In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans

Nat Commun. 2014 Sep 18;5:4974. doi: 10.1038/ncomms5974.

Abstract

Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans Proteins / metabolism*
  • Calcium / metabolism
  • Calcium Channels / metabolism
  • Cell Membrane / metabolism
  • Diffusion
  • Dystrophin / genetics*
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / metabolism
  • Homeostasis
  • Membrane Proteins / metabolism
  • Microscopy, Fluorescence
  • Mutation*
  • Recombinant Fusion Proteins / metabolism
  • Sarcolemma / metabolism

Substances

  • Caenorhabditis elegans Proteins
  • Calcium Channels
  • Dystrophin
  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Calcium