Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site

Abstract

Resealing of a disrupted plasma membrane requires Ca2+-regulated exocytosis. Repeated disruptions reseal more quickly than the initial wound. This facilitated response requires both Ca2+ and protein kinase C (PKC), and is sensitive to brefeldin A. There is also evidence that this response is polarized to the site where the cell membrane had previously been disrupted. Observations of GFP-tagged α-tubulin and end-binding protein 1 (EB1) revealed that membrane disruption initially induced disassembly of microtubules around the wound site, followed by elongation of microtubules toward the wound site. Recruitment of EB1 to microtubules required Ca2+ influx, but was independent of PKC. NBD C6-ceramide, a probe for the Golgi apparatus and Golgi-derived lipids, initially stained the perinuclear region, and a portion of the probe was translocated to the wound site 5 minutes after wounding. Translocation of the lipids required microtubules and PKC activity, and was suppressed by low temperature. On the other hand, constitutive traffic of the lipid was still normal in the presence of a PKC inhibitor. These findings suggest that membrane disruption stimulates regulated vesicle traffic from the region of the trans-Golgi network to the wound site along rearranged microtubules in a PKC-dependent manner.

Short-term potentiation of membrane resealing in neighboring cells is mediated by purinergic signaling

“…As described previously [13,14], cell membrane disruption was indicated by a decrease in fluorescence intensity (arrows in Fig. 1a).…”

Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

“…Dysferlincontaining vesicles are thought to play a critical role in muscle resealing, but there have been very few studies attempting to directly examine the behavior of dysferlin-containing vesicles in livemuscle cells in the context of cellular wounding. Studies from non-muscle model systems suggest that microtubule-based transport of intracellular vesicles is critical for resealing (15,21), but the role of microtubules and kinesin motors in dysferlin-mediated membrane repair in muscle remains unexplored. Therefore, the aim of this study was to examine the behavior of dysferlincontaining vesicles under normal conditions and following cellular wounding in muscle cells, and test the hypothesis that microtubules and kinesin are required for dysferlin-containing vesicle function in muscle cells.…”

“…Current knowledge of membrane resealing is largely derived from studies in the sea urchin egg and fibroblast model systems, which demonstrated that fusion of intracellular vesicles with the plasma membrane is critical for resealing (15)(16)(17)21). Dysferlin localizes to the plasma membrane and intracellular vesicles in developing myotubes, and interacts with numerous proteins involved in membrane transport, including caveolin-3 (22,23), annexin-4 (5), annexin-6 (24), enlargeosomal marker AHNAK (25) and tubulin (26), but the exact contribution of dysferlincontaining vesicles to resealing following wounding remains elusive, as few studies have examined the behavior of dysferlincontaining vesicles in live cells following cellular wounding.…”

Effects of extracellular calcium on cell membrane resealing in sonoporation

“…Importantly, the removal of extracellular calcium blocked healing and prevented recruitment of F-actin and myosin-II [43]. Studies in cultured fibroblast cells [44] showed similarly that membrane disruption stimulates cytoskeletal response that is Ca 2+ -dependent, including disassembly of microtubules (MTs) around the wounding site, recruitment of end binding protein (EB1) to the MTs especially around the wound site, and subsequent elongation of MTs towards the wounding site. Such cytoskeletal response was found to require calcium at concentrations above 0.4 mM and has a higher rate at higher levels of [Ca 2+ ].…”