Showing papers on "Plasma membrane repair published in 1999"

The mechanism of facilitated cell membrane resealing

Abstract: Disruption of the plasma membrane evokes an exocytotic response that is required for rapid membrane resealing. We show here in Swiss 3T3 fibroblasts that a second disruption at the same site reseals more rapidly than the initial wound. This facilitated response of resealing was inhibited by both low external Ca2+ concentration and specific protein kinase C (PKC) inhibitors, bisindolylmaleimide I (BIS) and Go-6976. In addition, activation of PKC by phorbol ester facilitated the resealing of a first wound. BIS and Go-6976 suppressed the effect of phorbol ester on resealing rate. Fluorescent dye loss from a FM1-43 pre-labeled endocytotic compartment was used to investigate the relationship between exocytosis, resealing and the facilitation of resealing. Exocytosis of endocytotic compartments near the wounding site was correlated with successful resealing. The destaining did not occur when exocytosis and resealing were inhibited by low external Ca2+ concentration or by injected tetanus toxin. When the dye loaded cells were wounded twice, FM1-43 destaining at the second wound was less than at the first wound. Less destaining was also observed in cells pre-treated with phorbol ester, suggesting newly formed vesicles, which were FM1-43 unlabeled, were exocytosed in the resealing at repeated woundings. Facilitation was also blocked by brefeldin A (BFA), a fungal metabolite that inhibits vesicle formation at the Golgi apparatus. Lowering the temperature below 20 degrees C also blocked facilitation as expected from a block of Golgi function. BFA had no effect on the resealing rate of an initial wound. The facilitation of the resealing by phorbol ester was blocked by pre-treatment with BFA. These results suggest that at first wounding the cell used the endocytotic compartment to add membrane necessary for resealing. At a second wounding, PKC, activated by Ca2+ entry at the first wound, stimulated vesicle formation from the Golgi apparatus, resulting in more rapid resealing of the second membrane disruption. Since vesicle pools were implicated in both membrane resealing and facilitation of membrane resealing, we reasoned that artificial decreases in membrane surface tension would have the same result. Decreases in surface tension induced by the addition of a surfactant (Pluronic F68 NF) or cytochalasin D facilitated resealing at first wounding. Furthermore, Pluronic F68 NF restored resealing when exocytosis was blocked by tetanus toxin. These results suggest that membrane resealing requires a decrease in surface tension and under natural conditions this is provided by Ca2+-dependent exocytosis of new membrane near the site of disruption.

The mechanism of cell membrane repair

Abstract: Disruption of plasma membranes is a widespread, common and normal event that occurs in many mechanically challenged tissues (McNeil & Steinhardt, 1997). After injury to the plasma membrane, rapid resealing of the membrane occurs with little loss of intracellular contents. Analysis of plasma membrane repair in the sea urchin egg and early embryos revealed a new model of the mechanism for plasma membrane repair. Resealing of disrupted plasma membranes required external Ca2+ that could be antagonised by Mg2+. Block of Ca2+/calmodulin kinase II, which regulates exocytotic vesicle availability at synapses (Llinas et al., 1991), inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins A, B, C1, and tetanus toxin, which disrupt SNARE vesicle docking/fusion proteins. Confocal microscopic observations of exocytotic events in sea urchin eggs and embryos during membrane resealing showed that inhibition of kinesin or myosin motor activity, which are believed to be required for vesicle transport (Goodson et al., 1997), also inhibited membrane resealing and delivery of vesicles to sites of membrane disruption. This pattern of inhibition indicates that membrane repair of micrometre-sized lesions requires vesicle delivery, docking and fusion, similar to the exocytosis of neurotransmitter (Steinhardt et al., 1994; Bi et al., 1995, 1997). The mechanism of resealing in eggs and embyros was found to be a general property of all cells (Steinhardt et al., 1994; Togo et al., 1999). It is now known that elevated intracellular Ca2+ triggers exocytosis in various types of cells (Dan & Poo, 1992; Coorssen et al., 1996), and that endosomal compartments such as lysosomes can behave as Ca2+-regulated exocytotic vesicles (Rodriguez et al., 1997).