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ARTICLE |
Correspondence to Alan Fein: afein{at}neuron.uchc.edu
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100-fold lower, while the membrane potential has depolarized from –80 to –30 mV and is now sensitive to the Cl– concentration but not to that of Na+, K+, or H+. We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mechanical trauma produces wounds that are sealed in vivo during eccentric exercise in muscle or at regions of turbulent flow in arteries (McNeil and Khakee, 1992; Yu and McNeil, 1992), indicating that plasma membrane wounding and repair is likely to be an important process in pathophysiology.
88 years ago, experimental studies of wound repair were begun by Chambers, who showed remarkable healing of gross wounds in echinoderm eggs made by glass needles (Chambers, 1917). Heilbrunn showed that healing of such wounds requires extracellular calcium in the millimolar range (Heilbrunn, 1930). Recent experimental studies of wound healing have been conducted on several different systems using a variety of methods for producing disruptions. These studies have shown that the time required for sealing, the involvement of intracellular organelles, and the requirement for calcium vary according to cell type and the means by which the disruptions are made (for review see McNeil and Steinhardt, 2003).
Small wounds made by electroporation reseal without requirement for extracellular Ca2+. Electroporation-induced wounds in red blood cells seal within a few seconds and clearly do so without the presence of intracellular membranes (Chang and Reese, 1990), and in sea urchin eggs electroporation-induced wounds reseal in much less than a second (Hibino et al., 1993).
Other wounds, generally thought to be larger than the electroporation-induced wounds, seem to involve the exocytosis of intracellular organelles and require calcium. For example, wounding of cultured mammalian cells by microneedles has been extensively studied. Repair occurs in 10–30 s, requires extracellular calcium, and fusion of intracellular organelles with the plasma membrane (exocytosis) is required (Steinhardt et al., 1994), probably accounting for the calcium involvement. The identity of the organelles required for membrane resealing remains unresolved (Reddy et al., 2001; Cerny et al., 2004; Shen et al., 2005) as does the specific identity of the molecules involved in the fusion process (Detrait et al., 2000b; Reddy et al., 2001; Bansal et al., 2003; Shen et al., 2005).
As another example, when large diameter axons of various invertebrates are transected, the cut end reseals completely only after about an hour. The evidence suggests that vesicles formed by endocytosis migrate to the transection site where they form a tight plug, first without any membrane fusion, but eventually fusing together and with the axonal membrane to form the final seal (Spira et al., 1993; Eddleman et al., 2000). As with mammalian cells, progress has been made in identifying the molecules that are involved in axon repair (Detrait et al., 2000a).
Lastly, large plasma membrane disruptions, of the type originally made by Chambers and Heilbrunn, have been made in echinoderm eggs by ripping off large fragments of membrane (Terasaki et al., 1997) or by local application of the detergent Triton X-100 (McNeil et al., 2000). Under these conditions, repair requires calcium and seems to occur within a few seconds through a calcium-dependent fusion of yolk platelets to each other and to the plasma membrane (for review see McNeil and Steinhardt, 2003). The yolk platelets are likely to be derived from endocytic membranes during oocyte development, as they are in other species such as frog (Opresko et al., 1980).
One approach that has not been exploited with oocytes is to monitor the plasma membrane by electrophysiological methods while it is undergoing disruption and repair. The experimental difficulty is that mechanical methods of wounding can disrupt the seal around the electrode, causing electrical artifacts. This can be circumvented by using femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation–induced disruptions of the plasma membrane (Galbraith and Terasaki, 2003). We have undertaken a combined electrophysiological and optical study of membrane disruption and repair using oocytes from the starfish Asterina miniata. These cells are well suited for electrophysiology due to their uniform size, and to their spherical geometry and low cytoplasmic resistance, which makes them easy to voltage clamp (Hagiwara et al., 1975).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 min, the ovary clumps were removed and discarded. The oocytes were monitored by light microscopy until most of the follicle cells were detached (<5 min) at which point 0.15 ml 1 M CaCl2 was added to restore normal calcium. After settling, the oocytes were transferred to natural seawater and kept at 16–20°C. Fully grown immature oocytes (170–180 µm diameters) were used for experiments, which were done at room temperature (20°C). For some experiments, the vitelline envelope was removed by incubating oocytes for 30 min at room temperature in 100 µg/ml Type XIV protease (P5147; Sigma-Aldrich).
We used a Carl Zeiss MicroImaging, Inc. LSM 510 NLO system on an inverted Axiovert 200 microscope. It was equipped with a Mira Ti-Sapphire laser pumped by an 8W Verdi pump laser (Coherent). The microscope was focused at the equator of the oocyte (i.e., 80–90 µm from the coverslip) using a Carl Zeiss MicroImaging, Inc. 25x multi-immersion plan-neofluar (N.A. 0.8) objective lens. The laser spot was scanned once in a line of length 10 µm, intersecting perpendicularly with the cell surface. At full laser power tuned at 800 nm, the energy at the focal plane was 100 mW, and when scanned at the slowest speed (205 µs/pixel), this invariably caused a large wound such as shown in Fig. 1 A. To make small wounds as exemplified in Fig. 1 B, we started with a low, nonwounding intensity at a faster scan speed (e.g., 25 µs/pixel). The laser intensity was increased in increments of 1% until the intensity was reached at which a wound occurred (as signaled by a membrane depolarization at the time of the line scan); if no wound occurred at full laser power, we decreased the scan speed and repeated the process.
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For single electrode experiments, a modified version of a microinjection chamber (Terasaki and Jaffe, 1993) allowed manipulation of the electrode in the plane of focus using a Narishige SM-20 micromanipulator. For voltage clamp experiments, which required two electrodes with access from above, we used an open chamber with a round coverslip bottom (25 mm; Fisher Scientific). A nylon mesh with 149 µm opening (Small Parts) was tacked down to the coverslip with silicon grease. Oocytes in a small drop of seawater were drawn by surface tension into the mesh openings, after which the chamber was filled with seawater.
Oocytes were impaled with either one or two single-barreled microelectrodes, which were formed on a P-97, Flaming/Brown micropipette puller (Sutter Instrument Co.). Electrodes were filled with 2M KCl and had a resistance of around 10 M
. An Axoclamp-2B current and voltage clamp amplifier (Axon Instruments) was used for current clamp recording of membrane potential or for recording membrane current when performing a two electrode voltage clamp. During current clamp recordings, membrane resistance was monitored by passing 1 nA of hyperpolarizing inward current through the membrane via the intracellular pipette. The voltage drop across the pipette resistance, resulting from the 1 nA current pulse, was balanced out using the amplifier's bridge balance. PClamp 9 was used to record experimental data by computer (Axon Instruments). Data from pClamp was output to Origin 7.5 (Microcal Software) and Coreldraw 12.0 (Corel Corp.) for preparation of figures. Unless otherwise specified, results were repeated in at least 10 cells, and when analyzed statistically, results are given as the mean ± SD.
Natural seawater (NSW) was obtained from the supply department of the Marine Biological Laboratory. Artificial seawater (ASW) was, in mM, 483 NaCl, 10 KCl, 11 CaCl2, 29 MgSO4, 27 MgCl2, 2.4 NaHCO3, pH 8.0. For calcium-free seawater (CFSW), NaCl replaced CaCl2, and for choline ASW, choline Cl replaced NaCl. For pH 7.0 ASW, 10 mM Hepes replaced NaHCO3 and the pH was set at 7.0. For 100 mM K+ ASW, NaCl was reduced to 393 mM and KCl increased to 100 mM. For Na isethionate ASW, NaCl was replaced by Na isethionate. ASW was mixed with Na isethionate ASW to make solutions having chloride concentrations in between that of ASW and Na isethionate ASW. Omitting MgCl2 from Na isethionate ASW made ASW with 32 mM Cl–. For low calcium experiments, a stock solution of 500 mM EGTA, pH 8.0, was used to make 1 mM EGTA in CFSW. To transfer the oocytes to this solution, 5 µl of oocytes in ASW were added to 2 ml of EGTA-buffered CFSW in a test tube; after settling, oocytes were transferred to the observation chamber, which was filled with additional EGTA-buffered CFSW.
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RESULTS |
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). Electrically, this type of wound was characterized by a rapid membrane depolarization of 60 mV after which the membrane potential, over several seconds, approached a value of about –30 mV. Additionally, the membrane resistance following the wound dramatically decreased to a value below what could be measured reliably by the bridge amplifier. The membrane slope resistance with the membrane potential voltage clamped at –30 mV decreased 100-fold after wounding to the range of 100 k to 1 M (unpublished data).
We also observed a transient response as shown for the cell in Fig. 1 B. The wound was characterized by a brief membrane depolarization of 60 mV, and the membrane potential and resistance after the wound were approximately the same as before wounding. Furthermore, the photomicrograph in Fig. 1 B shows no obvious residual membrane damage in the region of the cell where the line scan of threshold light intensity occurred. At this magnification, it would not have been apparent that a wound had occurred, without electrical recording of membrane potential (however see Figs. 4 and 7). By starting with a low, nonwounding laser intensity then increasing the laser power in 1% increments, we were able to produce the transient response of Fig. 1 B. However, even with this threshold protocol, many oocytes only gave the response shown in Fig. 1 A, indicating that the energy range for causing the transient response is relatively narrow. First we will consider wounds of the type caused by high laser intensities, afterward we will consider the wounds produced by threshold light intensities.
Wounds Caused by High Laser Intensities
As mentioned in INTRODUCTION, extracellular calcium is required to repair large wounds similar to that in Fig. 1 A. Accordingly, we compared the wound response in the presence or absence of extracellular Ca2+ (Fig. 2). For 10 cells, the membrane potential, after wounding in NSW, was –29.5 ± 2.5 mV. Following wounding of cells bathed in 0 Ca2+ 1 mM EGTA ASW, the membrane potential goes toward 0 mV and the membrane resistance is below the resolution of the bridge amplifier (Fig. 2 B). For 13 cells, the membrane potential, after wounding in 0 Ca2+ 1 mM EGTA ASW, was –1.4 ± 0.52 mV. These findings are in agreement with the earlier work that extracellular Ca2+ is required for wound healing to occur.
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; Eddleman et al., 2000). Another possibility is that the plasma membrane has been sealed by fusion of intracellular membranes, and the depolarization results from a change in the ionic permeability of the plasma membrane. To distinguish between these possibilities, we performed ion substitution experiments since a membrane plug would not be expected to exhibit ionic selectivity. Substituting choline for sodium, decreasing the pH to 7.0, or increasing K+ from 10 to 100 mM in the solution bathing the cells, had no significant effect (Student's t test, P < 0.01) on the membrane potential following wounding (Fig. 3 B). In contrast, replacing 483 mM NaCl with Na isethionate resulted in a +5 mV membrane potential after wounding (Fig. 3, A and B), indicating high Cl– permeability of the plasma membrane. To further characterize the Cl– permeability of the plasma membrane, for these wounds, we wounded cells bathed in solutions with different Cl– concentrations. The data for the membrane potential after wounding (Fig. 3 C) is fit by a straight line with a slope of –33.2 mV per decade for Cl–. This is well below the ideal slope of –58 mV per decade for a membrane perfectly selective to Cl–.
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We monitored wound healing in a different way by imaging cells in the presence of a fluorescent probe in the sea water. Cells were bathed in a saturated solution of calcein, a bright fluorescein analogue (623 D) that is impermeant to membranes, and were impaled with an electrode in order to simultaneously monitor membrane potential and membrane resistance. The Ti-Sapphire laser was used to wound and to image; a slightly different scanning procedure was used in order to obtain images rapidly after wounding (see MATERIALS AND METHODS). As expected, when cells were bathed in calcium-free sea water (0 Ca2+ added, 1 mM EGTA), calcein entered the wound site and diffused throughout the cytoplasm (unpublished data, n = 5 cells). In sea water, calcein began to enter but was stopped at the boundary of the hemispherical depression previously seen by transmitted light (Fig. 4). Although it takes a few seconds for calcein to completely fill the depression, the movement of calcein in a direction parallel to the plasma membrane along the sides of the depression is blocked as early as 1 s. This suggests that internal membranes had already fused with the plasma membrane at the edge of the wound by that time.
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60 mV, and the membrane resistance after the wound was approximately the same as before wounding. Moreover, at this magnification, there was no obvious residual membrane damage in the region of the cell where the line scan occurred (unpublished data). This indicates that wounds caused by threshold light intensities do not require the presence of extracellular Ca2+ in order to heal. To estimate the size of these wounds, we recorded from oocytes held under voltage clamp (Fig. 6 B). Cells were bathed in 0 Ca2+ 1 mM EGTA ASW and impaled with two microelectrodes and voltage clamped at –30 mV. The membrane potential was repeatedly ramped from –35 to –25 mV in order to monitor the slope resistance of the plasma membrane at –30 mV (Fig. 6 B, top). As can be seen in the lower current trace of Fig. 6 B, membrane disruption resulted in an inward current of 6 nA in magnitude. In addition, there was no change in the membrane currents evoked by the voltage ramps as a result of wounding, indicating that there was no significant change in the slope resistance of the membrane. For eight cells, the magnitude of the peak inward current measured in 0 Ca2+ 1 mM EGTA ASW was 4.5 ± 3.5 nA. Assuming the wound forms a 10-nm-long cylinder through the lipid bilayer, filled with CFSW, the diameter of the cylinder would need to be 40 nm to produce a current of 4.5 nA. Remember from Fig. 1 B that this type of wound is also seen in the presence of calcium in the NSW. The magnitude of the peak inward current of these wounds measured under voltage clamp in NSW was (154 ± 90 nA, n = 10). The diameter of the wound in NSW would need to be 1.0 µm to sustain the larger current.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; McNeil and Steinhardt, 2003). We now provide data on the speed with which repair occurs and on resultant changes in plasma membrane ionic permeability.
Wounds Caused by High Laser Intensities
Within a few seconds of a laser-induced disruption, the membrane potential stabilizes at –30 mV (Fig. 1 A), and a barrier to extracellular fluorescent dye entry is established (Fig. 4). For oocytes with the vitelline envelope removed, we were unable to detect calcein entry into the original confines of the cell as early as 0.985 s after wounding (Fig. 5). This time course is significantly faster than the 10–30 s required for sealing in cultured mammalian cells. Echinoderm eggs are normally shed and fertilized in the open ocean, so perhaps they have evolved an unusually efficient membrane repair capability to survive in that hazardous environment.
After the wound is resealed, the plasma membrane resistance has decreased 100-fold and the membrane is highly permeable to Cl–. Two possibilities might explain how the plasma membrane became permeable to Cl–. First, the molecules responsible for the Cl– permeability may have been present in the plasma membrane before wounding but with the permeability inactivated so that their presence was undetectable before wounding. The plasma membrane of mature and immature starfish oocytes has been reported to exhibit an inwardly rectifying K+ current and a regenerative action potential due to Na+ and Ca2+ (for example see Shen and Steinhardt, 1976) with no evidence for a Cl– permeability. If there is an inactivated Cl– permeability in the plasma membrane, then the localized wound would have to generate some type of signal, other than membrane depolarization, to activate the permeability.
The second possibility is that the Cl– permeability is present originally in the yolk platelets and inserted into the plasma membrane at the time of repair. The presence of Cl– permeability in yolk platelets, an organelle that is likely to be derived from endocytic membranes, is consistent with a variety of evidence, indicating that endosomal organelles and lysosomes are Cl– permeable (Gunther et al., 1998; Debska et al., 2001; Demaurex, 2002; Nilius and Droogmans, 2003). Since the wound site comprises <5% of the total oocyte surface area, the Cl– permeability would likely be present at a high density in order for it to dominate the membrane potential after insertion. The on cell patch clamp technique might be used to detect the Cl– permeability if the debris on the surface of the wounded membrane (Fig. 5) could somehow be removed.
If the Cl– permeability is indeed localized to the wound site, then one would predict that there should be a positive injury current flowing into the cell (carried by Cl– flowing out of the cell) at the site of injury. This hypothetical oocyte injury current would be analogous to injury currents that flow out of wounded regions of epithelium, and which are thought to play a role in healing the wounded epithelium (Nuccitelli, 2003). Likewise, the hypothetical injury current in the oocyte may play a role in the steps of healing the wound that follow the initial sealing of the hole by exocytosis.
Lastly, we observed large differences in the shape of the resealed plasma membrane surface after wounding, depending on whether the vitelline envelope extracellular matrix was present or not; it was concave in its presence but convex in its absence. It is possible that oocyte cytoplasm tends to flow outwards from regions where the actin cortex is damaged, and that this outwards flow is prevented by the presence of the vitelline envelope, which traps the yolk platelet contents released during resealing and causes the repaired region to take on a concave shape. The effect of the vitelline envelope suggests that other factors in addition to membrane fusion can have a large effect on the shape that the repaired region takes on.
Wounds Caused by Threshold Laser Intensities
The discussion so far pertains to wounds easily made using high laser intensities. There was a second type of wound response that could be produced intentionally by a threshold protocol, i.e., by starting with low nonwounding laser intensity and increasing the intensity in very small steps. Threshold wounds often failed to occur even with this protocol, and thus must occur within a small window of laser power, suggesting that this type of disruption might be equally difficult to produce in natural conditions or by other experimental methods.
The threshold wound response was characterized by a brief depolarization indicative of a disruption, followed by a return of the membrane potential to its prewound level. From the instantaneous peak current in NSW, the disruption diameter was estimated to be 1 µm. The same type of response could be produced in the absence of extracellular Ca2+. However, the instantaneous peak current was smaller; consistent with a wound diameter that was 25x smaller, suggesting that the resealing process involved is somewhat more efficient in the presence of extracellular Ca2+.
In the absence of extracellular Ca2+, some of the threshold wounds sealed without any visible change in the surface by fluorescence microscopy with extracellular calcein (8 of 14 cells). This appears to be similar to brief, transient disruptions induced by electroporation, where sealing does not require extracellular Ca2+ (Chang and Reese, 1990; Hibino et al., 1993), and which have been interpreted as spontaneous sealing of the plasma membrane without the involvement of internal membranes.
However, some threshold wounds in the absence of extracellular Ca2+ resulted in a small hemispherical depression visible by fluorescence microscopy with extracellular calcein (6 of 14 cells), which seems inconsistent with simple plasma membrane closure. We suggest that this results from the exocytosis of internal vesicles to the plasma membrane; this membrane fusion apparently does not require extracellular Ca2+, perhaps there is some Ca2+ contribution resulting from laser-induced disruption of cortical granules, which contain a large amount of Ca2+ (Gillot et al., 1989).
Several possibilities could explain this finding. First, the amount of membrane added during threshold wound sealing does not add enough chloride permeability to change the membrane potential. Another possibility is that threshold wounds are resealed primarily using cortical granules while high intensity wounds use primarily yolk platelets. One would then have to suppose that cortical granules have a much lower Cl– permeability than yolk platelets. A third possibility is that the Cl– permeability is present in the plasma membrane before wounding but with the permeability inactivated and that the threshold wound does not produce a large enough signal to activate the permeability. This would imply that a 1-µm hole in the plasma membrane does not generate a large enough signal to activate the Cl– permeability.
In summary, we have used electrophysiological and optical methods to demonstrate very robust plasma membrane repair mechanisms in the starfish oocyte. Large disruptions are sealed within a few seconds at most, possibly by Ca2+-dependent fusion of chloride-permeable intracellular organelles with the plasma membrane.
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ACKNOWLEDGMENTS |
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This work was partially supported by R01 GM060389-04 to M. Terasaki. The Ti-sapphire microscope was supported by National Institutes of Health 5P41-RR 13186 and 1FI0-RR 15705.
Olaf S. Andersen served as editor.
Submitted: 29 March 2005
Accepted: 24 June 2005
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