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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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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.
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Figure 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.

Mentions: 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.


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

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

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.
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Figure 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.
Mentions: 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.

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