Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

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 ∼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 endomembrane requirement for cell surface repair

“…NIH 3T3 fibroblasts were harvested by trypsinization from logarithmic-phase cultures. Eggs were obtained from Strongylocentrotus purpuratus (Marinus, Long Beach, CA) as described (6) and used in experiments within 4 h. Artificial seawater was prepared, with or without a 10 mM CaCl 2 addition, as described (6). Primary cultures of hippocampal neuron cultures were prepared as described (13).…”

“…The principal function proposed for this extracellular Ca 2ϩ is as signal-triggering homotypic and heterotypic membrane fusion responses. Thus, Ca 2ϩ -dependent exocytotic (cytoplasmic vesicle-plasma membrane) and homotypic (vesicle-vesicle) fusion events have clearly been demonstrated to correlate spatially, temporally, and quantitatively with the resealing response in fibroblasts and eggs (4)(5)(6). If these cytoplasmic membrane-dependent fusion events are required for, and are not simply correlates of, rapid resealing, then a simple prediction follows: a cell lacking cytoplasmic membrane (endomembrane) will be incapable of rapid resealing.…”

Embryo stability and vulnerability in an always changing world

“…The protective mechanism is calcium-activated membrane sealing (15). When the membrane is broken, the influx of extracellular calcium ions triggers fusion of intracellular vesicles at the site of injury (15,16). The added vesicle membranes plug and fuse with the injured membrane so that embryo integrity is maintained.…”

“…Embryologists make use of this whenever they microinject eggs or embryos; the cells survive, and the embryos develop normally. The protective mechanism is calcium-activated membrane sealing (15). When the membrane is broken, the influx of extracellular calcium ions triggers fusion of intracellular vesicles at the site of injury (15,16).…”

Plasma Membrane Ordering Agent Pluronic F-68 (PF-68) Reduces Neurotransmitter Uptake and Release and Produces Learning and Memory Deficits in Rats

“…Third, increased PMO brought about by either cholesterol enrichment of the plasma membrane or exposure to a membrane ordering agent results in an increase in mechanical shear-induced plasma membrane wounding (Clarke and McNeil 1992;Clarke et al 1995). It is interesting to note that the cellular processes required for membrane resealing of a plasma membrane wound (i.e., a calcium-dependent mobilization of an intracellular membrane pool that fuses with the plasma membrane at the site of the membrane wound) (Miyake and McNeil 1995;Terasaki et al 1997) have been shown to be similar to those associated with synaptic vesicle fusion during neuronal transmission (Steinhardt et al 1994). These data suggest that conditions that inhibit membrane wound resealing and hence increase susceptibility to mechanically induced plasma membrane wounding (i.e., increased PMO) may also inhibit synaptic transmission.…”