles, identified on the basis of
the timing of their exocytosis and on their susceptibility to myosin/kinesin
inhibitors, are required for resealing in urchin eggs
(Bi et al., 1997
). This is
indirect evidence that, in the sea urchin egg, resealing might use both yolk
granules and cortical granules, as well as other, as-yet-unidentified
organelles. It also remains possible that, in other `specialized' cell types,
organelle compartments other than, or in addition to, lysosomes are mobilized
for resealing.
![]() |
Rescue by lysosomes: a dangerous expedient? |
---|
![]() |
Making way for lysosomes: disruption-induced cortical depolymerization |
---|
Earlier studies suggested that the cytoskeleton is an aid, as well as an
obstacle, to resealing. Antibodies to kinesin and a general inhibitor of
myosins, both inhibited resealing (Bi et
al., 1997; Steinhardt et al.,
1994
). One possible role for these motors might be to move
vesicles into the disruption site and hence to promote contacts leading to the
vesicle-vesicle and exocytotic fusion events required for resealing. The
remarkable concentration of vesicles observed by electron microscopy to occupy
cortical disruption sites in endothelial cells
(Miyake and McNeil, 1995
) is
consistent with this conjecture, but, so far, the disruption-induced vesicular
movements that might be powered by kinesin and/or myosin have not been
directly observable.
![]() |
Continuing repairs and defensive preparations |
---|
|
After resealing a disruption, and repairing associated cortical damage, can
a cell then prepare itself to better withstand future injury? Fibroblasts
reseal a second plasma membrane disruption more rapidly than a first made ten
minutes earlier (Togo et al.,
1999). Drug inhibitor and activator experiments suggest that this
`facilitated resealing' is dependent on enhanced protein kinase C activity and
enhanced vesicle production by the Golgi apparatus. Such an enhancement of
vesicle production might target lysosomes, which are, of course, supplied with
membrane (and enzymes) by Golgi-derived shuttle vesicles. An increase in the
size and/or number of lysosomes should enhance resealing (see above) and
might, therefore, constitute the mechanistic basis of the facilitated
resealing response.
![]() |
Future challenges |
---|
![]() |
References |
---|
Armant, D. R., Carson, D. D., Decker, G. L., Welply, J. K. and Lennarz, W. J. (1986). Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata. Dev. Biol. 113,342 -355.[Medline]
Bakker, A. C., Webster, P., Jacob, W. A. and Andrews, N. W.
(1997). Homotypic fusion between aggregated lysosomes triggered
by elevated [Ca2+]i in fibroblasts. J. Cell
Sci. 110,2227
-2238.
Bement, W. M., Mandato, C. A. and Kirsch, M. N. (1999). Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. Curr. Biol. 9, 579-587.[Medline]
Bi, G.-Q., Alderton, J. M. and Steinhardt, R. A. (1995). Calcium-regulated exocytosis is required for cell membrane resealing. J. Cell Biol. 131,1747 -1758.[Abstract]
Bi, G. Q., Morris, R. L., Liao, G., Alderton, J. M., Scholey, J.
M. and Steinhardt, R. A. (1997). Kinesin- and myosin-driven
steps of vesicle recruitment for Ca2+-regulated exocytosis.
J. Cell Biol. 138,999
-1008.
Chambers, R. and Chambers, E. L. (1961).Explorations into the nature of the living cell . Cambridge, Massachusetts: Harvard University Press.
Chernomordik, L. V., Melikyan, G. B. and Chizmadzhev, Y. A. (1987). Biomembrane fusion: a new concept derived from model studies using two interacting planar lipid bilayers. Biochim. Biophys. Acta 906,309 -352.[Medline]
Clarke, M. F. C., Khakee, R. and McNeil, P. L.
(1993). Loss of cytoplasmic basic fibroblast growth factor from
physiologically wounded myofibers of normal and dystrophic muscle.
J. Cell Sci. 106,121
-133.
Clarke, M. S., Caldwell, R. W., Chiao, H., Miyake, K. and
McNeil, P. L. (1995). Contraction-induced cell wounding and
release of fibroblast growth factor in heart. Circ.
Res. 76,927
-934.
Clarke, M. S. F. and McNeil, P. L. (1994). Syringe loading: a method for inserting macromolecules into cells in suspension. In Cell Biology: A Laboratory Handbook., vol. 3 (ed. J. E. Celis), pp.30 -36. San Diego, CA: Academic Press.
Detrait, E., Eddleman, C. S., Yoo, S., Fukuda, M., Nguyen, M. P., Bittner, G. D. and Fishman, H. M. (2000a). Axolemmal repair requires proteins that mediate synaptic vesicle fusion. J. Neurobiol. 44,382 -391.[Medline]
Detrait, E. R., Yoo, S., Eddleman, C. S., Fukuda, M., Bittner, G. D. and Fishman, H. M. (2000b). Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin. J. Neurosci. Res. 62,566 -573.[Medline]
Geppert, M. and Sudhof, T. C. (1998). RAB3 and synaptotagmin: the yin and yang of synaptic membrane fusion. Annu. Rev. Neurosci. 21,75 -95.[Medline]
Granger, B. L., Green, S. A., Gabel, C. A., Howe, C. L.,
Mellman, I. and Helenius, A. (1990). Characterization and
cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.
J. Biol. Chem. 265,12036
-12043.
Heilbrunn, L. V. (1930). The surface precipitation reaction of living cells. Proc. Am. Philos. Soc. LXIX,295 -301.
Heilbrunn, L. V. (1956). Dynamics of Living Protoplasm. New York: Academic Press.
Mandato, C. A. and Bement, W. M. (2001).
Contraction and polymerization cooperate to assemble and close actomyosin
rings around Xenopus oocyte wounds. J. Cell
Biol. 154,785
-797.
Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J.,
Fowler, K. and Andrews, N. W. (2000). Synaptotagmin VII
regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts.
J. Cell Biol. 148,1141
-1149.
Mayorga, L. S., Beron, W., Sarrouf, M. N., Colombo, M. I.,
Creutz, C. and Stahl, P. D. (1994). Calcium-dependent fusion
among endosomes. J. Biol. Chem.
269,30927
-30934.
McNeil, P. L. (1993). Cellular and molecular adaptations to injurious mechanical force. Trends Cell Biol. 3,302 -307.
McNeil, P. L. and Baker, M. M. (2001). Cell surface events during resealing visualized by scanning electron microscopy. Cell Tissue Res. 304,141 -146.[Medline]
McNeil, P. L. and Khakee, R. (1992). Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am. J. Pathol. 140,1097 -1109.[Abstract]
McNeil, P. L. and Terasaki, M. (2001). Coping with the inevitable: how cells repair a torn surface membrane. Nat. Cell Biol. 3,E124 -E129.[Medline]
McNeil, P. L., Vogel, S. S., Miyake, K. and Terasaki, M.
(2000). Patching plasma membrane disruptions with cytoplasmic
membrane. J. Cell Sci.
113,1891
-1902.
Miyake, K. and McNeil, P. L. (1995). Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J. Cell Biol. 131,1737 -1745.[Abstract]
Miyake, K., McNeil, P. L., Suzuki, K., Tsunoda, R. and Sugai,
N. (2001). An actin barrier to resealing. J. Cell
Sci. 114,3487
-3494.
Parsegian, V. A., Rand, R. P. and Gingell, D. (1984). Lessons for the study of membrane fusion from membrane interactions in phospholipid systems. Ciba Found. Symp. 103,9 -27.[Medline]
Raikhel, A. S. (1987). The cell biology of mosquito vitellogenesis. Mem. Inst. Oswaldo Cruz. 82 Suppl. 3,93 -101.[Medline]
Rappaport, R. (1976). Furrowing in altered cell surfaces. J. Exp. Zool. 195,271 -278.[Medline]
Raucher, D. and Sheetz, M. P. (1999).
Characteristics of a membrane reservoir buffering membrane tension.
Biophys. J. 77,1992
-2002.
Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 106,157 -169.[Medline]
Rodriguez, A., Webster, P., Ortego, J. and Andrews, N. W.
(1997). Lysosomes behave as Ca2+-regulated eoxcytotic
vesicles in fibroblasts and epithelial cells. J. Cell
Biol. 137,93
-104.
Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B. R. and Montecucco, C. (1992). Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359,832 -835.[Medline]
Steinhardt, R. A., Bi, G. and Alderton, J. M. (1994). Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263,390 -393.[Medline]
Terasaki, M., Miyake, K. and McNeil, P. L.
(1997). Large plasma membrane disruptions are rapidly resealed by
Ca2+-dependent vesicle-vesicle fusion events. J. Cell
Biol. 139,63
-74.
Togo, T., Alderton, J. M., Bi, G. Q. and Steinhardt, R. A.
(1999). The mechanism of facilitated cell membrane resealing.
J. Cell Sci. 112,719
-731.
Togo, T., Krasieva, T. B. and Steinhardt, R. A.
(2000). A decrease in membrane tension precedes successful
cell-membrane repair. Mol. Biol. Cell
11,4339
-4346.
von Figura, K. and Hasilik, A. (1986). Lysosomal enzymes and their receptors. Annu. Rev. Biochem. 55,167 -193.[Medline]
Wallace, R. A., Opresko, L., Wiley, H. S. and Selman, K. (1983). The oocyte as an endocytic cell. Ciba Found. Symp. 98,228 -248.[Medline]
Woolley, K. and Martin, P. (2000). Conserved mechanisms of repair: from damaged single cells to wounds in multicellular tissues. BioEssays 22,911 -919.[Medline]
Related articles in JCS: