期刊
JOURNAL OF CELL BIOLOGY
卷 193, 期 2, 页码 365-380出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.201101035
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资金
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research [11J07828, 21370086, 22121501] Funding Source: KAKEN
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 similar to 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 stimulusinduced 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-beta 4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.
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