Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells.
The mechanism of bulk membrane uptake at the synapse remains poorly defined, although exocytosis of synaptic vesicles is followed by compensatory membrane retrieval into both small vesicles and large cisternas or vacuoles. We investigated bulk retrieval in the presynaptic terminal of retinal bipolar cells. Fluorescence imaging of the membrane dye FM1-43 indicated that Ca2+-triggered exocytosis was followed by endocytosis into small vesicles and larger vacuoles that could be selectively labeled using large fluorescent dextrans. Disruption of actin filaments with cytochalasin D or latrunculin B inhibited the formation and transport of vacuoles, but exocytosis and endocytosis continued at normal rates. Bulk retrieval was linked to remodeling of the actin network, and both processes were inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). The regulation of F-actin dynamics by Ca2+ and PI 3-kinase therefore played an important role in compensatory endocytosis at this synapse, but this role was confined to bulk membrane uptake. Capacitance measurements demonstrated that fast endocytosis and refilling of the rapidly releasable pool of vesicles were not dependent on F-actin or PI 3-kinase activity. The basic properties of bulk membrane retrieval at this synapse were very similar to macropinocytosis described in non-neural cells. Bulk retrieval did not play an essential role in maintaining the vesicle cycle during maintained stimulation, but we suggest that it may play a role in the structural plasticity of this synaptic terminal.
Holt
Matthew
M
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.
Cooke
Anne
A
Wu
Minnie M
MM
Lagnado
Leon
L
D008841:Actin Cytoskeleton / Q000502:physiology; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003911:Dextrans / Q000378:metabolism; D004705:Endocytosis; D006054:Goldfish; D007700:Kinetics; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010873:Pinocytosis; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D012160:Retina / Q000166:cytology* / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism
D003911:Dextrans; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium