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J. Biol. Chem., Vol. 280, Issue 2, 1652-1660, January 14, 2005

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Molecular Regulation of Membrane Resealing in 3T3 Fibroblasts^*

Sheldon S. Shen, Ward C. Tucker¶||, Edwin R. Chapman¶**, and Richard A. Steinhardt

From the Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011, the ^¶Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, and the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Received for publication, September 3, 2004 , and in revised form, November 8, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Membrane resealing in mammalian cells after injury depends on Ca²⁺-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca²⁺ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca²⁺-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Puncture of the plasma membrane invokes a rapid resealing response that depends on a source of intracellular membrane and delivery of this membrane to the plasma membrane by exocytosis to ensure cell survival (1). Repair signaling pathways are initiated by the sudden rise in cytosolic Ca²⁺ due to influx through the puncture site (2). When cells are wounded twice, the second wound generally reseals more quickly. Two different signaling responses, facilitated and potentiated, have been described and depend on the site of the second wound in relation to the first. The facilitated response is characterized by a faster resealing response to a second wound at the same initial wound site and depends on the generation of new vesicles in a protein kinase C and Golgi-dependent manner (3). Cells also have a globally increased exocytotic response to a second wound that increases the rate of repair to a second wound at a different site from the initial wound. This potentiated response does not depend on the generation of new vesicles (4). The global increase in exocytosis and repair is cAMP- and protein kinase A-dependent in the first few minutes, becomes dependent on protein synthesis within a few hours, and with CREB (cAMP-response element-binding protein) activation can last for 24 h or more (4).

The molecular mechanism mediating Ca²⁺-dependent exocytosis has been extensively investigated and appears to require the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)¹ (5–7). The fusion event requires coupling of the plasma membrane-localized heterodimer target SNAREs, syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle-localized SNARE, synaptobrevin 2 (syb2) (8, 9). However, fusion of purified SNAREs in an in vitro vesicle-vesicle fusion assay occurs with slow (minutes) kinetics and is Ca²⁺-independent (10). The addition of soluble synaptotagmin I (syt I) markedly enhanced both the rate and extent of vesicle fusion in a Ca²⁺-dependent manner (11). Thus, synaptotagmin and SNAREs probably comprise the minimal proteins required for Ca²⁺-mediated exocytosis.

syt is a large family of at least 15 identified isoforms that are expressed at different levels in a variety of tissues and implicated in numerous types of vesicular traffic (7, 12). All syts share the structural organization of tandem C2 domains (13). The C2 domain is a motif of 130 residues, initially identified in protein kinase C and later in more than 100 proteins (14, 15). The tandem C2 domains of syt are separated into the membrane proximal C2 domain called C2A, and the membrane distal C2 domain is called C2B. Similar to its function in some protein kinase C isoforms, the C2AB domain of syt I (C2AB1) binds Ca²⁺ ions and phospholipids and is thought to act as the Ca²⁺ sensor for regulating secretion (11, 16). Although C2AB domains from nearly half of the identified isoforms of syts do not bind Ca²⁺/phospholipids, nearly all do bind the target SNARE heterodimer (17); thus, syts may trigger Ca²⁺-dependent exocytosis either as the Ca²⁺ sensor or in collaboration with an additional Ca²⁺ sensor.

Participation of SNARE proteins in membrane repair has been suggested by the use of neurotoxins, recombinant protein fragments, and inhibitory antibodies (2, 3, 18–20). Membrane resealing of 3T3 fibroblast cells is inhibited by the action of clostridial neurotoxins via proteolytic cleavage of SNAREs. Cleavage of SNAP-25 by botulinum neurotoxin A (21) or syb2 by either botulinum neurotoxin B or tetanus toxin (22) inhibited 3T3 membrane resealing after micropuncture of the plasma membrane (2, 3). Recombinant syt I and VII C2 domains and also an antibody against syt VII C2A (C2A7) domain have been tested on plasma membrane repair in NRK fibroblasts. Recombinant fragments of C2A7, as well as the antibody against this domain blocked plasma membrane repair, whereas recombinant C2A1 had no effect (20). Because these C2 domains also had differential effects on Ca²⁺-dependent exocytosis of secretory lysosomes (23), it has been proposed that lysosomal fusion is the sole source of membrane for membrane resealing (20, 24). An important experimental support for this proposal was that C2B7 did not block lysosome exocytosis (23) or membrane resealing (24). These results are surprising since the two C2 domains share many interactions with other molecules (16). An exclusive use of lysosomes in membrane repair would also appear to conflict with earlier evidence of the sensitivity of resealing and exocytosis to botulinum neurotoxin B and tetanus toxin and the sensitivity of the facilitated response to brefeldin-A (3). These sensitivities are also unexpected if enlargosomes are exclusively used in membrane repair (25).

Here we have extended the use of recombinant protein domains in tests of membrane resealing of 3T3 fibroblasts with the goal of better understanding which intracellular compartments are used in repair and to find which functional properties of the C2 domains are essential for successful resealing. We focused mainly on tests of competitive inhibition using recombinant C2 domains of syts I and VII, mutant variants of these domains that are unable to bind Ca²⁺, and the cytoplasmic domain of syb2 (cd-syb2). Our results support the view that the pool of membrane vesicles for membrane repair is a heterogeneous population rather than a specific sub-type, that the Ca²⁺ binding properties of syt C2 domains are essential for inhibition of membrane resealing, and that different vesicle pools have different sensitivities to exogenous syt C2 domains.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Preparation and Reagents—Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 8% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 50 µg/ml normocin (Invitrogen). Cells for experiments were plated on glass coverslip-inserted plastic dishes (35 x 10 mm) and grown for 1–3 days before use. Fura-2 acetoxymethyl ester (fura-2 AM) was purchased from Molecular Probes (Eugene, OR).

Preparation of Recombinant Fragments—cDNA encoding rat syt I (26) and mouse syt VII (27) were kindly provided by T. C. Sudhof (University of Texas Southwestern Medical Institute, Dallas, TX) and M. Fukuda (Institute of Physical and Chemical Research, Saitama, Japan), respectively. The C2B (248–421) and C2A-C2B (96–421) domains of syt I and the C2A domain (residues 134–262) of syt VII were prepared as previously described (28–31). The C2B domain (residues 243–403) of syt VII was subcloned into pGEX 2T-1 (Amersham Biosciences) as previously described (32). Ca²⁺ binding mutants of syt I C2AB (D230N,D232N,D363N,D365N), syt VII C2A (D225N,D227N), and syt VII C2B (D357N,D359N) domains and a control mutant of syt VII C2B (K320N,K321N) domain were generated by PCR. All recombinant syt constructs were confirmed by DNA sequencing and expressed as glutathione S-transferase fusion proteins. Proteins were purified by affinity chromatography with glutathione-Sepharose 4B (Amersham Biosciences) and washed with nucleases and high salt buffer to remove bacterial contaminants as described in Wu et al (33). Beads were washed twice more in buffer A (12.4 mM Hepes-NaOH (pH 7.4), 138 mM NaCl, and 2.4 mM KCl) before generating soluble syt fragments by thrombin cleavage (32). The cytoplasmic domain (residues 1–94) of synaptobrevin 2 (9) was expressed as His₆ fusion proteins and purified by nickel-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA). The proteins were step-eluted with 400 mM imidazole and dialyzed against buffer A. All proteins were stored on ice and used within 7–10 days.

Assay of Membrane Resealing—Membrane resealing was monitored by measuring the fluorescence of the calcium-sensitive dye fura-2 as described previously (2). Fura-2 was introduced into the cells by AM-ester loading of 1 µM fura-2-AM at 25 °C for 1 h and washed with Ringer's solution containing 1.8 mM Ca²⁺_. During experiments the cells were maintained in 1.8 mM Ca²⁺ Ringer's solution. Ca²⁺-free Ringer's solution contained 138 mM NaCl, 2.7 mM KCl, 1.06 mM MgCl₂, 5.6 mM D-glucose, and 12.4 mM HEPES (pH 7.25). A stock solution of 100 mM CaCl₂ was used to adjust the concentration of Ca²⁺ to 1.8 mM in all experiments. Before the wounding experiments, the purified recombinant fragments, which ranged in concentration from 44 to 160 µM in buffer A, were diluted into the Rodent Ringer's to a final concentration of 10, 20, or 30 µM, and Ca²⁺ was adjusted to 1.8 mM. 10 µM recombinant syt C2 domains maximally inhibited exocytosis in PC12 cells.² Similar concentrations have previously been tested on secretory lysosome exocytosis (23) and membrane resealing after glass bead wounding (20) in NRK fibroblasts. Fura-2-loaded cells were wounded with a 0.5-µm-tipped diameter solid glass needle pulled with a PE-2 vertical puller (Narishige, Tokyo) and manipulated using an Eppendorf 5242 microinjector and 5170 manipulator (Brinkmann Instruments, Westbury, NY) mounted on a Zeiss IM-35 inverted microscope. The time setting for wounding was 0.3 s. Resealing was monitored by photometric measurements of fura-2 fluorescence at 500–520 nm. Wounding was marked by a persistent decrease in the Ca²⁺-insensitive 357-nm excited fluorescent intensity (as an indicator of dye loss) together with an increase in the ratio of fluorescent intensity excited by 357/385-nm light (as an indicator of increased intracellular Ca²⁺ activity). Membrane resealing was marked by a cessation of dye loss. The duration between wounding and cessation of dye loss is the resealing time, and the resealing rate is defined as the inverse of the resealing time in seconds. Thus, a faster resealing time is reflected as a greater resealing rate. For cells that failed to reseal after the second wounding, the rate was defined as zero.

In these experiments the recombinant proteins were added to the bath before wounding. Because diffusion of these fragments into the cell after micropuncture occurs in tens of milliseconds and membrane resealing requires tens of seconds, the addition of fragments in the bath before wounding should not significantly alter their effects on the kinetics of membrane resealing events. Exocytosis in cracked PC12 cells was inhibited regardless of the addition of recombinant fragments before, during, or after the addition of Ca²⁺ to stimulate fusion (31). All experiments were performed at 23–25 °C.

Data Analysis—All data are indicated as the means ± S.E. Statistics were calculated by Student's t test for paired data and Mann-Whitney (nonparametric) test for unpaired data using InStat 2.00 (GraphPad Software, San Diego, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Swiss 3T3 fibroblasts rapidly reseal after puncture of the plasma membrane. To analyze membrane resealing, fura-2-loaded cells were wounded as previously described (2). Wounding (arrows in Fig. 1) by a 0.3-s jab of a microneedle is indicated by a sharp rise in the Ca²⁺-sensitive fura-2 ratio (lower trace) and loss of fura-2 dye, which results in a decrease in the fluorescent intensity of fura-2 (upper trace) excited at a Ca²⁺-insensitive wavelength (357 nm). When the cell resealed, the decrease in fluorescence intensity stopped (bars in Fig. 1). In the control, 1.8 mM Ca²⁺ Ringer's, the average resealing time for an initial wound was 15.12 ± 0.88 (n = 51) s, or an average resealing rate of 0.077 ± 0.004 s^-1 (Table I). Swiss 3T3 fibroblasts are able to withstand multiple punctures and usually reseal even faster after a second wounding. The faster resealing to a second wounding at the same site is called a facilitated response (3). In the example shown in Fig. 1A the resealing time is the 27 s between the initial wound (first arrow) and the cessation of fura-2 dye loss (first bar). The second wounding (arrow) required only 16 s to reseal (bar). Each 0.3-s poke stimulated a rise in intracellular Ca²⁺, which is seen as a rise in ratio of the 520 emission to excitation at 357 and 385 nm. When cells were wounded at the same site, the average initial resealing rate of 0.08 ± 0.006 (n = 29) was significantly (p < 0.0001, Student's t test) increased to a resealing rate of 0.132 ± 0.009 for the second wound. When plotted as the rate of membrane resealing for the second wound on the y axis to the rate of membrane resealing for the first wound on the x axis, most of the data fall above the diagonal (Fig. 1B, ). A second wounding at a different site also shows a faster resealing rate but uses a different pathway and is called a potentiated response (4). When cells were wounded at different sites, the average initial resealing rate of 0.074 ± 0.007 (n = 22) was observed to significantly (p < 0.003) increase to 0.118 ± 0.013 for the second wound. When plotted as the rate of the second membrane resealing to the rate of the first membrane resealing, most of the data fall above the diagonal (Fig. 1B, •), reflecting the faster resealing response to the second wound. The average ratios of the second with respect to the first resealing rates of control cells for facilitation and potentiation were 1.81 ± 0.16 (n = 29) and 1.9 ± 0.14 (n = 22), respectively.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Facilitated and potentiated membrane resealing responses in untreated control NIH 3T3 cells. A, a typical cell was micropunctured by a 0.3-s jab of a microneedle at the first arrow. The membrane wound caused a loss in fura-2 dye, detected as a drop in intensity at the Ca2+-insensitive = 357 nm (top trace), and a rise in intracellular Ca2+ activity, detected as an increase in the emission ratio of fura-2 dye excited at 357 and 385 nm (bottom trace). Resealing of the membrane causes a cessation of dye loss (bar), and the rate of membrane resealing is the inverse of the duration of dye loss. In this example the duration of dye loss after the initial wound was 27 s or an initial rate of resealing of 0.037 s-1. The cell was wounded a second time (second arrow) at the same site, which required only 16 s to reseal. The faster resealing to a second wounding at the same or different site is called a facilitated or potentiated response, respectively. B, plot of the rate of membrane reseal for the second wound on the y axis to the rate of membrane reseal for the first wound on the x axis for the same () or different (•) sites.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Ca2+ binding-dependent inhibition of the facilitated response by syt I C2AB. Cells were micropunctured in the presence of 30 µM C2AB1 (), non-Ca2+ binding C2AB1 mutant (•), or C2B1 (). The presence of C2AB1 blocked facilitation, but C2B1 and Ca2+ binding mutant of C2AB1 had no effect.

The role of synaptotagmins in membrane fusion depends on their function as Ca²⁺ binding modules mediating Ca²⁺-dependent interactions with other fusion proteins (16). Disruption of the Ca²⁺ binding properties of syt I impairs synaptic exocytosis (39–43). The effect of C2AB1 on the facilitated membrane resealing response was dependent on its Ca²⁺ binding properties. The syt I Ca²⁺ ligand mutant C2AB (D230N, D232N,D363N,D365N) (30) did not inhibit the facilitated membrane resealing response. The second resealing rates of 3T3 cells treated with 30 µM syt I C2AB (D230N,D232N,D363N,D365N) was significantly (p < 0.0002) increased from the first resealing rates (Fig. 2, •) and were 0.168 ± 0.018 and 0.097 ± 0.022 (n = 28), respectively. These resealing rates were similar to those of control cells.

Synaptotagmin VII C2 domains have been previously used to conclude that lysosomes are exclusively used in membrane repair (24). We repeated and extended tests of syt VII C2 domains and reached different conclusions. Recombinant C2AB7 fragment had no measurable effect on membrane resealing to an initial wounding but did block the facilitated response. The initial resealing rate of 3T3 cells in 30 µM C2AB7 was 0.078 ± 0.004 (n = 32), which is similar to the untreated control cell resealing rate (Table I). But the rate of membrane resealing in the presence of C2AB7 after a second wounding at the same site was significantly (p < 0.03) decreased such that the second resealing rate fell to 0.06 ± 0.009. Furthermore, 7 of 32 cells failed to reseal after the second wounding.

We then examined separately the effects of recombinant fragments of the two Ca²⁺ binding domains. We observed that the addition of 30 µM C2A7 to the medium did not inhibit the rate of membrane resealing to an initial wound (0.077 ± 0.004, n = 59), which was similar (p = 0.78) to the rate of resealing in untreated control cells (Table I). This observation was in contrast to the partial inhibition of membrane resealing after glass bead-wounding in NRK fibroblasts (20). However, membrane resealing to a second wound at the same site was significantly impaired in 3T3 fibroblasts. Half of the cells were unable to reseal to a second wound at the same site (23 of 46 cells), and the resealing rate of cells that did reseal was significantly (p < 0.0001) decreased to 0.025 ± 0.007 (n = 23) from the initial membrane resealing rate of 0.073 ± 0.004 (n = 46). In the example shown in Fig. 3A, the membrane resealed 17 s after cell wounding, but a significantly longer time of 51 s was necessary for membrane resealing after a second wounding. The reduction of the rate of membrane resealing is seen as data points below the diagonal (Fig. 3B, •) and a second/first resealing ratio of 0.036 ± 0.09 (n = 46). The presence of C2A7 did not block membrane resealing to a second wound at a different site (Fig. 3B, ). 13 of 13 cells resealed after a second wound, but potentiation of the response was not observed. The rates of membrane sealing to the first and second wounds were 0.09 ± 0.012 and 0.092 ± 0.014 (n = 13), respectively. Plots of the second and first resealing rates at different sites of C2A7-treated cells were scattered on both sides of the diagonal (Fig. 3B, ) and a second/first resealing ratio of 1.28 ± 0.23 (n = 13). The effect of C2A7 was dependent on its Ca²⁺ binding function since a putative non Ca²⁺ binding mutant, syt VII C2A (D225N,D227N), did not block the facilitated response (Fig. 3B, ), and the resealing rate (0.134 ± 0.016, n = 28) was significantly (p < 0.001) increased in response to a second wounding at the same site when compared with the initial resealing rate of 0.087 ± 0.007 (n = 28).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Ca2+ binding dependent inhibition of the facilitated and potentiated responses by syt VII C2A. A, the resealing record of a 3T3 cell in 30 µM C2A7. Membrane resealing after the initial wound was similar to untreated control cells, and in this case the membrane resealed 17 s after the initial wounding. Half of the cells wounded a second time at the same site failed to reseal, and the rate was defined as zero. In most cases that did reseal, the resealing time was significantly longer, and in this case the membrane resealed 51 s after the second wounding. B, plot of the second and first resealing rates at the same sites (•) for cells treated with C2A7. All cells wounded a second time at a different site () did reseal, but 5 of 13 cells had a slower rate of membrane resealing than that of the initial wound. Cells treated with the Ca2+ binding mutant of C2A7 () showed a facilitated response to a second wounding.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Ca2+ binding dependent inhibition of initial resealing, facilitated, and potentiated responses by syt VII C2B. A, the rate of membrane resealing to first and second woundings of 3T3 cells in 30 µM C2B7 at same () and different (•) sites were significantly inhibited. Many cells failed to reseal after an initial micropuncture. Of those that did reseal the rate of membrane resealing was significantly increased. More than half of these cells failed to reseal after a second wounding at the same or different sites. B, the inhibitory effect of the recombinant C2B7 peptide was dependent on Ca2+ binding. Cells treated with the deficient Ca2+ binding mutant fragment () resealed normally after the initial wounding, and the response to a second wounding at the same site was facilitated. A control mutant fragment (•) still inhibited membrane repair after the initial micropuncture, and 8 of 24 cells that resealed after the initial wounding failed to reseal after a second wounding at the same site.

Other Ca²⁺-dependent or -independent effects of syt VII C2 domains are suggested by comparing syt VII C2B-(K320A,K321A) (Fig. 4B, •) with C2B7 (Fig. 4A, ) or syt I C2AB Ca²⁺ mutant (Fig. 2, •) with either syt VII C2A Ca²⁺ mutant (Fig. 3B, ) or syt VII C2B Ca²⁺ mutant (Fig. 4B, ). The inhibition of facilitation by syt VII C2B-(K320A,K321A) appears to be less than that by C2B7, although both fragments would have intact Ca²⁺ binding. Similarly the syt VII C2 Ca²⁺ mutants appear to still slightly inhibit facilitation when compared with syt I C2AB Ca²⁺ mutant despite the loss of Ca²⁺ binding by the fragments. However, Mann-Whitney statistical analysis of these data sets did not show significance in their differences.

Membrane vesicle fusion in neurons is dependent on the formation of the SNARE complex (8), which can be blocked by the cytoplasmic domain of synaptobrevin 2 (cd-syb2) (9, 11). Syb2 is ubiquitously expressed (45) and can be specifically cleaved by botulinum neurotoxin B or tetanus toxin, both of which inhibit resealing in a site specific manner (2, 3); thus, we investigated the effect of cd-syb2 on membrane resealing. Cd-syb2 at 10 µM (•) and 20 µM () were tested with no statistical significant differences between the two concentrations; thus, the data were combined (Fig. 5A). Cd-syb2 significantly (p = 0.0001) inhibited membrane resealing after an initial wound (Table I). The membrane resealing rate in cd-syb2 was 0.043 ± 0.005 (n = 46), which was significantly reduced from that of untreated control cells. The membrane resealing rate after a second wound at the same site was also decreased when compared with that of untreated control cells, but facilitation was often still observed (25 of 39 recordings are above the diagonal in Fig. 5A). As seen in the example in Fig. 5B after an initial wounding (arrow), the membrane resealed after 54 s (bar), but a second wounding (arrow) resealed in only 14 s (bar). The membrane resealing rate was significantly (p < 0.02) increased after the second wounding at the same site in cd-syb2-treated 3T3 cells to 0.062 ± 0.008 (n = 44) or a second/first resealing ratio of 1.874 ± 0.309.

View larger version (14K): [in this window] [in a new window]   FIG. 5. The rate of membrane resealing, but not the facilitated response, was inhibited by cd-syb2. A, the rate of membrane resealing to first and second woundings of 3T3 cells in 10 (•)or20() µM cytoplasmic domain of synaptobrevin 2 (cd-syb2) at the same sites was significantly reduced. However, a facilitated response was still observed in these cells. B, an example of membrane resealing of a 3T3 cell in cd-syb2. After the initial wounding (first arrow) the membrane resealed (first bar) after 54 s. A second wounding (second arrow) resealed (second bar) in 14 s.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

syts have been proposed to act as the Ca²⁺ sensor for a variety of Ca²⁺-dependent exocytotic events including synaptic transmission (46, 50), PC12 secretion (29, 31, 49, 51), and endocrine secretion (47, 48). Our results suggest syts may regulate Ca²⁺-dependent membrane resealing after micropuncture woundings. The inhibition of membrane resealing by C2AB1 (Fig. 2), C2A7 (Fig. 3B), and C2B7 (Fig. 4B) were all dependent on the Ca²⁺ binding property of the recombinant fragment. In all cases the non-Ca²⁺ binding mutants of inhibitory C2 domains no longer blocked membrane resealing. The amino acid sequences of the C2AB domains of syt I and VII show only a 44% identity and 65% similarity, but both tandem C2 domains conserve the acidic residues (17, 52) required in coordinating Ca²⁺ (15). Neutralization of these conserved acidic residues disrupts the putative Ca²⁺ binding activity of the C2 domains and inhibition of membrane resealing, which suggests the syt fragments act either directly or indirectly as the Ca²⁺ sensors for membrane resealing. Inhibition by syt VII C2 fragments may not implicate particular syt isoforms in normal function. Recombinant fragments of syt VII C2 domains are potent inhibitors of PC12 secretion (31, 53), yet PC12 cells express syt VII at very low levels (31). A recent gene silencing approach suggested syt IX, not syt VII, is indispensable for PC12 exocytosis (51). Other studies suggest that syt VII C2 domains are non-selective due to binding with a variety of other molecules (31, 49, 52), including oligomerization with other subclasses of syts (29, 54, 55) and competition with native syts for binding to effectors, such as SNARE proteins and phosphatidylinositol 4,5-bisphosphate (31). These other binding properties of syt VII C2 domains may also be important in membrane repair.

The source of intracellular compartments for membrane repair is uncertain. The lysosome compartment has been suggested as the vesicular source of membrane for resealing (24) due to a correlation of C2A7 fragment inhibition of lysosome exocytosis (23) and membrane resealing of glass bead-wounded NRK fibroblasts (20) and failure of both C2B7 and C2A1 fragments to inhibit either lysosome exocytosis (23) or membrane resealing (20). We have found that the C2A7 fragment does not inhibit membrane repair after the initial wound (Table I) and the C2B7 fragment is an even more potent inhibitor than C2A7 fragment of both membrane resealing after the first wounding (Table I) and the facilitated or potentiated response after the second wounding. We do not know why C2B7 inhibited membrane resealing in 3T3 fibroblasts but not in NRK fibroblasts (20). syt VII has been shown to partially co-localize to lysosomes in both 3T3 (37) and NRK (23) fibroblasts. The C2B domain engages a wider array of effector molecules than the C2A domain (16, 44). At least in 3T3 fibroblasts, C2B7 cannot be used as supporting evidence that lysosomes are the exclusive compartment for resealing. This conclusion is corroborated by the recent report that syt VII knockout mice exhibit enhanced lysosome fusion (56) but show a somewhat reduced membrane repair response (57). In addition, vacuolin-1, which can completely block Ca²⁺-dependent exocytosis of lysosomes, did not affect the rate of membrane resealing (58). Thus, lysosomes do not appear to be essential for the process of membrane resealing, and inhibition by syt VII C2 fragments does not now appear to be consistent with a major role for lysosomes in membrane repair.

From studies of Ca²⁺-triggered exocytosis in chromaffin cells, intracellular vesicles have been proposed to be subdivided into separate pools of "rapidly releasable," "slowly releasable," and "unprimed" vesicles (59). A proposed distinguishing difference between the pools is the formation of the SNARE complex between vesicle-localized SNARE and target SNARE proteins and interacting partners such as synaptotagmins (59). The role of syts in the fusion process is unclear, but our studies suggest that vesicle pools used during membrane resealing may also be similarly subdivided. The differential effects of C2A7 and C2B7 suggest different effectors for syts in membrane fusion of releasable and unprimed vesicles. C2A7 had no effect on the initial rate of membrane resealing (Table I) but significantly prevented both facilitation and potentiation (Fig. 3B). An important difference in C2A7 inhibition of facilitation and potentiation was that 65% of the second woundings at the same site either failed to reseal or had a second resealing rate less than 0.01 s^-1, whereas all the second woundings at a different site resealed. Because facilitation, but not potentiation, requires recruitment of new vesicles (3, 4), these results suggest that C2A7 did not block membrane resealing by releasable vesicles but the recruitment of newly synthesized or unprimed vesicles. Facilitation also depends on protein kinase C activity (3), which enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool (60). In contrast to C2A7, C2B7 significantly inhibited the initial rate of membrane resealing (Table I). The rate of membrane resealing for a second wounding at either the same or different site was further reduced. More than 60% of the second wounding at same or different sites had a second resealing rate less than 0.01 s^-1. A possible explanation is that C2B7 was able to disrupt the fusion of vesicles in releasable pools, with the plasma membrane as well as the recruitment of unprimed vesicles. These observations suggest that the C2B fragment engages a wider array of effectors than the C2A fragment, which is similar to a more crucial role for C2B in synaptic transmission (44). A difference in vesicle pools seen with syt VII C2 domain inhibitions of first and second wounds can also be detected by syt I C2 domains. We found that CAB1 had no effect on the rate of resealing to an initial wound but significantly inhibited both the facilitated and potentiated responses to a second wound. The different sensitivities of first and second wounds again suggest that more than one vesicle pool is used in membrane repair.

The dependence of the initial wound resealing upon vesicles of the releasable pool is further supported by our observations with the recombinant cd-syb2, which would be expected to disrupt the coupling between vesicle-localized SNARE and target SNARE (9). The addition of cd-syb2 caused a dramatic delay in membrane repair such that the rate of membrane resealing after the initial wound was significantly decreased from that of control cells (Table I). Interestingly, membrane resealing after a second wounding at the same site was also significantly delayed when compared with that of control, but nonetheless, facilitation was observed. The ratio of second/first resealing rates at the same site for cd-syb2-treated cells was similar to that of control cells, 1.874 ± 0.309 (n = 43) and 1.814 ± 0.164 (n = 29), respectively. This implies that cd-syb2 interferes with the trans-SNARE complex formation and vesicle-membrane fusion required for membrane repair but does not prevent the recruitment of new vesicles required for the facilitated response.

Wounding the plasma membrane evokes a series of membrane trafficking events at a distinct time and place ranging from the immediate Ca²⁺-dependent exocytotic fusion of docked vesicles to the generation, recruitment, and transport of new ones. Our studies of resealing with recombinant syt and syb2 fragments reveal a high degree of similarity with the fusion mechanisms of neurosecretion and suggest that membrane repair will be an important adjunct to understanding the molecular events in vesicle trafficking. The use of different vesicle pools for initial, facilitated, and potentiated membrane resealing responses is suggested by the differential effects of the recombinant syt and syb2 fragments on these physiological processes. Membrane resealing can be useful for further characterization of the molecular properties of the different vesicle pools.

	FOOTNOTES

 
^* This study was supported in part by National Institutes of Health (NIH) Grant EY 13436 (to R. A. S.) and NIGMS, NIH Grant GM 56827 and NIMH NIH Grant MH 61876 (to E. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported by a Postdoctoral Individual National Research Service Award from the National Institutes of Health.

^** A Pew Scholar in the Biomedical Sciences.

To whom correspondence should be addressed. Tel.: 510-520-1073; Fax: 510-643-6791; E-mail: rsteinha{at}socrates.berkeley.edu.

¹ The abbreviations used are: SNARE, N-ethylmaleimide-sensitive factor attachment protein receptor; syb2, synaptobrevin 2; SNAP-25, synaptosome-associated protein of 25 kDa; syt, synaptotagmin; NRK, normal rat kidney.

² W. C. Tucker and E. R. Chapman, unpublished observations.

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