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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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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.
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Figure 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.

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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139822&req=5

Figure 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.
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