§
* Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032; The Marine Biological
Laboratory, Woods Hole, Massachusetts 02543; § Laboratory of Neurobiology, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Bethesda, Maryland 20892;
Fukushima Medical College, Second Department of Anatomy,
Fukushima 960-12 Japan; and ¶ The Medical College of Georgia, Department of Cellular Biology and Anatomy, Augusta,
Georgia 30912
A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ~40 by 10 µm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (~5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ~3 mM Ca2+ (SW is ~10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.
LIVING, nucleated cells respond to microneedle punctures of their plasma membranes by rapidly (within
sec) resealing the breach created. The mechanism
used is Ca2+ dependent and hypothesized to be an active
process governed by specific protein-protein interactions
that result in a local exocytotic reaction. As a result of this
exocytotic response, new membrane is added locally to the
site of cell surface injury (for review see McNeil and Steinhardt, 1997 An extensive literature documents examples of resealing on a far larger scale than that required for repair of a
microneedle prick. For example, we know that transected
neurons and skeletal muscle cells reseal after transection,
since they are capable of surviving this injury (Yawo and
Kuno, 1985 The capacity of the echinoderm egg/oocyte for surviving
disruptions, large and small, is well established. Heilbrunn
and others, for example, described a "surface precipitation
reaction" that occurs when large portions of the sea urchin
surface are torn off and showed that egg survival, that is,
retention of intracellular components, depended on a
Ca2+-initiated event (Heilbrunn, 1930a Here we severely challenge the cytoplasm of starfish oocytes and sea urchin eggs to seal itself off from its normal
external environment, sea water (SW).1 We find these
cells have a remarkable capacity for rapidly resealing plasma membrane disruptions. We demonstrate that the
Ca2+ present in SW induces the rapid fusion of cytoplasmic
organelles with one another to form de novo an impermeant, cell surface membrane barrier. This vesicle-vesicle
fusion response separates intracellular from extracellular
domains, and can occur in the complete absence of plasma
membrane involvement. We propose that this intracytoplasmic membrane fusion reaction, coupled with cytoplasmic membrane fusion with the plasma membrane, is essential for resealing large plasma membrane disruptions.
Oocytes and Eggs
Starfish (Asterina miniata) were obtained from Marinus, Inc. (Venice,
CA), and sea urchins (Lytechinus variegatus) were obtained from T. Andacht (Duke University Marine Lab, Beaufort, NC) or S. Decker (independent collector, Davie, FL). They were maintained in running SW at
the Marine Biological Laboratory (Woods Hole, MA) or in a SW aquarium at the University of Connecticut (Farmington, CT). Starfish gametes
were obtained by using a small sample corer (Fine Science Tools Inc., Foster City, CA). Sea urchin eggs were obtained by injection of a small
amount of 0.5 M KCl to spawn eggs from a single gonad at a time (0.15 ml
for the North Carolina animals and 0.3 ml for the Florida animals; Fuseler,
1973 Wounding Procedure
Sea urchin eggs were dejellied with either Ca2+ Mg2+-free SW for 1.5 min
(Detering et al., 1977 Microscopy
An upright microscope (Axioskop; Carl Zeiss, Inc., Thornwood, NY) was
coupled with a scanning confocal microscope (MRC 600; Bio-Rad Laboratories, Cambridge, MA). To make the recordings shown in Figs. 1, 2, 4,
5, 9, and 10, the confocal microscope was set to scan continuously at one
or two frames per second, and each frame was recorded on an optical
memory disk recorder (OMDR; Panasonic 3038F; Secaucus, NJ). In early
experiments, the frames were recorded manually as the scan reached the
bottom of the monitor screen, but in later experiments, automatic recording was accomplished by means of a trigger circuit using a sync signal from
the confocal microscope (described in detail at http://www.uchc.edu/~terasaki/trigger.html).
Microinjection
The eggs were quantitatively injected using mercury-loaded pipets (Hiramoto, 1962 Solutions and Reagents
Solutions requiring SW were made using Marine Biological Laboratory
(MBL) artificial SW (9.25 mM Ca2+, 48.4 mM Mg2+, 425 mM Na+, 9.0 mM
K+, 25.5 mM SO42 Electron Microscopy
Sea urchin eggs attached to polylysine-coated coverslips were injected
with SW as described above and then fixed 10-20 min later by immersion
in 1% glutaraldehyde, which was made by diluting 8% glutaraldehyde in
SW (Electron Microscopy Sciences, Gibbstown, NJ). After ~1 h, the eggs
were changed to SW and then postfixed for 1 h with 1% OsO4 and 0.8%
potassium ferricyanide in 0.1 M sodium cacodylate, pH 7.4. The eggs were
rinsed thoroughly in distilled water and stained in 0.5% aqueous uranyl
acetate for 1 h. They were dehydrated and embedded in Poly/Bed (Polysciences Inc., Warington, PA). Ultra thin sections were cut by using an ultramicrotome (MT6000-XL; Research and Manufacturing Co., Inc., Tucson, AZ) and stained with a fresh mixture of equal parts acetone and
saturated uranyl acetate, stained a second time with lead citrate, and then
examined in a transmission electron microscope (JEM-1200EX; JEOL
LTD., Tokyo, Japan).
Stratification of Sea Urchin Eggs
Approximately 1 ml of dilute egg suspension was layered on top of 50 µl
of 18% ficoll (70,000 mol wt; Sigma Chemical Co.) in a 1.5-ml microfuge
tube, and was centrifuged at 12,000 g for 15-30 min.
Large Wounds in Sea Urchin Eggs
Are Resealed Rapidly
Echinoderm eggs are large (~100-200 µm), relatively easy
to inject, and optically clear, and thus have several experimental advantages for investigating large-scale membrane
wound healing. To understand the reaction of eggs to surface disruptions, it is necessary to recall that the surface of
unfertilized eggs is poised to undergo large changes at fertilization. Lining the interior of the plasma membrane is a
dense monolayer of docked cortical granules (each ~1 µm
in diameter). Exterior to the plasma membrane is the vitelline layer, a thin proteinaceous coat (Chandler and Heuser, 1980 The vitelline layer of unfertilized sea urchin eggs adheres strongly to glass coated with polycationic macromolecules such as protamine sulfate or polylysine (Steinhardt
et al., 1971 We observed this wounding process directly using a confocal microscope. Eggs were immersed in SW containing
either fluorescein dextran, 10,000-70,000 mol wt (FDx) or
fluorescein stachyose, mol wt 1,146 (FS), both of which are
inert, impermeant fluorescent markers. The confocal microscope was set to scan continuously at the rate of either
one or two scans per second as the wound was being made.
Very little fluorescent marker entered the wounded egg,
indicating that the breach in the permeability barrier of the cell surface was repaired rapidly (Fig. 1). In contrast,
when eggs were wounded in CFSW (Ca2+-free SW), no
sharp boundary formed, and the fluorescent marker entered the egg (not shown). Also, the cytoplasm leaked out
steadily, as reported by Heilbrunn (1930a Because so little (undetectable amounts) of FDx or FS
leaked into the egg during rip-off, we could not determine
from this experiment how fast resealing occurred. To further characterize the time course of resealing, eggs were
injected with the fluorescent Ca2+ indicator, calcium green
dextran, so that we could record the duration of Ca2+ influx into the wounded cell. A local Ca2+ rise was recorded
at the wound site, but this local elevation persisted for only
a few seconds and did not propagate throughout the cell
(Fig. 2). Since intracellular Ca2+ concentration is thought
to be ~0.1 µM, compared to the extracellular SW Ca2+
concentration of 9.3 mM, this shows that very little Ca2+
enters from the outside and suggests resealing of these
large disruptions is complete within a few seconds after
disruption. The lack of fertilization envelope elevation is a
biological indication that Ca2+ entry is restricted.
In the original sea urchin egg cortical preparation (Vacquier, 1975
Since even a small marker such as FS was not permeable
to healed eggs, and since influx of Ca2+ is rapidly halted in
large wounds, it is very likely that the healing consists of a
Ca2+-dependent formation of a membrane barrier rather
than a dense precipitation of proteins. Small wounds in unfertilized sea urchin eggs are thought to be healed by exocytosis of cortical granules (Steinhardt et al., 1994 Intracellular Injection of Ca2+ Causes Fusion of
Intracellular Membranes
When Ca2+ is injected directly into eggs, it can activate
them, but only when the injected solution contains millimolar total Ca2+ buffered at free concentrations near 1 µM (Hamaguchi and Hiramoto, 1981 Injections of CFSW and SW (9.3 mM Ca2+) with fluorescent markers were observed using confocal microscopy.
When CFSW was injected, the fluorescent marker spread
throughout the cytoplasm (Fig. 4). When SW was injected,
a wound vesicle containing apparently all of the injected
fluorescence formed rapidly in both starfish oocytes and
sea urchin eggs (Fig. 4). Since 1-kD FS as well as FDx is contained in such experiments, it seems very likely that a
membrane is formed at the boundary. When SW was injected into an egg containing Ca2+ indicator, there was a
transient increase near the wound vesicle that was detectable with this indicator for no longer than 6 s (Fig. 5). As
was the case in the large disruption created by a rip-off, a
Ca2+ impermeant barrier was rapidly formed when cytoplasm was exposed to SW by injecting it. However, in this
case, there could be no involvement of the plasma membrane, suggesting that vesicle-vesicle fusion was responsible.
In transmitted light images, the wound vesicle was often
seen to have two boundaries, a sharper inner boundary
and finer outer boundary. At other times, there was only
one sharp boundary. The presence of one or two boundaries seemed to be related to yolk platelets. Yolk platelets
are 1-2-µm-sized organelles that are distributed abundantly throughout the egg cytoplasm. Nile red is a fluorescent dye that stains lipid-rich organelles (Greenspan et al.,
1985
The wound vesicle was examined by thin section EM
(Fig. 7). A cross section through the center of a wound
vesicle showed that the injected SW was surrounded by a
donut-shaped domain corresponding to the more continuously stained Nile red region of a two-boundary wound
vesicle (Fig. 6). High magnification revealed that an electron-dense boundary is present continuously around the
injected SW (Fig. 7) in a location consistent with the innermost boundary described above (Fig. 6) as limiting FS diffusion and stainable with DiI. This confirms that the
wound vesicle is a membrane-bounded inclusion. The donut-shaped domain contains abnormal cytoplasm that may
be the result of massive fusion of organelles. Outside of
the donut-shaped domain, the cytoplasm appeared normal, except for the presence of large organelles that appeared to be the result of fusion of several yolk platelets.
Thus, alterations in normal cytoplasmic structure occur
beyond the membrane boundary that excludes the SW.
SW of varying Ca2+ concentration was injected to determine the threshold for the formation of the wound vesicle.
SW containing 1.9 mM Ca2+ did not form a stable wound
vesicle, as indicated by the declining ratio measurement of
Fig. 8. Indeed, 1.9 mM Ca2+ yielded a response that was
indistinguishable from that elicited by artificial SW to
which no Ca2+ was added. SW containing 3 mM Ca2+
formed a more stable vesicle than did 1.9 mM, but one that
was quantitatively (Fig. 8) and qualitatively (it could often
be observed to break up into numerous smaller vesicles)
less stable than those formed with 4.9 mM or normal SW
(~10 mM Ca2+).
Mg2+, a group II divalent cation, was present at 48.4 mM
in the Ca2+-free SW used above, and so we knew this divalent did not induce wound vesicle formation or allow resealing of large disruptions. Strontium and barium are also
group II divalent cations, and sometimes have similar
physiological effects as Ca2+ (Hille, 1992 The wound vesicle was generally very stable. In one experiment, eight starfish immature oocytes were injected
with a 2% volume of SW. The oocytes were then matured
by the hormone 1-methyladenine and fertilized; the wound
vesicles remained through the meiotic divisions as well as
fertilization and first mitosis in all eight eggs (cleavage was
normal in five out of eight eggs). In another experiment,
12 starfish immature oocytes were injected with a 2% volume of SW and cultured for 3 d. Eight out of 12 oocytes survived, and in each, the wound vesicle was still intact.
Extrusion of Cytoplasm into SW Causes Fusion of
Intracellular Membranes
We devised another way to test whether Ca2+ causes intracellular membranes to fuse and form boundaries. Mature
starfish eggs were first injected with a fluorescent marker
(200 µg/ml 70-kD Fl FDx final concentration). After the
fluorescent marker had diffused throughout the cytosol,
egg cytoplasm was removed by suction using a microneedle. The cytoplasm was then extruded into solutions with
or without calcium. When cytoplasm was extruded into
CFSW or an intracellular buffer, the fluorescence diffused away; in contrast, when cytoplasm was extruded into SW, a
large amount of the fluorescence remained with the extruded
cytoplasm (Fig. 9). This is consistent with Ca2+-induced fusion of intracellular membranes which encloses the cytosol
as it is extruded. When cytoplasm was extruded into 1 mM
Ca2+, significantly less of the dye was retained. This concentration is similar to the threshold for creating wound
vesicles, and suggests that this process is similar to what
occurs when Ca2+ solutions are injected into the cytoplasm.
Which Intracellular Membranes Are Involved?
The Nile red staining described above suggested that the
yolk granules fused with one another around the SW injection site (Fig. 6). To determine whether there is a functional requirement for the yolk platelet compartment in
wound vesicle formation, we made use of stratified sea urchin eggs. Centrifugation at 12,000 g causes the yolk platelets to sediment within the egg, leaving a clear area of cytoplasm at the other (centripetal) end of the egg (Fig. 10;
Harvey, 1956 FS was injected into stratified eggs, then cytoplasm from
either the clear area or the yolk-containing area was removed by micropipet and extruded into SW. There was
significantly more dye retained by the cytoplasm containing yolk platelets compared to the clear cytoplasm (Fig.
10), suggesting that yolk platelets are involved in sealing
off cytoplasm and that ER membranes by themselves cannot.
SW was injected into fertilized sea urchin eggs because
there is evidence that ER membranes become transiently
disrupted at fertilization (Jaffe and Terasaki, 1993 Plasma membrane disruption is a normal and common
form of cell injury in many mammalian tissues (McNeil,
1993 Why are disruptions sometimes lethal? The answer is
not known, but one possibility is failure to adequately prevent entry of potentially toxic levels of Ca2+ (Trump and
Berezesky, 1995 What is the mechanism for preventing Ca2+ influx or
protein escape? First of all, Ca2+ itself is the signal to repair a disruption. Heilbrunn discovered that healing of
large surface wounds does not occur in the absence of extracellular Ca2+ in sea urchin eggs and in many other cells
(Heilbrunn, 1930a We know of no example of a boundary composed solely
of proteins that can exclude small molecules (<10,000 mol
wt). Cytosol, even when gelled by elevation of Ca2+ to >5
µM for extended periods, does not significantly restrict the
mobility of 10-kD FDx (Luby-Phelps, 1994 The simplest mechanism of resealing, used by erythrocyte ghosts and liposomes, occurs spontaneously as the energetically favored outcome of disruption-induced exposure of the hydophobic residues of phospholipid molecules
to water (for review see McNeil and Steinhardt, 1997 Recently, plasma membrane disruption was found to
evoke rapid fusion of internal, vesicular membrane with
the plasma membrane (Fig. 11 A) (Steinhardt et al., 1994
We here show that large disruptions (>10 µm2) can be
rapidly resealed on approximately the same time scale as
much smaller, microneedle-induced disruptions (Steinhardt et al., 1994 This second model for rapid resealing (Fig. 11 B) suggests that in addition to exocytosis, organelle-organelle
fusion is induced by disruption and is crucial for rapid resealing. If this were the case, then cytoplasm could be separated from the plasma membrane and still form a barrier
when exposed to the high Ca2+ in SW. We achieved this
separation experimentally in the following two ways: by
injecting SW into cytoplasm and by injecting cytoplasm into SW. In both cases, a de novo barrier formed that
could restrict for hours the diffusion of FDx, FS, or Ca2+.
EM of the SW-induced barrier demonstrated that this
boundary displayed the electron-dense morphology expected of a cell membrane. Moreover, this boundary could
be stained with DiI, a lipophilic dye that stains membranes.
We propose that exposure of cytoplasm to high Ca2+
caused by a wound results in massive fusion of internal
vesicles with each other and with the plasma membrane.
These fusion reactions proceed rapidly until a patch of
membrane continuous with the plasma membrane has
formed, preventing further Ca2+ entry. There are other reactions in response to injury which are likely to be secondary to fusion-mediated resealing, such as alterations in the
actin-based cytoskeleton (Jeon and Jeon, 1975 In some cases, the mechanism we propose may not be
sufficient to make a rapid seal, whereupon the cell must
depend on more slowly acting mechanisms. For instance,
transection of large diameter axons (>70 µm) from earthworm or crayfish takes minutes to seal (Krause et al., 1994 In echinoderm eggs, where rapid sealing occurs, there
are many new structures in the vicinity of the healed
plasma membrane boundary. Outside of the wound created by a rip-off, there are numerous, spherically shaped
domains of various sizes (Fig. 3), and on the cytoplasmic
side of the SW injection vesicle there are many large membrane-bounded compartments (Fig. 7, A and B, indicated
by solid dots). We propose that these are also results of
Ca2+-induced fusion events, and that they represent the
membranes that did not become incorporated into the
plasma membrane. In this view, at least some of the large
spherical domains outside of the cell are regions of cytoplasm that are surrounded by a new membrane boundary,
whereas the compartments on the cytoplasmic side of the
wound are vesicles derived from fusion of smaller compartments.
A wound vesicle formed by vesicle-vesicle fusion in response to a focal source of elevated Ca2+ (e.g., a SW injection) is predicted (Fig. 11) to consist of two major domains: (a) an innermost domain, consisting of the high calcium source (injected SW); and (b) a surrounding domain, consisting of the contents of the vesicle population
used (probably the yolk granules, in the model studied
here). In fluorescence micrographs of SW-injected eggs
stained with Nile red, both of these domains are in fact
present: an unstained, SW-filled, innermost domain surrounded by a Nile red-stained outer domain that, in turn,
is surrounded by normal appearing cytoplasm. In electron
micrographs, too, these two domains are evident. However, a further prediction is that the outer domain, containing the contents of multiple vesicle lumina, would be
delimited on each of its boundaries with a continuous
membrane. EM performed on eggs fixed 20 min after SW
injection confirmed this prediction only partially: a continuous membrane was observed on the SW but not the cytoplasmic side of the outer domain. Its absence may be artifactual in the limited sample examined by EM. Or, if real,
its absence suggests that in addition to simple vesicle-vesicle fusion reactions, other membrane-transforming events
are also occurring which destabilize or degrade the outer
domain's cytoplasmic membrane boundary.
The egg's rapid, de novo formation of a continuous
membrane sheet is Ca2+ dependent, but displays an exceptionally high threshold (~3 mM) in the SW injection experiment. In this it resembles the Ca2+ threshold previously reported for resealing of small plasma membrane disruptions (Steinhardt et al., 1994 In echinoderm eggs, the yolk platelet appears to be the
organelle principally involved in the fusion events leading
to formation of the protective membrane sheet, whereas
the ER is probably not involved. In the vicinity of a wound
vesicle, the yolk platelets often appear to have fused or disintegrated. Moreover, when the yolk platelet-rich cytoplasm
of the centrifuged egg was injected into SW, it was capable,
like whole cytoplasm, of trapping dissolved FDx, whereas the
ER-rich cytoplasmic domain was not. It is interesting to
note that Ca2+ causes isolated sea urchin egg yolk platelets
to fuse with each other (Vogel, S.S., unpublished results)
whereas Ca2+ does not cause isolated ER microsomes to
fuse with each other (Paiement and Bergeron, 1991 Preliminary evidence (Terasaki, M., and P. McNeil, unpublished results) suggests that the wound vesicle is an
acidic compartment as soon as 1 min after its formation.
This is an indication that the wound vesicle is at least partially formed from the fusion of endocytic or lysosomal
compartments. Such an involvement can be rationalized
on the grounds that endocytic membranes are derived from the plasma membrane. There is considerable recent
evidence from other systems consistent with an endocytic
membrane involvement in wound healing. Fusion of the
endosomal/lysosomal compartment vesicles with the plasma
membrane, and with one another, is induced by Ca2+ elevation in broken cell preparations (Mayorga et al., 1994 The molecular components of the disruption-induced
exocytotic response (Bi et al., 1995 There is, however, very little data to suggest how the
vesicle-vesicle fusion induced by disruptions is engineered
at the molecular level. In broken cell preparations of the
egg, Ca2+-dependent, vesicle-vesicle fusion can be observed but appears to use an unorthodox mechanism. Homotypic fusion of isolated cortical granule vesicles is a trypsin-
and N-ethyl maleimide (NEM)-sensitive reaction (Vogel
et al., 1992 In summary, we propose that a replacement patch of bilayer is erected in the cytoplasm bordering on a plasma
membrane disruption. Ca2+-regulated vesicle-vesicle membrane fusion events are responsible for this rapidly completed event. Exocytotic events, e.g., vesicle-plasma membrane fusions occurring at the same time, join the bilayer patch to the plasma membrane discontinuity. We speculate that disruption-induced vesicle-vesicle and vesicle-
plasma membrane fusion events may use a primitive, and
relatively indiscriminate molecular mechanism, possessing
an exceptionally high threshold for Ca2+ (~3 mM). The
vesicle population mobilized is most likely the endosomal/
lysosomal compartment, but could involve others as well.
Vesicle-vesicle fusion-promoting mechanisms may have
initially evolved for the purpose of repairing mechanically
initiated damage to the cell surface, rather than moving
molecules from one compartment to another. If so, resealing may involve a basic or bare bones fusion apparatus, to
which numerous additions have been made in evolution as
new and more complex functions were assumed. Investigation of the molecular apparatus of resealing may, therefore, provide a better understanding of more complex and
difficult-to-study membrane-membrane fusion systems,
such as those involved in neurotransmission (Rothman
and Sollner, 1994) and intracellular protein trafficking (Rothman and Wieland, 1996).
; Casademont et al., 1988
; Krause et al., 1994
).
Resealing on this scale requires the replacement of many
square micrometers of surface barrier. Unless the exocytotic fusion events induced by disruption are far more numerous than have been detected (Bi et al., 1995
; Miyake
and McNeil, 1995
), it does not seem possible that exocytosis alone could replace the amount of surface membrane
requisite for large-scale (many square micrometers) resealing.
,b). Indeed, echinoderm eggs are favored subjects of microinjection experiments, not only because they are large, easily obtained,
and safely manipulated at room temperature, but also because they are adept at surviving plasma membrane disruptions induced by this experimental manipulation (Chambers and Chambers, 1961
).
MATERIALS AND METHODS
). Microscope observations were done at room temperature. Starfish
oocytes were kept in an incubator at 18-20°C for long-term experiments.
), or by extensive SW washes, resuspended in SW,
and attached for 1 min to a polylysine-coated coverslip (500,000 mol wt;
coverslips were treated with 1 mg/ml polylysine in water for 5 min followed by several washes in SW; Sigma Chemical Co., St. Louis, MO). The
coverslip was used as the top part of a microinjection chamber. A microinjection needle tip was immersed in 1 mg/ml polylysine for 3 min and then
mounted in a Narishige SM-20 micromanipulator so that the needle was in
the plane of focus of the microscope (Narishige Scientific Instruments
Laboratory, Tokyo, Japan). The needle was moved up against the side of
an egg for ~1 min and was then rapidly moved away to generate the wound.
Fig. 1.
Survival and rapid
healing of a large wound in
the plasma membrane. A
polylysine-coated microneedle was maneuvered so that it contacted the surface of an
immobilized sea urchin egg
and then was rapidly moved
away from the surface (the
movement was in the plane
of focus of the microscope).
This ripped off the contacted
surface and created a wound
~40 by 10 µm. (Top) (A) an
apparent sharp boundary
formed immediately (within
1-2 s) at the interface of cytoplasm and SW produced by the rip-off. After addition of sperm, the wounded egg became fertilized (B; arrow denotes fertilization envelope) and underwent several rounds of division (C; shown 2.5 h after fertilization). This shows that the wounded egg healed successfully after the rip-off. (Bottom) A sea urchin egg was immersed in 100 µg/ml FS in SW and observed by confocal microscopy during the
rip-off wound procedure. No entry of FS was observed after a rip-off, indicating that the wound had resealed rapidly. Images were obtained at 1-s intervals; consecutive images are shown except the last image, which was 40 s after wounding. Bars: (top) 20 µm; (bottom)
10 µm.
[View Larger Version of this Image (62K GIF file)]
Fig. 2.
Cytosolic Ca2+ during the healing of a wound. A
sea urchin egg was injected
with the fluorescent Ca2+ indicator calcium green dextran (10 kD; 20 µM final concentration). As in Fig. 1, a
polylysine-coated microneedle was used to create a wound. There is a local rise in Ca2+ during wounding, but the rise diminishes rapidly. In view of the large gradient of Ca2+ from SW to cytosol, this is further indication of a rapid healing reaction. Images were obtained at 1-s intervals; consecutive
images are shown except the last image, which was 15 s after wounding. The small dark circle near the center of the egg is an oil drop resulting from the calcium green dextran injection. Bar, 10 µm.
[View Larger Version of this Image (30K GIF file)]
Fig. 4.
Injected SW is contained at the injection site. FDx (10 mg/ml; 70 kD) dissolved in CFSW or in SW was injected into a
starfish oocyte. The micropipet contained an oil cap at the tip to
prevent mixing of the injectate with chamber SW. The oil cap
comes out first during the injection and forms a spherical droplet
in the cytoplasm. The injections were observed by simultaneous
scanning transmission and fluorescence imaging. (A) When
CFSW containing FDx was injected, the fluorescence spread
throughout the cytoplasm. (B) When SW containing FDx was injected, the fluorescence did not spread from the injection site and
was contained within a wound vesicle that is visible in the transmitted light image. These results suggest that high Ca2+ causes fusion of intracellular membranes, creating a boundary that prevents spreading of high Ca2+ throughout the cytoplasm. Images
were obtained at 1-s intervals; consecutive images are shown except that last image of the CFSW sequence was 125 s after injection, and the last image of the SW sequence was 306 s after injection. Bar, 20 µm.
[View Larger Version of this Image (80K GIF file)]
Fig. 5.
Cytosolic Ca2+ during SW
injection rises only briefly. A mature starfish egg was injected with
the fluorescent Ca2+ indicator calcium green dextran (20 µM final
concentration) then was imaged by
simultaneous scanning transmission and fluorescence confocal microscopy during an injection of SW.
There was a brief rise in fluorescence around the injection site
which then decreased rapidly. The
oil drop in the first transmitted light
image is from the calcium green
dextran injection. Images were obtained every 0.5 s; consecutive images are shown except the last
frame which is 6 s after the beginning of the sequence. Bar, 20 µm.
[View Larger Version of this Image (42K GIF file)]
Fig. 9.
Cytoplasm extruded into SW retains a fluorescent
marker in the cytosol. Starfish oocytes were injected with a final
concentration of 0.2 mg/ml FS. After allowing the FS to diffuse
throughout the oocyte, cytoplasm was removed by micropipet
(2% of the oocyte volume). The cytoplasm was then extruded
while observing by simultaneous scanning transmission and fluorescence confocal microscopy. When extruded into CFSW (left),
the fluorescent marker diffused away, indicating lack of a boundary formation. When extruded into SW (right), the fluorescent
marker was retained, indicating that the Ca2+ caused fusion of intracellular membranes, trapping the marker. Images were obtained at 1-s intervals; consecutive images are shown except the
last image, which was 30 s after the sequence began. (bottom) Sea
urchin eggs were injected as described above with FS and their
cytoplasm was then extruded into CFSW (bottom left) or SW
(bottom right). As was the case for starfish, when sea urchin egg
cytoplasm was extruded into CFSW, the FS diffused away
whereas the FS became trapped when cytoplasm was extruded into SW. Both images were taken 45 s after extrusion. Bars, 10 µm.
[View Larger Version of this Image (53K GIF file)]
Fig. 10.
Behavior of extruded cytoplasm from centrifuged
eggs. Sea urchin eggs were centrifuged to stratify the cytoplasm
(top); the clear region is devoid of yolk granules. Centrifuged
eggs were injected with a final concentration of 0.2 mg/ml 70-kD
FDx. Cytoplasm was removed by micropipet (2% of the total cytoplasmic volume) and then extruded into SW and observed by
simultaneous scanning transmission and fluorescence microscopy. (bottom left) When yolk granule-containing cytoplasm was
extruded, the fluorescence was retained. (bottom right) When
clear cytoplasm was extruded, the fluorescence diffused away.
These results suggest that yolk granule membranes are involved
in the Ca2+-dependent fusion of intracellular membranes. Images
were obtained at 1-s intervals; consecutive images are shown except for the last images, which were both taken at 40 s. Bar, 10 µm.
[View Larger Version of this Image (41K GIF file)]
). Briefly, a micropipet was back-loaded with a small drop of
mercury, which was pushed to the micropipet tip. The injectate was front-loaded using a microscope with an eyepiece reticle for calibrating the volume injected. A small amount of oil (dimethylpolysiloxane, 100 cps;
Sigma Chemical Co.) was drawn up as an oil cap. The injection chamber
was as described previously (Kiehart, 1982
).
, 489 mM Cl
) or CFSW (same formulation without
Ca2+). Fluorescent reagents were all obtained from Molecular Probes, Inc. (Eugene, OR). Calcium green-1 dextran (10 kD) was made as a solution of 1 mM in 100 mM potassium glutamate, 10 mM Hepes, pH 7, and
injected to a final concentration of 20 µM. Nile red was dissolved at 0.5 mg/ml in acetone and used at 0.5 µg/ml in SW. Intracellular buffer contained 350 mM potassium glutamate, 350 mM glycine, 3 mM MgCl2, 0.57 mM CaCl2, 10 mM EGTA, 20 mM Hepes, pH 6.7. DiI (DiIC16[3])-saturated oil was made using soybean oil (Wesson; Hunt-Wesson, Inc., Fullerton, CA).
RESULTS
). The egg's natural environment is SW,
which contains ~10 mM Ca2+. At fertilization, the sperm
initiates a wave of Ca2+ release from the ER, resulting in a
rise of cytosolic Ca2+ concentration from ~0.1 µM to ~2-3
µM (Steinhardt et al., 1977
; Hafner et al., 1988
). The elevated Ca2+ triggers exocytosis of the cortical granules,
which releases proteases and other enzymes that act on
the vitelline layer, resulting in formation of the fertilization envelope (Kay and Shapiro, 1985
).
; Mazia et al., 1975
). When attached eggs are
subjected to a sudden fluid force, they are sheared off,
leaving a large circular patch of egg cortex (including the
cortical granules) attached to the glass (Vacquier, 1975
).
To make controlled wounds in the egg surface, we maneuvered a polylysine-coated microneedle so that it lay
against the side of an immobilized egg. A quick movement
of the microneedle away from the egg ripped off a patch of
cortex. The size of this rip-off zone is estimated, based on
needle diameter and estimated cell contact length, to be 40 by 10 µm. By transmitted light, an apparent sharp boundary formed rapidly at the wound site (Fig. 1 A). Some cytoplasm always leaked out beyond this apparent boundary and appeared to be transformed into many small spheres
of varying sizes. The wounding process usually did not result in artificial activation, i.e., fertilization envelope elevation. Most of the eggs could be fertilized and undergo apparently normal cleavage (Fig. 1, B and C).
,b). This confirms that Ca2+ is required for sealing the wound, and the
effectiveness of dye exclusion suggested that sealing occurs very rapidly.
), attached eggs are sheared, leaving an ~50-µm-diam cortical patch on the coverslip. Since this patch
comes directly from the egg surface, the sheared eggs must
have an equivalently sized wound. We sheared eggs in SW
to see if this very large wound can be repaired. By confocal
microscopy, the sheared eggs excluded FDx in SW from
the cytosol, showing that the wound had been sealed (Fig.
3). Most of the sheared eggs, however, had a fertilization envelope. Most likely, enough of the extracellular Ca2+
had entered into the egg to activate the processes that are
normally triggered by Ca2+ at fertilization. In this case, the
wound healing was only partially successful, because it did
not prevent Ca2+ from activating an inappropriate response for the cell.
Fig. 3.
Sea urchin eggs are able to heal very large wounds in
their surface. Eggs were attached to a polylysine-coated coverslip
and then sheared in SW. This process leaves a ~50-µm patch of
cortex on the coverslip (Vacquier, 1975), and creates an equivalently sized wound in the sheared egg surface. After 3 min, the
sheared eggs were transferred to SW containing 0.3 mg/ml 10-kD
FDx and then were imaged by scanning transmission and fluorescence confocal microscopy. The fluorescence image shows that
this very large wound has healed, since the FDx is excluded from
the egg interior. Part of a low fertilization envelope is seen in the
transmitted light image (arrow), showing that the wound healing
was not able to prevent a partial activation of the egg by Ca2+ entry. Bar, 10 µm.
[View Larger Version of this Image (93K GIF file)]
; Bi et
al., 1995
). However, the cortical granules at the wound site
are probably removed with the plasma membrane with the
type of wounds we have made, so it seemed likely that
some of the intracellular membranes had participated in
the membrane fusion events that sealed the breach between intra- and extracellular spaces.
). Rapid injection of
unbuffered solutions of millimolar Ca2+ concentration
does not activate eggs; instead, it causes formation of an
inclusion that appears to contain the injectate (Hiramoto, 1965
; Kiehart, 1981
). For example, it is a very common observation that an inadvertent injection of SW causes formation of an inclusion. We realized that this phenomenon
might be relevant for plasma membrane wound healing, so
we investigated it more closely.
). It is apparently the active component of Nile blue used for staining yolk platelets in frog eggs (Danilchik and
Gerhart, 1987
), and we found that it stains yolk platelets in
sea urchin eggs as well. In cases where the wound vesicle
had two apparent boundaries, Nile red stained individual
yolk platelets outside of the fine outer boundary, but it
stained the region between the two boundaries more uniformly, as if the yolk platelets had fused together or disintegrated (Fig. 6). Moreover, in the two-boundary vesicle,
FS injected with the SW was restricted by the innermost of
these two boundaries (Fig. 6). This innermost boundary
was stained with DiI, a lipophilic fluorescent dye that
stains membrane bilayers (Haugland, 1996
), providing
strong evidence that the boundary is a membrane (Fig. 6).
When the wound vesicle had only one apparent boundary,
there was no intermediate zone between the normal appearing yolk platelets and the FS-containing wound vesicle.
Fig. 6.
Yolk platelet and membrane staining near the wound
vesicle. SW containing 0.5 mg/ml FS was injected into a sea urchin egg whose yolk platelets were stained by Nile red. The egg
was imaged using dual channel fluorescence confocal microscopy.
(A) Fluorescein channel imaging, showing that the FS had been
contained within the wound vesicle. (B) Rhodamine channel imaging showing the fluorescence from Nile red. Unaltered yolk
platelets are seen throughout most of the cytoplasm. A more uniformly stained region surrounds the SW injectate that is marked
by the FS seen in A. This domain seems to represent yolk platelets that have fused together or disintegrated, and corresponds
with the region surrounding the SW in the electron micrographs
in Fig. 7. By transmitted light, the wound vesicle often was seen
to have a sharp inner boundary and a finer, less distinct outer
boundary. The inner boundary corresponds to the sea water injectate interface, and the outer boundary corresponds to the
boundary between the disrupted and normal appearing yolk platelets. (C and D) Composition of the wound vesicle boundary. A SW injection was made using DiI-saturated oil for the oil cap in the microinjection needle. DiI spreads in membranes contacted by the oil drop (Terasaki and Jaffe, 1991). 15 min after injection, the wound vesicle was viewed by (C) scanning transmission and (D) fluorescence confocal imaging. The fluorescence
image shows that DiI has labeled the wound vesicle boundary,
providing strong evidence that the boundary is a membrane. Also
labeled are free-floating vesicles that are often observed in the
wound vesicle lumen. Bar, 10 µm.
[View Larger Version of this Image (120K GIF file)]
Fig. 7.
Electron micrographs of the SW injection site. (A) Low
magnification view of a section through the SW injection site. An
empty central region or vesicle lumen (VL) at the injection site is surrounded by a shell of abnormal cytoplasm (AC) and then,
abruptly, by normal appearing cytoplasm (Arrow points to
plasma membrane). (B) At higher magnification, the shell of abnormal cytoplasm surrounding the vesicle lumen (VL; arrows indicate boundary to SW) is seen to be devoid of organelles, and
appears to consist predominantly of a course granular material.
Vesicles (dots) smaller than the wound vesicle but larger than any
normally seen in egg cytoplasm, presumably also formed during
the SW injection, are common in the immediate vicinity of the injection site. (C) At the interface of the VL with the abnormal cytoplasm is a continuous electron-dense boundary (arrows), suggesting that this is the site of SW vesicle's permeability barrier.
These images were from eggs fixed in glutaraldehyde ~10-20 min
after the SW injection. Bars, (A) 10 µm; (B and C) 1 µm.
[View Larger Version of this Image (166K GIF file)]
Fig. 8.
Ca2+ dependence for forming a wound vesicle. SW containing FS and with varying Ca2+ concentration was injected into
starfish oocytes and imaged by confocal microscopy. The ratio of
fluorescence at the injection site compared with cytoplasmic fluorescence far from the injection site was determined from measurements of the average fluorescence brightness in a small region at these two sites. A high ratio indicates containment of the
SW in a wound vesicle whereas a ratio of 1.0 indicates uniform
spreading throughout the cytoplasm. Under these conditions,
there is a threshold concentration of ~3 mM Ca2+ for forming
stable wound vesicles.
[View Larger Version of this Image (17K GIF file)]
). When 10 mM
SrCl2 added to CFSW was injected, a wound vesicle did
not form, but instead the eggs became partially activated
as indicated by fertilization envelope elevation near the injection site (three out of three sea urchin eggs; 2% volume injection). Precipitates formed when 10 mM BaCl2 was
added to CFSW, so Ba2+ was injected instead in 500 mM
NaCl. This did not form a wound vesicle nor cause fertilization envelope elevation (three out of three eggs; as control, 10 mM CaCl2 in 500 mM NaCl formed a wound vesicle in three of three eggs). Therefore, wound vesicle formation appears to be regulated specifically by Ca2+, as
opposed to divalent cations in general.
). The ER is concentrated in the clear area,
though there is some ER present among the yolk platelets (Henson et al., 1989
). At a time when knowledge of intracellular organelles was fragmentary at best, Heilbrunn
(1930b)
reported that surface wounds healed better in the
yolk platelet end of the egg. We confirmed this (data not
shown), which suggests that the yolk platelets are required
for healing of large wounds, and that the ER is not sufficient.
; Terasaki
et al., 1996
). When SW was injected at 1-2 min after the
fertilization envelope first began to rise, the wound vesicle
still formed (three out of three eggs). This also suggests
that the ER is not involved in wound vesicle formation.
DISCUSSION
), and possibly also in other mechanically challenging
cellular environments as well. It is an injury that compromises a crucial barrier function, one that is often, but not
always, survived by cells in culture and in vivo.
). As Ca2+ is present outside the cell at an
~104-fold higher concentration than in the cytosol, it is not
surprising that whole cell or population measurements reveal a rapid but transient wound-induced rise in cytosolic
Ca2+ concentration (McNeil et al., 1985
; Steinhardt et al.,
1994
). Another possibility is that loss of crucial cellular
constituents through the disruption is fatal.
,b; summarized in Heilbrunn, 1958
). He
termed this Ca2+-dependent process the "surface precipitation reaction" and likened it to blood clotting, another
process that requires Ca2+. Thus, he envisioned this reaction as involving a "coagulation" of cytoplasmic components
particularly protein filaments
which, once formed,
plugged the disruption site, and thereby repaired the surface defect, allowing the cell to survive. Due to uncertainties about the nature of the cell surface, Heilbrunn did not
advocate the possibility of Ca2+-induced membrane fusion.
). Blood clots
slow protein diffusion by only ~20% relative to water
alone (Blinc and Francis, 1996
). Proteinaceous, Ca2+-induced
precipitates of cytosol formed in vitro are readily redissolved by incubation in EGTA, showing that this membrane impermeant molecule can diffuse into such precipitates (Nakajo et al., 1984
). Lipid bilayers are the only
demonstrated biological barrier to molecule diffusion of
the kind described here in the egg response to plasma membrane disruption.
).
The torn membrane edges must rejoin and then, as a final
step at least, a fusion event must occur. Resealing in erythrocytes is slow (minutes to hours) and is apparently a Ca2+-independent process. By contrast, most nucleated
cells rapidly reseal small disruptions (within sec) by a Ca2+-dependent mechanism (Steinhardt et al., 1994
), suggesting
that nucleated and nonnucleated cells lacking internal
membranes use fundamentally different mechanisms.
;
Bi et al., 1995
). Such fusion events, which constitute an
exocytotic response, appear to be required for resealing,
but how exactly they facilitate the resealing process has
not been addressed experimentally. An alternative but potentially related mechanism that involves rapid resealing was proposed by Wohlfarth-Bottermann and Stockem
(1970)
, who removed large portions of Physarum plasma
membrane, and then prepared this giant, unicellular organism for EM at short (1-10 s) intervals thereafter. The
electron micrographs indicated that extensive vesicle-vesicle fusion was rapidly induced at the interface of naked cytoplasm with the external medium, and the result was the formation across the disruption gap of a new, continuous
plasma membrane sheet from this enlarging vesicle.
Fig. 11.
Mechanisms for
rapid resealing. (A) Small
disruptions evoke vesicle
transport to the breached
site, followed by an exocytotic reaction at this site; vesicle-plasma membrane fusion predominates. (B) A
large plasma membrane disruption evokes the rapid formation of a large membrane
sheet across the breach site,
followed by exocytotic joining of this sheet with the
plasma membrane; vesicle- vesicle fusion predominates.
See text for further discussion of this model.
[View Larger Version of this Image (16K GIF file)]
). The echinoderm egg or oocyte is able,
within 5-10 s after suffering a large disruption (~40 by 10 µm), to completely prevent the further influx of exogenous molecules, including 10-kD FDx, ~1-kD FS, and
Ca2+, down a steep concentration gradient into cytosol.
We suggest that large disruptions such as these cannot be
rapidly resealed merely by exocytotic fusion events. We
propose instead (Fig. 11 B) that Ca2+ entering through a
large disruption initiates vesicle-vesicle fusion. This forms
a large vesicular sheet of membrane, as originally envisioned by Wohlfarth-Bottermann and Stockem (1970; see
above). Fusion of this sheet with the plasma membrane,
that is, vesicle-plasma membrane fusion, then completes
resealing.
; Taylor et
al., 1980
).
;
Eddleman et al., 1997
). These axons are densely packed
with cytoskeletal structures and may not support the fusion of enough vesicles to rapidly form a complete membrane boundary. In support of this possibility, an injection
of SW is not encapsulated in the squid giant axon
(Terasaki, M., unpublished observations). Vesicles (>1 µm)
are induced by transection (Fishman et al., 1990
), and are
found to plug the wound (Krause et al., 1994
; Gallant et
al., 1995
). Vesicles may be derived from glia membranes
or from Ca2+ stimulated endocytosis from the axolemma
(Eddleman et al., 1997
). Constriction of the axonal sheath
probably also facilitates wound healing (Gallant, 1988
).
). Perhaps this high
threshold insures that the disruption-induced membrane-
membrane fusion mechanism is only activated in case of
emergency, when the cytoplasm is exposed to the potentially lethal environment of the outside world. Inadvertent
activation would waste organelles and could possibly create abnormal, and hence potentially disruptive internal boundaries.
).
), and in intact fibroblast and endothelial cells experiencing
plasma membrane disruptions (Miyake and McNeil, 1995
)
and calcium ionophore treatments (Rodriguez et al., 1997
)
that elevate cytosolic Ca2+. In axons, endocytosis is apparently induced by transection, and this newly formed vesicle population is suggested to participate in repair (Eddleman et al., 1997
). There is much uncertainty about the
origin of echinoderm egg yolk platelets (e.g., Smiley, 1990
) but it is likely that they are similar to frog egg yolk platelets, which are a derivative of endocytic membranes (Wallace and Jared, 1976
) and which have a low pH (Fagotto
and Maxfield, 1994
). Clearly, it is necessary to find out
more about echinoderm yolk platelets, because it will help
to determine in what ways these experiments with eggs are
applicable to somatic cells.
; Miyake and McNeil,
1995
), and of vesicle mobilization to wound sites before
exocytosis, are now partially characterized. Steinhardt et
al. microinjected into fertilized urchin eggs and fibroblasts
various toxins known to specifically target membrane
docking/fusion proteins, antibodies capable of blocking kinesin function, and peptides that competitively inhibit
multifunctional Ca2+/calmodulin kinase activity (Steinhardt et al., 1994
). Resealing of microneedle punctures
was inhibited by all of these reagents. It was suggested,
based on the functional requirement for this set of proteins, that resealing of small disruptions is accomplished by a docking/fusion mechanism similar in its molecular
composition to that used in neurotransmitter release at the
synapse. In addition to docking and fusion events, disruption-induced exocytosis is postulated to involve active vesicle movement to and accumulation at the disruption site.
In fact, an extremely high density of vesicles has been visualized by EM to surround disruption sites (Miyake and
McNeil, 1995
; Eddleman et al., 1997
).
). Surprisingly, however, when only one of two
vesicle populations is trypsinized, fusion is still possible
(Vogel et al., 1992
). Furthermore, in distinction from other
described mechanisms, this fusion event does not require ATP or GTP as cofactors. Yolk platelets, too, undergo homotypic fusion in vitro in the presence of Ca2+ and absence of ATP or GTP (Vogel, S.S., unpublished communication).
), to name just two of many
possible examples. New strategies for promoting the survival of plasma membrane disruptions, with the aim, for
example, of improving neuronal recovery after traumatic
injury or muscle regeneration in a wasting disease such as
Duchenne muscular dystrophy, may be possible based on
a better understanding of the wound healing process.
Address correspondence to Mark Terasaki Tel.: (860) 679-2695. Fax: (860) 679-1661. E-mail: terasaki{at}panda.uchc.edu or Paul McNeil Tel.: (706) 721-3065. Fax: (706) 721-8732. E-mail: pmcneil{at}mail.mcg.edu.
Received for publication 13 May 1997 and in revised form 28 July 1997.
Quicktime movies of most of the figures are available at http://www.uchc.edu/~terasaki/resealing.htmlWe thank A. Hand (University of Connecticut Health Center) for help with EM, J. Galbraith (Duke University, Durham, NC) for making the OMDR trigger, D. Serwanski (University of Connecticut Health Center) for technical assistance, and L.A. Jaffe (University of Connecticut Health Center) for loan of equipment. We also thank S. Vogel (Medical College of Georgia), J. Heuser (Washington University Medical School, St. Louis, MO), L.F. Jaffe (Marine Biological Laboratory), D. Kiehart (Duke University, Durham, NC), B. Ehrlich (Yale University, New Haven, CT), and B. Kaminer (Boston University Medical School, Boston, MA) for useful discussions, and L.A. Jaffe (University of Connecticut Health Center) for reading the manuscript. Most of this work was done at the Marine Biological Laboratories; we thank T. Reese (National Institutes of Health) for generously providing laboratory space and facilities.
This work was supported by grants to P.L. McNeil from the Muscular Dystrophy Association and the National Institutes of Health (48091) and by a grant to M. Terasaki from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.
CFSW, Ca2+-free sea water; Fdx, fluorescein dextran; FS, fluorescein stachyose; SW, sea water.
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