Reduced protein diffusion rate by cytoskeleton in vegetative and polarized Dictyostelium cells
Reduced protein diffusion rate by cytoskeleton in vegetative and polarized Dictyostelium cells
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DOI：
10.1016/s0006-3495(01)75851-1
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发表时间：
2001-10-01
影响因子：
3.4
通讯作者：
Wiersma, DA
中科院分区：
文献类型：
--
作者：
Potma, EO;de Boeij, WP;Wiersma, DA
进行高空间分辨率光漂白测量后的荧光恢复，以阐明肌动蛋白细胞骨架对盘基网柄菌阿米巴水域中绿色荧光蛋白 (GFP) 翻译迁移率的影响。在盘基网柄菌营养细胞中，相对于稀水溶液，GFP 分子的平移迁移率降低了 3.6 倍。在使用 latrunculin-A 破坏肌动蛋白丝状网络时，完整的肌动蛋白细胞骨架网络显示出 1.36 cP 的有效粘度，占 GFP 受限分子扩散的 53%。其余 47% 的蛋白质运动受阻归因于其他机械障碍和细胞液体的粘度。此外，根据不同渗透条件下的测量，建立了肌动蛋白网络的密度与其对蛋白质扩散的限制作用之间的直接相关性。在高度运动的极化细胞中，肌动蛋白丝状网络的阻碍作用在细胞的非皮质区域下降至 0.46 cP。我们的结果表明，肌动蛋白丝的网络构成了蛋白质扩散的主要机械屏障，并且网络的任何明显重组都伴随着细胞内蛋白质流动性的改变。
Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.