Clathrin dependence of synaptic-vesicle formation at the Drosophila neuromuscular junction.
BACKGROUND
Among the most prominent molecular constituents of a recycling synaptic vesicle is the clathrin triskelion, composed of clathrin light chain (Clc) and clathrin heavy chain (Chc). Remarkably, it remains unknown whether clathrin is strictly necessary for the stimulus-dependent re-formation of a synaptic vesicle and, conversely, whether clathrin-independent vesicle endocytosis exists at the neuronal synapse.


RESULTS
We employ FlAsH-FALI-mediated protein photoinactivation to rapidly (3 min) and specifically disrupt Clc function at the Drosophila neuromuscular junction. We first demonstrate that Clc photoinactivation does not impair synaptic-vesicle fusion. We then provide electrophysiological and ultrastructural evidence that synaptic vesicles, once fused with the plasma membrane, cannot be re-formed after Clc photoinactivation. Finally, we demonstrate that stimulus-dependent membrane internalization occurs after Clc photoinactivation. However, newly internalized membrane fails to resolve into synaptic vesicles. Rather, newly internalized membrane forms large and extensive internal-membrane compartments that are never observed at a wild-type synapse.


CONCLUSIONS
We make three major conclusions. (1) FlAsH-FALI-mediated protein photoinactivation rapidly and specifically disrupts Clc function with no effect on synaptic-vesicle fusion. (2) Synaptic-vesicle re-formation does not occur after Clc photoinactivation. By extension, clathrin-independent "kiss-and-run" endocytosis does not sustain synaptic transmission during a stimulus train at this synapse. (3) Stimulus-dependent, clathrin-independent membrane internalization exists at this synapse, but it is unable to generate fusion-competent, small-diameter synaptic vesicles.
Heerssen
Heather
H
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158, USA.
Fetter
Richard D
RD
Davis
Graeme W
GW
D000818:Animals; D033941:Clathrin Light Chains / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism*; D004594:Electrophysiology; D004705:Endocytosis / Q000502:physiology; D005452:Fluoresceins; D007814:Larva / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000648:ultrastructure; D009942:Organometallic Compounds; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D033941:Clathrin Light Chains; D005452:Fluoresceins; D009942:Organometallic Compounds