Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction.
Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for beta-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.
Department of Physiology and Biophysics/C-240, University of Colorado Medical School, 4200 East Ninth Avenue, Denver, CO 80262, USA.
D A
DA
Richards
S O
SO
Rizzoli
W J
WJ
Betz
D000109:Acetylcholine / Q000378:metabolism; D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects; D004594:Electrophysiology; D004705:Endocytosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000502:physiology; D008839:Microelectrodes; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009045:Motor Endplate / Q000187:drug effects / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000648:ultrastructure; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017729:Presynaptic Terminals / Q000187:drug effects; D011894:Rana pipiens; D015398:Signal Transduction / Q000187:drug effects; D013844:Thiazoles / Q000494:pharmacology*; D053778:Thiazolidines; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013844:Thiazoles; D053778:Thiazolidines; D000109:Acetylcholine; C037067:latrunculin A; D000077191:Wortmannin