1 Second Department of Anatomy, School of Medicine, Fukushima Medical University, Fukushima 960-1295, Japan
2 Institute of Molecular Medicine and Genetics, Department of Cellular Biology and Anatomy, The Medical College of Georgia, Augusta, GA 30912-2000, USA
* Present address: Institute of Molecular Medicine and Genetics, Department of Cellular Biology and Anatomy, The Medical College of Georgia, Augusta, GA 30912-2000, USA
Author for correspondence (e-mail: kmiyake{at}mail.mcg.edu; pmcneil{at}mail.mcg.edu)
Accepted June 20, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Plasma membrane, Disruption, Resealing, Exocytosis, Actin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rapid resealing of a disruption limits influx of potential toxins such as Ca2+ and/or prevents excessive loss of essential cytosolic constituents, permitting survival. Survival is possible even when disruptions occur on a massive scale: the sea urchin egg, for example, can restore plasma membrane continuity within approximately 5 seconds after thousands of square microns of its plasma membrane are ripped off or dissolved away; such eggs can then be fertilized and will go on to divide (McNeil et al., 2000; Terasaki et al., 1997). This remarkable capacity for resealing represents an essential animal cell adaptation, and its early evolutionary development in the ancestral eukaryote may have been the crucial first step, after loss of the cell wall, towards exploitation by wall-less eukaryotes of natures many mechanically challenging environments (McNeil and Terasaki, 2001).
Rapid resealing has an absolute requirement for physiologic levels of external Ca2+ (Heilbrunn, 1930b; Steinhardt et al., 1994). Exocytosis is evoked locally at disruption sites in both sea urchin eggs and mammalian cells, and quantitatively correlates in extent with disruption size (Bi et al., 1995; Miyake and McNeil, 1995). Moreover, botulinum toxins, which cleave/inactivate SNARE family protein components of the fusion machinery, inhibit resealing in both eggs and fibroblasts (Steinhardt et al., 1994). Finally, depletion of cytoplasmic vesicles from egg peri-disruption cortex blocks resealing (Terasaki et al., 1997). Thus one hypothesized role of Ca2+ in resealing is to evoke vesicle-plasma membrane (exocytotic) fusion events.
The function in resealing of these fusion events, leading to deposition of internal membrane into the plasma membrane, is proposed, in the case of smaller disruptions (1 µm or less in diameter), to be the lessening of surface tension and hence the promotion of lipid flow over the disruption (Togo et al., 1999; Togo et al., 2000). For larger disruptions, we propose that resealing occurs by a patch mechanism (McNeil et al., 2000; Terasaki et al., 1997). Ca2+ entering through a plasma membrane disruption initiates vesicle-vesicle and vesicle-plasma membrane (exocytotic) fusion events locally in peri-disruption cell cortex. In this way, large patch vesicles are created and joined, by exocytotic fusion events, to plasma membrane surrounding the defect site. The disruption is, we propose, patched over with internally derived membrane. The strongest evidence supporting this hypothesis is that cytoplasm (of the sea urchin egg) can, by itself, rapidly (seconds) erect extensive membrane boundaries in response to Ca2+ (Terasaki et al., 1997), as can an isolated vesicle population (yolk granules) (McNeil et al., 2000).
Importantly, recent work has established that it is an undocked vesicle population (lysosomes in mammalian cells (Andrews, 2000; Reddy et al., 2001), yolk granules in eggs (McNeil and Terasaki, 2001)), that is available for these homotypic and exocytotic fusion events. Therefore, a rapid mechanism for bringing the relevant membranes into intimate physical contact must be activated by a disruption. Vesicle transport powered by myosin and kinesin is hypothesized to be required for resealing, since resealing is inhibited by function blocking antibodies against these motor proteins (Bi et al., 1997; Steinhardt et al., 1994). Disruption-induced vesicle transport may promote resealing not only by bringing vesicles into intimate contact with one another and/or the plasma membrane, but also by recruiting additional vesicles, for use in fusion events, into peri-disruption cytoplasm (Miyake and McNeil, 1995).
We hypothesize that the cytoskeleton itself, especially the actin-filament-rich, gel-like cortical cytoskeleton of mammalian epithelial cells, can present a physical obstacle to the vesicle transport and contact events required for resealing. Previous observations have failed to resolve this question. Thus, treatment of fertilized sea urchin eggs and embryonic cells with cytochalasin is reported to inhibit resealing (Bi et al., 1997), whereas cytochalasin treatment of fibroblasts promotes resealing (Togo et al., 2000). Here we test several predictions of this hypothesis by variously manipulating the actin polymerization state in living, gastric epithelial cells and measuring the effect on resealing.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
The rat gastric mucosal epithelial cell line, RGM1, established by H. Matsui from normal Wistar rat gastric epithelium (Kobayashi et al., 1996), was kindly supplied by the Riken Cell Bank (Tsukuba, Japan). RGM1 cells were cultured to confluence in a 1:1 mixture of Dulbeccos modified eagle medium and Hams F12 medium supplemented with 20% fetal calf serum, and used after their 25th passage.
Plasma membrane disruption
For microscope experiments with adherent cultures, cells (20,000) were plated onto sterile coverslips or into wells of Teflon coated slides (Eric Scientific, Portsmouth, NH) and allowed to attach overnight. Cell monolayers (confluent) were scratched with sterile 18-gauge needle (Swanson and McNeil, 1987), or alternatively, treated with glass beads (McNeil and Warder, 1987), to induce plasma membrane disruptions, and then processed for microscopy at various intervals afterwards as described below.
For flow cytofluorometric analysis of phalloidin staining, sub-confluent RGM1 cultures were scraped from 100 mm diameter culture plates using a soft rubber policeman as previously described (McNeil et al., 1984).
For luciferin-based assays of disruption survival, plasma membrane disruptions were induced in an automated syringe loading device designed to inflict a reproducible level of mechanical stress on cells (Clarke and McNeil, 1992). Briefly, cells (5x105 per ml) in 1 ml of culture medium were drawn up into and expelled from a sterile 1 ml syringe through a sterile 30 gauge needle (Becton-Dickinson, Ruterford, NJ) by the action of a foot activated two-way valve under a constant pressure of 1 kg/cm2.
F-actin staining
For rapid fixation for microscopy, cells were immersed in PBS containing formaldehyde-glutaraldehyde based fixative (Ito and Karnovsky, 1968) for 10 seconds, washed in ice cold PBS and then fixed for a further 10 minutes in 4% paraformaldehyde (freshly generated). For flow cytometery, cells were fixed with paraformaldehyde (2.5%) only. Fixed cells were washed three times in PBS, permeabilized in 0.2% (vol/vol) Triton X-100 in PBS for 10 minutes, incubated in 10 mM FITC, TRITC or Alexa 633 phalloidin dissolved in PBS at room temperature for 20 minutes and then washed thoroughly in PBS before microscopic analysis by confocal laser microscopy (Olympus, Fluorview System, Tokyo, Japan) or by flow cytofluorometry.
Semi-quantitative evaluation of cell F-actin content by flow cytofluorometry
Flow cytometric analysis was performed on a FACS flow cytometer (Becton Dickinson) using a 488 or 632 nm argon ion laser excitation line and appropriate band-pass filters. Forward angle scatter and fluorescence intensities were recorded from 10,000 cells, and dead cells eliminated from data sets based on their forward angle scatter.
Electron microscopy
Cells were syringed (taken up into and expelled twice from a 30 gauge needle) in the presence of 1 ml of culture medium containing HRP (10 mg/ml) and fixed 15 seconds later by immersion in the formaldehyde-glutaraldehyde fixative for 10 minutes at room temperature. HRP reaction product was developed using diaminobenzedine as a substrate and the cells were then postfixed in 1% OsO4 at room temperature for 1 hour. Ultra thin sections (cut on an MT1 ultramicrotome, Sorval) were examined in a JOEL (1210, Tokyo, Japan) transmission electron microscope.
Luminometer quantification of cell injury
This assay is based on one previously described (Miyake and McNeil, 1998) in which release of ATP from cells is used as an index of damage. After cells (20,000) were syringed or otherwise manipulated, 50 µl of a luciferin/luciferase solution (from the Toyo kit) were added to the cell suspension, and the cuvette containing this mixture then immediately inserted into a luminometer (Yamato Scientific, Compactlumi VS500, Tokyo, Japan) for measurement of light intensity.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
We first examined the effect of cytochalasin on resealing, as previously it had been reported to both enhance and inhibit resealing (Bi et al., 1997; Togo et al., 2000). Monolayers of RGM1 cells were scratched with a sharp implement and, as above, this was done in the presence of FDx, so that cells incurring and resealing a plasma membrane disruption could subsequently be identified microscopically by virtue of their cytosolic staining with this marker. In monolayers pre-treated with the actin depolymerizing agent, cytochalasin B, actin depolymerization was evident by TRITC-phalloidin staining as a loss of cortical actin and stress fiber staining (Fig. 4a,b) and, by phase-contrast imaging, as a failure of wound closure (Fig. 4c,d). However, the densities of FDx-positive cells along scratch sites made with or without cytochalasin B were qualitatively (Fig. 4e,f) and quantitatively (Fig. 4g) indistinguishable. In a positive control, monolayers were pre-treated with EGTA to reduce extracellular Ca2+ levels below the 0.1 mM threshold required for resealing (Steinhardt et al., 1994). FDx-positive cell density was reduced by approximately twofold in the absence of Ca2+ relative to control (normal Ca2+) values (Fig. 4g).
|
|
The inhibitory effects of phalloidin and jasplakinolide could be ascribed to nonspecific, drug-induced alterations unrelated to actin polymerization state. Owing to the high affinity constants of phalloidin and jasplakinolide for filamentous actin, reversing these treatments was not a feasible option (Ayscough, 2000). Therefore, for this purpose we used a biologically induced increase in filamentous actin, namely, that initiated in certain mammalian cells by monolayer scratch wounding (Bement et al., 1999; Martin and Lewis, 1992). Fig. 6a shows that RGM1 cells, too, lining a scratch wound are heavily stained in their cortices with TRITC-phalloidin. Many of the most heavily labeled (with TRITC-phalloidin) of these had suffered plasma membrane disruptions during the scratch, as indicated by their staining with FDx (Fig. 6b). Moreover, flow cytofluorometric analysis of TRITC -phalloidin staining intensity of cells from RGM1 cultures wounded with glass beads clearly revealed a quantitative increase, relative to unwounded controls, in staining with this specific probe for filamentous actin (data not shown). Therefore, we next made a first, actin polymerization-inducing round of disruptions by scratching RGM1 monolayers in presence of FDx, so that those cells in which disruptions were produced by this first scratch event could subsequently be identified by virtue of their labeling with this green fluorescence dye. Alternatively, we induced a first round of disruptions using glass beads. This first round of disruptions served to load membrane impermeant DNase1 into cells. Two hours later a second round of scratches was made on the slides, or a second bead treatment was performed, this time in the presence of Texas Red-labeled dextran (TRDx). In combined fluorescein and Texas Red fluorescence images, yellow cells are those that incurred and survived two plasma membrane disruptions, and hence retained both the green (first disruption) and red (second disruption) fluorescent tracers (Fig. 6c,d). Therefore the proportion of yellow/green will quantitatively reflect whether successful resealing of the second disruption was modulated by conditions present during the first disruption. In cytochalasin-treated (Fig. 6c) or DNase1-loaded (not shown) cultures, the proportion of yellow cells was apparently greatly enhanced, compared with controls (Fig. 6d). Fig. 6e,f presents a quantitative analysis of this difference: the presence of cytochalasin B, or DNase1, during the first disruption increased the yellow/green proportion by seven- to eightfold. Thus, the resealing inhibitory effect of a biologically induced increase in filamentous cortical actin was readily reversible with two different depolymerizing agents.
|
Our results suggest that a second key function of Ca2+ during resealing is to regulate actin assembly state. First, we confirmed the prediction that resealing can be facilitated by experimental manipulations favoring filamentous actin dissolution (e.g. by loading the actin-depolymerizing enzyme, DNase1, into RGM1 cells). Our failure using RGM1 cells to confirm the enhancing effect of cytochalasin treatment on resealing, previously reported in fibroblasts (Togo et al., 2000), may reflect differences between these two cell types and/or the methods used for wounding and the evaluation of resealing. It is, in any case, worth noting that cytochalasin, which binds to barbed ends of actin filaments, is generally considered to be a less effective depolymerizing agent than other drugs, such as latruculin or DNase1, which display a high affinity for monomeric actin (Wakatsuki et al., 2001). Second, we confirmed the previously untested and crucial prediction that resealing can be inhibited by manipulations favoring filamentous actin stabilization (e.g. using two different actin stabilizing agents, phalloidin and jasplakinolide). Third, we confirmed the prediction that a resealing deficit, mediated by a biologically induced increase in filamentous actin, can be reversed by cell treatment with filamentous actin destabilizing agents (e.g. cytochalasin and DNase 1). Fourth, we showed that filamentous actin content is quantitatively reduced in a Ca2+-dependent fashion shortly (15 seconds) after wounding. Our results parallel those of many studies that have similarly provided structural and functional evidence that actin filament disassembly is required for various other exocytotic responses (Muallem et al., 1995; Sontag et al., 1988; Trifaro et al., 2000). They can moreover be understood in terms of the well-documented capacity, at both the cell and molecular level (Taylor and Fechheimer, 1982; Yin et al., 1981), of Ca2+ to stimulate actin depolymerization.
The requirement for actin depolymerization may be a particularly strong in the case of resealing-based homotypic fusion and exocytosis. Resealing is a crisis response to an event that does not occur with predictable timing or location. It uses vesicles, lysosomes (Andrews, 2000; Reddy et al., 2001) and possibly endosomes (Bi et al., 1995; Miyake and McNeil, 1995), which are not pre-docked with their appropriate fusion partners; yet fusion of these partners must occur rapidly (seconds). Active transport (Bi et al., 1997) of endosome/lysosomes, most of which are >100 nm in diameter, into cell cortex bordering on a disruption, may function to rapidly concentrate internal membrane locally when and where needed (Miyake and McNeil, 1995). However, direct physical contact of these vesicles with one another is then required for the ensuing fusion events that form an enlarged (many >1 µm diameter) patch vesicle population (Miyake and McNeil, 1995; Terasaki et al., 1997). Similarly, exocytotic fusion of these large patch vesicles cannot occur until they make direct physical contact with the plasma membrane. The mean pore diameter of cell cytosol is estimated to be 30-40 nm, and the filamentous actin component of the cytoskeleton contributing to this size limit on particle diffusion is strikingly enriched in the cortex of most cells (Luby-Phelps, 2000). Based on these theoretical considerations and the experimental data presented here, we propose a new element of the patch hypothesis for explaining how mammalian cells possessing an actin-rich cortex reseal: Ca2+-regulated disassembly of the actin-based cortical cytoskeletal barrier is an essential prelude to vesicle docking and fusion beneath the disruption site (Fig. 7).
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrews, N. W. (2000). Regulated secretion of conventional lysosomes. Trends Cell Biol. 10, 316-321.[Medline]
Ayscough, K. R. (2000). Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton. Curr. Biol. 10, 1587-1590.[Medline]
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.
Bubb, M. R., Senderowicz, A. M., Sausville, E. A., Duncan, K. L. and Korn, E. D. (1994). Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 269, 14869-14871.
Burgoyne, R. D. and Morgan, A. (1998). Calcium sensors in regulated exocytosis. Cell Calcium 24, 367-376.[Medline]
Cano, M. L., Cassimeris, L., Fechheimer, M. and Zigmond, S. H. (1992). Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes. J. Cell Biol. 116, 1123-1134.[Abstract]
Clarke, M. S. F. and McNeil, P. L. (1992). Syringe loading introduces macromolecules into living mammalian cell cytosol. J. Cell Sci. 102, 535-541.
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.
Gimlich, R. L. and Braun, J. (1985). Improved fluorescent compounds for tracing cell lineage. Dev. Biol. 109, 509-514.[Medline]
Heilbrunn, L. V. (1930b). The surface precipitation reaction of living cells. Proc. Am. Philos. Soc. LXIX, 295-301.
Hitchcock, S. E., Carisson, L. and Lindberg, U. (1976). Depolymerization of F-actin by deoxyribonuclease I. Cell 7, 531-542.[Medline]
Ito, S. and Karnovsky, M. J. (1968). Formaldehyde-glutaraldehyde fixative containing trinitro compounds (Abstr.). J. Cell Biol. 39, 168a.
Kobayashi, I., Kawano, S., Tsuji, S., Matsui, H., Nakama, A., Sawaoka, H., Masuda, E., Takei, Y., Nagano, K., Fusamoto, H. et al. (1996). RGM1, a cell line derived from normal gastric mucosa of rat. In Vitro Cell Dev. Biol. Anim 32, 259-261.[Medline]
Lengsfeld, A. M., Low, I., Wieland, T., Dancker, P. and Hasselbach, W. (1974). Interaction of phalloidin with actin. Proc. Natl. Acad. Sci. USA 71, 2803-2807.[Abstract]
Luby-Phelps, K. (2000). Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. 192, 189-221.[Medline]
Martin, P. and Lewis, J. (1992). Actin cables and epidermal movement in embryonic wound healing. Nature 360, 179-183.[Medline]
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. and Ito, S. (1989). Gastrointestinal cell plasma membrane wounding and resealing in vivo. Gastroenterology 96, 1238-1248.[Medline]
McNeil, P. L. and Ito, S. (1990). Molecular traffic through plasma membrane disruptions of cells in vivo. J. Cell. Sci. 96, 549-556.[Abstract]
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 Steinhardt, R. A. (1997). Loss, restoration and maintenance of plasma membrane integrity. J. Cell. Biol. 137, 1-4.
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. and Warder, E. (1987). Glass beads load macromolecules into living cells. J. Cell Sci. 88, 669-678.[Abstract]
McNeil, P. L., Murphy, R. F., Lanni, F. and Taylor, D. L. (1984). A method for incorporating macromolecules into adherent cells. J. Cell Biol. 98, 1556-1564.[Abstract]
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. and McNeil, P. L. (1998). A little shell to live in: evidence that the fertilization envelope can prevent mechanically induced damage of the developing sea urchin embryo. Biol. Bull. 195, 214-215.
Muallem, S., Kwiatkowska, K., Xu, X. and Yin, H. L. (1995). Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J. Cell Biol. 128, 589-598.[Abstract]
Mulroy, M. J., Henry, W. R. and McNeil, P. L. (1998). Noise-induced transient microlesions in the cell membranes of auditory hair cells. Hear. Res. 115, 93-100.[Medline]
Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell 106, 157-169.[Medline]
Sontag, J. M., Aunis, D. and Bader, M. F. (1988). Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells. Eur. J. Cell Biol. 46, 316-326.[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]
Swanson, J. A. and McNeil, P. L. (1987). Nuclear reassembly excludes large macromolecules. Science 238, 548-550.[Medline]
Taylor, D. L. and Fechheimer, M. (1982). Cytoplasmic structure and contractility: the solationcontraction coupling hypothesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 299, 185-197.[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.
Trifaro, J., Rose, S. D., Lejen, T. and Elzagallaai, A. (2000). Two pathways control chromaffin cell cortical F-actin dynamics during exocytosis. Biochimie 82, 339-352.[Medline]
Wakatsuki, T., Schwab, B., Thompson, N. C. and Elson, E. L. (2001). Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114, 1025-1036.
Yin, H. L., Albrecht, J. H. and Fattoum, A. (1981). Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues. J. Cell Biol. 91, 901-906.
Yu, Q. C. and McNeil, P. L. (1992). Transient disruptions of aortic endothelial cell plasma membranes. Am. J. Pathol. 141, 1349-1360.[Abstract]