Disruption of the plasma membrane evokes an exocytotic response that is required for rapid membrane resealing. We show here in Swiss 3T3 fibroblasts that a second disruption at the same site reseals more rapidly than the initial wound. This facilitated response of resealing was inhibited by both low external Ca2+ concentration and specific protein kinase C (PKC) inhibitors, bisindolylmaleimide I (BIS) and Go-6976. In addition, activation of PKC by phorbol ester facilitated the resealing of a first wound. BIS and Go-6976 suppressed the effect of phorbol ester on resealing rate. Fluorescent dye loss from a FM1-43 pre-labeled endocytotic compartment was used to investigate the relationship between exocytosis, resealing and the facilitation of resealing. Exocytosis of endocytotic compartments near the wounding site was correlated with successful resealing. The destaining did not occur when exocytosis and resealing were inhibited by low external Ca2+ concentration or by injected tetanus toxin. When the dye loaded cells were wounded twice, FM1-43 destaining at the second wound was less than at the first wound. Less destaining was also observed in cells pre-treated with phorbol ester, suggesting newly formed vesicles, which were FM1-43 unlabeled, were exocytosed in the resealing at repeated woundings. Facilitation was also blocked by brefeldin A (BFA), a fungal metabolite that inhibits vesicle formation at the Golgi apparatus. Lowering the temperature below 20 degrees C also blocked facilitation as expected from a block of Golgi function. BFA had no effect on the resealing rate of an initial wound. The facilitation of the resealing by phorbol ester was blocked by pre-treatment with BFA. These results suggest that at first wounding the cell used the endocytotic compartment to add membrane necessary for resealing. At a second wounding, PKC, activated by Ca2+ entry at the first wound, stimulated vesicle formation from the Golgi apparatus, resulting in more rapid resealing of the second membrane disruption. Since vesicle pools were implicated in both membrane resealing and facilitation of membrane resealing, we reasoned that artificial decreases in membrane surface tension would have the same result. Decreases in surface tension induced by the addition of a surfactant (Pluronic F68 NF) or cytochalasin D facilitated resealing at first wounding. Furthermore, Pluronic F68 NF restored resealing when exocytosis was blocked by tetanus toxin. These results suggest that membrane resealing requires a decrease in surface tension and under natural conditions this is provided by Ca2+-dependent exocytosis of new membrane near the site of disruption.

REFERENCES

Angleson
J. K.
,
Betz
W. J.
(
1997
).
Monitoring secretion in real time: capacitance, amperometry and fluorescence compared.
Trends Neurosci
20
,
281
287
Bennett
M. K.
(
1997
).
Ca2+and the regulation of meurotransmitter secretion.
Curr. Opin. Neurobiol
7
,
316
322
Benfenati
F.
,
Valtorta
F.
,
Rubenstein
J. L.
,
Gorelick
F. S.
,
Greengard
P.
,
Czernik
A. J.
(
1992
).
Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.
Nature
359
,
417
420
Bi
G.-Q.
,
Alderton
J. M.
,
Steinhardt
R. A.
(
1995
).
Calcium-regulated exocytosis is required for cell membrane resealing.
J. Cell Biol
131
,
1747
1758
Bi
G.-Q.
,
Morris
R. L.
,
Liao
G.
,
Alderton
J. M.
,
Scholey
J. M.
,
Steinhardt
R. A.
(
1997
).
Kinesin-and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.
J. Cell Biol
138
,
999
1008
Billiard
J.
,
Koh
D.-S.
,
Babcock
D. F.
,
Hille
B.
(
1997
).
Protein kinase C as a signal for exocytosis.
Proc. Nat. Acad. Sci. USA
94
,
12192
12197
Binz
T.
,
Blasi
J.
,
Yamasaki
S.
,
Baumeister
A.
,
Link
E.
,
Sudhof
T. C.
,
Jahn
R.
,
Niemann
H.
(
1994
).
Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.
J. Biol. Chem
269
,
1617
1620
Blasi
J.
,
Chapman
E. R.
,
Yamasaki
S.
,
Binz
T.
,
Niemann
H.
,
Jahn
R.
(
1993
).
Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.
EMBO J
12
,
4821
4828
Buccione
R.
,
Bannykh
S.
,
Santone
I.
,
Baldassarre
M.
,
Facchiano
F.
,
Bozzi
Y.
,
Di Tullio
G.
,
Mironov
A.
,
Luini
A.
,
De Matteis
M. A.
(
1996
).
Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study.
J. Biol. Chem
271
,
3523
3533
Ceccaldi
P. E.
,
Grohovaz
F.
,
Benfenati
F.
,
Chieregatti
E.
,
Greengrad
P.
,
Valtorta
F.
(
1995
).
Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.
J. Cell Biol
128
,
905
912
Coorssen
J. R.
,
Schmitt
H.
,
Almers
W.
(
1996
).
Ca2+-triggers massive exocytosis in Chinese hamster ovary cells.
EMBO J
15
,
3787
3791
Dai
J.
,
Sheetz
M. P.
(
1998
).
Cell membrane mechanics.
Meth. Cell Biol
55
,
157
171
Dan
Y.
,
Poo
M.
(
1992
).
Quantal transmitter secretion from myocytes loaded with acetylcholine.
Nature
359
,
733
736
Deeney
J. T.
,
Cunningham
B. A.
,
Chheda
S.
,
Bokvist
K.
,
Juntti-Berggren
L.
,
Lam
K.
,
Korchak
H. M.
,
Corkey
B. E.
,
Berggren
P.
(
1996
).
Reversible Ca2+-dependent translocation of protein kinase C and glucose-induced insulin release.
J. Biol. Chem
271
,
18154
18160
De Matteis
M. A.
,
Santini
G.
,
Kahn
R. A.
,
Di Tullio
G.
,
Luini
A.
(
1993
).
Receptor and protein kinase C-mediated regulation of ARF binding to the Golgi complex.
Nature
364
,
818
821
Donaldson
J. G.
,
Klausner
R. D.
(
1994
).
ARF: a key regulatory switch in membrane traffic and organelle structure.
Curr. Opin. Cell Biol
6
,
527
532
Gillis
K. D.
,
Möβner
R.
,
Neher
E.
(
1996
).
Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules.
Neuron
16
,
1209
1220
Goda
Y.
(
1997
).
SNAREs and regulated vesicle exocytosis.
Proc. Nat. Acad. Sci. USA
94
,
769
772
Goodson
H. V.
,
Valetti
C.
,
Kreis
T. E.
(
1997
).
Motors and membrane traffic.
Curr. Opin. Cell Biol
9
,
18
28
Hanson
P. I.
,
Heuser
J. E.
,
Jahn
R.
(
1997
).
Neurotransmitter release—four years of SNARE complexes.
Curr. Opin. Neurobiol
7
,
310
315
Haruta
T.
,
Takami
N.
,
Ohmura
M.
,
Misumi
Y.
,
Ikehara
Y.
(
1997
).
Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex.
Biochem. J
325
,
455
463
Hochmuth
R. M.
,
Shao
J.-Y.
,
Dai
J.
,
Sheetz
M. P.
(
1996
).
Deformation and flow of membrane into tethers extracted from neuronal growth cones.
Biophys. J
70
,
358
369
Klausner
R. D.
,
Donaldson
J. G.
,
Lippincott-Schwartz
J.
(
1992
).
Brefeldin A: insights into the control of membrane traffic and organelle structure.
J. Cell Biol
116
,
1071
1080
Ktistakis
N. T.
,
Brown
H. A.
,
Waters
M. G.
,
Sternweis
P. C.
,
Roth
M. G.
(
1996
).
Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles.
J. Cell Biol
134
,
295
306
Liu
J. P.
(
1997
).
Protein phosphorylation events in exocytosis and endocytosis.
Clin. Exp. Pharmacol. Physiol
24
,
611
618
Llinás
R.
,
Gruner
J. A.
,
Sugimori
M.
,
McGuinness
T. L.
,
Greengard
P.
(
1991
).
Regulation by synapsin I and Ca2+-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse.
J. Physiol
436
,
257
282
Matlin
K. S.
,
Simons
K.
(
1983
).
Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation.
Cell
34
,
233
243
Martiny-Baron
G.
,
Kazanietz
M. G.
,
Mischak
H.
,
Blumberg
P. M.
,
Kochs
G.
,
Hug
H.
,
Marme
D.
,
Schächtele
C.
(
1993
).
Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976.
J. Biol. Chem
268
,
9194
9167
McNeil
P. L.
,
Steinhardt
R. A.
(
1997
).
Loss, restoration, and maintenance of plasma membrane integrity.
J. Cell Biol
137
,
1
4
Miyake
K.
,
McNeil
P. L.
(
1995
).
Vesicle accumulation and exocytosis at sites of plasma membrane disruption.
J. Cell Biol
131
,
1737
1745
Rodríguez
A.
,
Webster
P.
,
Ortego
J.
,
Andrews
N. W.
(
1997
).
Lysosomes behave as Ca2+-regulated exocytotic vesicles in fibroblasts and epithelial cells.
J. Cell Biol
137
,
93
104
Sabatini
D. D.
,
Adesnik
M.
,
Ivanov
I. E.
,
Simon
J.-P.
(
1996
).
Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission.
Biocell
20
,
287
300
Saraste
J.
,
Kuismanen
E.
(
1984
).
Pre-and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface.
Cell
38
,
535
549
Schiavo
G.
,
Benfenati
F.
,
Poulain
B.
,
Rossetto
O.
,
Polverio de Laureto
P.
,
DasGupta
B. R.
,
Montecucco
C.
(
1992
).
Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin.
Nature
359
,
832
835
Schiavo
G.
,
Santucci
A.
,
DasGupta
B. R.
,
Mehta
P. P.
,
Jontes
J.
,
Benfenati
F.
,
Wilson
M. C.
,
Montecucco
C.
(
1993
).
Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.
FEBS Lett
335
,
99
103
Simon
J.-P.
,
Ivanov
I. E.
,
Adesnik
M. A.
,
Sabatini
D. D.
(
1996
).
Theproduction of post-Golgi vesicle requires a protein kinase C-like molecule, but not its phosphorylating activity.
J. Cell Biol
135
,
355
370
Steinhardt
R. A.
,
Bi
G.-Q.
,
Alderton
J. M.
(
1994
).
Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release.
Science
263
,
390
393
Sudhof
T. C.
(
1995
).
The synaptic vesicle cycle: a cascade of protein-protein interactions.
Nature
375
,
645
653
Terasaki
M.
,
Miyake
K.
,
McNeil
P. L.
(
1997
).
Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.
J. Cell Biol
139
,
63
74
Toullec
D.
,
Pianetti
P.
,
Coste
H.
,
Bellevergue
P.
,
Grand-Perret
T.
,
Ajakane
M.
,
Baudet
V.
,
Boissin
P.
,
Boursier
E.
,
Loriolle
F.
,
Duhamel
L.
,
Charon
D.
,
Kirilovsky
J.
(
1991
).
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem
266
,
15771
15781
Tsai
M. A.
,
Frank
R. S.
,
Waugh
R. E.
(
1994
).
Passive mechanical behavior of human neutrophils: effect of cytochalasin B.
Biophys. J
66
,
2166
2172
Vitale
M. L.
,
Seward
E. P.
,
Trifaro
J.-M.
(
1995
).
Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis.
Neuron
14
,
353
363
Westermann
P.
,
Knoblich
M.
,
Maier
O.
,
Lindschau
C.
,
Haller
H.
(
1996
).
Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles.
Biochem. J
320
,
651
658
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