Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension.
Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension.
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DOI：
10.1083/jcb.201101035
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发表时间：
2011-04-18
期刊：
影响因子：
--
通讯作者：
Mizuno K
中科院分区：
文献类型：
--
作者：
Kiuchi T;Nagai T;Ohashi K;Mizuno K
G-肌动蛋白的细胞质浓度是确定刺激诱导的 G-肌动蛋白组装和细胞延伸程度的关键参数。为了了解G-肌动蛋白浓度在细胞迁移过程中刺激诱导的肌动蛋白组装和片状足延伸中的细胞内作用，我们开发了一种量化活细胞中G-肌动蛋白浓度时空变化的新技术，包括连续测量Dronpa标记的肌动蛋白光激活后荧光衰减（FDAP）。细胞刺激后，细胞质 G 肌动蛋白浓度立即下降约 40%，此后细胞面积扩大。刺激诱导的 G-肌动蛋白丢失和细胞延伸的程度与未刺激细胞中的 G-肌动蛋白浓度呈线性相关，即使浓度远高于肌动蛋白丝的临界浓度，表明细胞质 G-肌动蛋白浓度是确定刺激诱导的 G-肌动蛋白组装和细胞延伸程度的关键参数。多点FDAP分析显示板状伪足中的G-肌动蛋白浓度与细胞体内的G-肌动蛋白浓度相当。我们还通过对 jasplakinolide 诱导的 G-肌动蛋白浓度随时间变化进行模型拟合，评估了游离 G-肌动蛋白、profilin 和胸腺素-β4 结合的 G-肌动蛋白以及肌动蛋白丝的游离带刺和尖端的细胞浓度。
The cytoplasmic concentration of G-actin is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. 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.
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