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Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

Terasaki M, Miyake K, McNeil PL - J. Cell Biol. (1997)

Bottom Line: We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle.This entrapment did not occur in Ca2+ -free SW (CFSW).This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington 06032, USA. terasaki@panda.uchc.edu

ABSTRACT
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 approximately 40 by 10 microm 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 (approximately 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 approximately 3 mM Ca2+ (SW is approximately 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.

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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.
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Figure 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.

Mentions: 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).


Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

Terasaki M, Miyake K, McNeil PL - J. Cell Biol. (1997)

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.
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Figure 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.
Mentions: 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).

Bottom Line: We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle.This entrapment did not occur in Ca2+ -free SW (CFSW).This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington 06032, USA. terasaki@panda.uchc.edu

ABSTRACT
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 approximately 40 by 10 microm 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 (approximately 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 approximately 3 mM Ca2+ (SW is approximately 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.

Show MeSH
Related in: MedlinePlus