Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I

Neuron. 2002 Dec 5;36(5):805-13. doi: 10.1016/s0896-6273(02)01068-1.

Abstract

We demonstrate a noninvasive technique for protein photoinactivation using a transgenically encoded tag. A tetracysteine motif that binds the membrane-permeable fluorescein derivative 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) was engineered into synaptotagmin I (Syt I4C). Neuronally expressed Syt I4C rescues the syt I null mutation, can be visualized after FlAsH labeling, and is normally distributed at the Drosophila neuromuscular synapse. Illumination of FlAsH bound Syt I4C at 488 nm decreases evoked release in seconds demonstrating efficient fluorophore-assisted light inactivation (FlAsH-FALI) of Syt I. The inactivation of Syt I is proportional to the duration of illumination and follows first-order kinetics. In addition, Syt I FlAsH-FALI is specific and does not impair Syt I-independent vesicle fusion. We demonstrate that Syt I is required for a post-docking step during vesicle fusion but does not function to stabilize the docked vesicle state.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Calcium / metabolism
  • Calcium-Binding Proteins*
  • Cysteine / genetics
  • Cysteine / metabolism
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / physiology
  • Electrophysiology
  • Epitopes / genetics
  • Epitopes / metabolism
  • Female
  • Fluoresceins / metabolism*
  • Indicators and Reagents / metabolism
  • Light*
  • Male
  • Membrane Fusion / physiology
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Membrane Potentials / physiology
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurons / physiology
  • Neurotransmitter Agents / metabolism
  • Organometallic Compounds / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Synapses / metabolism*
  • Synaptic Transmission / physiology
  • Synaptic Vesicles / metabolism
  • Synaptotagmin I
  • Synaptotagmins
  • Transgenes

Substances

  • 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein
  • Calcium-Binding Proteins
  • Drosophila Proteins
  • Epitopes
  • Fluoresceins
  • Indicators and Reagents
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Neurotransmitter Agents
  • Organometallic Compounds
  • Recombinant Fusion Proteins
  • Synaptotagmin I
  • Synaptotagmins
  • Cysteine
  • Calcium