Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension

J Cell Biol. 2011 Apr 18;193(2):365-80. doi: 10.1083/jcb.201101035.

Abstract

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ∼40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. Multipoint FDAP analysis revealed that G-actin concentration in lamellipodia was comparable to that in the cell body. We also assessed the cellular concentrations of free G-actin, profilin- and thymosin-β4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / drug effects
  • Actin Cytoskeleton / physiology
  • Actins / physiology*
  • Antineoplastic Agents / pharmacology
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Cell Movement / physiology
  • Depsipeptides / pharmacology
  • Humans
  • Profilins / physiology
  • Pseudopodia / drug effects
  • Pseudopodia / physiology*
  • Thymosin / physiology

Substances

  • Actins
  • Antineoplastic Agents
  • Depsipeptides
  • Profilins
  • jasplakinolide
  • thymosin beta(4)
  • Thymosin