Membrane resealing in cultured rat septal neurons after neurite transection: evidence for enhancement by Ca(2+)-triggered protease activity and cytoskeletal disassembly.
Neurites of cultured septal neurons were transected with a laser under sterile conditions, and the subsequent membrane resealing was assayed using a dye exclusion method. In agreement with findings in other preparations, Ca2+ enhanced resealing: in normal culture medium the percentage of lesioned neurons that resealed within 20-30 min after transection increased with increasing bath [Ca2+] over the range 10(-7) to 2 x 10(-3) M; about 75% of cells resealed in 2 mM Ca2+. Mn2+ and Sr2+ also enhanced resealing, but Mg2+ inhibited it. The percentage of resealing neurons was sensitive to agents known to alter the stability of cytoskeletal components. Agents that tend to disassemble microtubules and/or neurofilaments (e.g., colchicine, low-ionic-strength media) strongly promoted resealing, whereas treatments that tend to stabilize microtubules (taxol, Mg2+) inhibited resealing. Addition of exogenous proteases (papain, trypsin, or dispase) enhanced resealing, whereas inhibitors of cysteine proteases (including a specific inhibitor of calpain, a Ca-activated neutral protease) strongly inhibited resealing. Calmodulin inhibitors inhibited resealing, consistent with reports that calmodulin facilitates calpain-mediated proteolysis of fodrin, a component of the cortical cytoskeleton. Based on these results, we hypothesize that one of the major mechanisms involved in resealing is activation of endogenous proteases by Ca2+ entry into the injured neurite. The resulting changes in the cellular cytoskeleton might promote fusion and resealing of the cut ends of the plasma membrane by enhancing membrane mobility and/or by removing structures that normally prevent membrane-membrane contact.
Department of Physiology and Biophysics, University of Miami School of Medicine, Florida 33101.
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D000818:Animals; D001369:Axons / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure; D003714:Denervation; D010450:Endopeptidases / Q000378:metabolism; D009474:Neurons / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012688:Septum Pellucidum / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure
D011480:Protease Inhibitors; D010450:Endopeptidases; D002118:Calcium