Reorganization of actin in depolarized synaptosomes.
Depolarization of whole brain synaptosomes, which stimulates transmitter release, also affects regulation of the assembly of actin microfilaments. Lysates of depolarized synaptosomes contain 20% less cytoskeletal actin than lysates of unstimulated synaptosomes. Parameters affecting the assembly of actin are modified before lysis, but release of actin from the Triton-insoluble cytoskeleton does not occur until after lysis. Actin released from the cytoskeleton is not precipitated with myosin, indicating that it consists of monomers and/or short oligomers. Synaptosomes were incubated for 12 sec in one of three solutions of identical ionic strength but of different salt mixtures: 75 mM KCl-2 mM CaCl2, 5 mM KCl-2mM CaCl2, or 75 mM KCl-0.1 mM EGTA. Synaptosomes were then lysed in an F-actin stabilizing buffer containing 1% Triton X-100. Control synaptosomes (no incubation) were lysed directly into the same lysis buffer containing one of the three different salt mixtures. The cytoskeletal and noncytoskeletal actin pools were separated 25 sec after lysis by centrifugation at 10(4) X g for 1 min, and the actin in each pool was quantitated by the DNase I inhibition assay. The drop in cytoskeletal actin induced by depolarization is maximized by including Ca2+ in the depolarizing buffer, and it is blocked completely by adding a neutral thiol protease inhibitor, leupeptin, to either the pre- or post-lysis buffer. The drop is also completely reversed by repolarizing the synaptosomes.
Bernstein
B W
BW
Bamburg
J R
JR
D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism; D004594:Electrophysiology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D011817:Rabbits; D013574:Synaptosomes / Q000201:enzymology / Q000502:physiology*
D000199:Actins; D018377:Neurotransmitter Agents; D007770:L-Lactate Dehydrogenase