Live Imaging for Two-photon microscopy: Membrane repair in Squid Gian Axon.

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Plasma membrane disruption is a common form of cell injury in mammalian tissues under physiological conditions. Cell survival depends on the initiation of a rapid (second time-scale) resealing response that is mounted only in the presence of physiological levels of extracellular Ca2+. Vesicle-vesicle and vesicle-plasma membrane fusion events occurring in cortical cytoplasm surrounding the defect are thought to be a crucial element of the resealing mechanism. Axolemmal repair has been also studied extensively in invertebrate giant axons because their large size facilitates the use of many techniques. However, it is not clear how to repair axolemmal injury which usually takes minutes to hours time scale. Therefore, we observed how to reseal axolemmal disruption with several fluorescent probes, FM dye, Calcein-AM, Rhodamin or FITC-Dextran(RhDx, FDx, 10kD) and fluo4-AM using multi-photon microscope. First , we examined individual narrow axons from the squid fin nerve bundle. FM1-43 was loaded by the cut ends of fin nerves to show internal vesicles, and then wounded the axolemma by two photon laser. The FM1-43-positive vesicles were recruited the way of the membrane -membrane contacts leading to the homotypic and very rapid (second time scale) exocytic fusion events required for membrane repair in the narrow fin nerve. Whereas, FM 1-43 or Ca2+by fluo4-AM did not get into the axoplasm of the giant axon through the axolemmal disruption. Furthermore, when natural sea water containing FDx (10kD) was injected, the fluorescence spread throughout the axoplasm. These results suggest that high Ca2+did not cause rapid fusion of intracellular membranes, creating a boundary that prevents spreading of FDx throughout the cytoplasm. We propose that the slow speed of calcium diffusion through axoplasm density leads to the slow axolemmal repair.