Clathrin Dependence of Synaptic-Vesicle Formation at the Drosophila Neuromuscular Junction

Curr Biol. 2008 Mar 25;18(6):401-9. doi: 10.1016/j.cub.2008.02.055.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Clathrin Light Chains / metabolism*
  • Drosophila / metabolism*
  • Electrophysiology
  • Endocytosis / physiology
  • Fluoresceins
  • Larva / metabolism
  • Neuromuscular Junction / metabolism*
  • Neuromuscular Junction / ultrastructure
  • Organometallic Compounds
  • Synaptic Membranes / metabolism*
  • Synaptic Membranes / ultrastructure
  • Synaptic Transmission / physiology
  • Synaptic Vesicles / metabolism*
  • Synaptic Vesicles / ultrastructure

Substances

  • 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein
  • Clathrin Light Chains
  • Fluoresceins
  • Organometallic Compounds