Vesicle accumulation and exocytosis at sites of plasma membrane disruption.

Abstract

Abstract. Plasma membrane disruptions are resealed by an active molecular mechanism thought to be composed, in part, of kinesin, CaM kinase, snap-25, and synaptobrevin. We have used HRP to mark the cytoplasmic site of a mechanically induced plasma membrane disruption. Transmission electron microscopy revealed that vesicles of a variety of sizes rapidly (s) accumulate in large numbers within the cytoplasm surrounding the disruption site and that microvilli-like surface projections overlie this region. Scanning electron microscopy confirmed that tufts of microvilli rapidly appear on wounded cells. Three assays, employing the membrane specific dye FM1-43, provide quantitative evidence that disruption induces Ca2+-dependent exocytosis involving one or more of the endosomal/lysosomal compartments. Confocal microscopy revealed the presence in wounded cells of cortical domains that were strikingly depleted of FM dye fluorescence, suggesting that a local bolus of exocytosis is induced by wounding rather than global exocytosis. Finally, flow cytometry recorded a disruption-induced increase in cell forward scatter, suggesting that cell size increases after injury. These results provide the first direct support for the hypothesis that one or more internal membrane compartments accumulate at the disruption site and fuse there with the plasma membrane, resulting in the local addition of membrane to the surface of the mechanically wounded cell.

Long-Term Potentiation of Exocytosis and Cell Membrane Repair in Fibroblasts

“…Ca 2ϩ -regulated exocytosis, which requires vesicle docking/ fusion SNARE proteins, has been shown to be essential for resealing of micrometer-sized membrane disruptions in mammalian cells and invertebrate embryos (Steinhardt et al, 1994;Bi et al, 1995Bi et al, , 1997Miyake and McNeil, 1995;Togo et al, 1999;Reddy et al, 2001).…”

“…If a cell experiences a membrane disruption in the micrometer diameter range, Ca 2ϩ influx at the wound site triggers exocytosis that is essential for successful cell membrane repair (Steinhardt et al, 1994;Bi et al, 1995Bi et al, , 1997Miyake and McNeil, 1995;Togo et al, 1999;Reddy et al, 2001). Both membrane repair and neurotransmission depend on Ca 2ϩ -regulated exocytosis that is inhibited by clostridial neurotoxins and tetanus toxin (Steinhardt et al, 1994;Bi et al, 1995;Togo et al, 1999).…”

Long-term Potentiation of Wound-induced Exocytosis and Plasma Membrane Repair Is Dependant on cAMP-response Element-mediated Transcription via a Protein Kinase C- and p38 MAPK-dependent Pathway

“…Small disruptions on the order of micron-diameter evoke the Ca 2ϩ -dependent exocytosis of vesicles near the wound site, which is essential for successful membrane resealing (2)(3)(4)(5)(6)(7)(8). The recruitment of vesicles to docking sites near the disruption is dependent on the motor proteins, kinesin and myosin, and the activity of Ca 2ϩ /calmodulin-dependent kinase (CaMK) 1 (2,4,9).…”

The endomembrane requirement for cell surface repair

“…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.…”