|
|
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§
* Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032; 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 ~40 by 10 µm 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 (~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 ~3 mM Ca2+
(SW is ~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.
The Marine Biological
Laboratory, Woods Hole, Massachusetts 02543; § Laboratory of Neurobiology, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Bethesda, Maryland 20892; Fukushima Medical College, Second Department of Anatomy,
Fukushima 960-12 Japan; and ¶ The Medical College of Georgia, Department of Cellular Biology and Anatomy, Augusta,
Georgia 30912
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
