title: Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension.
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.
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan. tai.kiuchi@kcl.ac.uk
authors.forename: Tai
authors.initials: T
authors.lastname: Kiuchi
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Nagai
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohashi
authors.forename: Kensaku
authors.initials: K
authors.lastname: Mizuno
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology; D000199:Actins / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D047630:Depsipeptides / Q000494:pharmacology; D006801:Humans; D051304:Profilins / Q000502:physiology; D011554:Pseudopodia / Q000187:drug effects / Q000502:physiology*; D013947:Thymosin / Q000502:physiology; D000099201:Thymosin beta(4)
chemical_list: D000199:Actins; D000970:Antineoplastic Agents; D047630:Depsipeptides; D051304:Profilins; D013947:Thymosin; D000099201:Thymosin beta(4); C057531:jasplakinolide