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

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

Author information

1
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan. tai.kiuchi@kcl.ac.uk

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.

PMID:
21502360
PMCID:
PMC3080261
DOI:
10.1083/jcb.201101035
[Indexed for MEDLINE]
Free PMC Article

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