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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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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.
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Figure 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.

Mentions: 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; 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.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 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.
Mentions: 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; 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.

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