Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139822&req=5

Figure 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.

Mentions: 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+).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 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.
Mentions: 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+).

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