Spatiotemporal dynamics of actin concentration during cytokinesis and locomotion in Dictyostelium.
Spatiotemporal dynamics of actin concentration during cytokinesis and locomotion in Dictyostelium.
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发表时间：
1998-08
影响因子：
4
通讯作者：
S. Yumura;Yoshio Fukui
中科院分区：
文献类型：
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作者：
S. Yumura;Yoshio Fukui
为了研究肌动蛋白细胞骨架的时空调控，我们分析了活盘基骨中肌动蛋白浓度的动态变化。荧光形态学分析相对肌动蛋白浓度对细胞行为的影响。我们将罗丹明-肌动蛋白电穿孔到盘状骨细胞中，获得了200-300毫秒的时间分辨率和大约250纳米的空间分辨率的图像。为了将荧光强度转化为肌动蛋白浓度，在近二维细胞上进行观察，并将肌动蛋白信号与体积标记物（FITC-BSA或GFP）进行比值。由于FITC和GFP的发射是pH依赖性的，我们首先测量了活细胞的细胞质pH，并确定假足的pHi与一般细胞质的pHi相同。在细胞质分裂过程中，卵裂沟中肌动蛋白的相对浓度明显高于一般细胞质。在迁移细胞中，肌动蛋白的募集速度惊人，尤其是在假足中。我们发现，当假足伸出或收缩时，高肌动蛋白浓度的区域相对于前缘移动。当假足收缩时，肌动蛋白密度在5秒内消散。我们还发现肌动蛋白在假足发育过程中以振荡的方式积累，并且这个时间与质心的前进相协调。这是首次揭示活细胞中肌动蛋白相对浓度的动态变化，并定量地将这些变化与变形虫的运动行为联系起来的研究。
To study the spatial and temporal regulation of the actin cytoskeleton, we have analyzed the actin concentration dynamics in live Dictyostelium. The relative actin concentration was analyzed with respect to cell behavior by fluorescence morphometry. We electroporated rhodamine-actin into Dictyostelium cells and acquired images with 200-300 millisecond temporal and approximately 250 nm spatial resolutions. To convert fluorescence intensity into actin concentration, the observation was made on nearly two-dimensional cells, and the actin signal was ratioed over a volume marker (FITC-BSA or GFP). Since the emission of FITC and GFP is pH-dependent, we first measured the cytoplasmic pH in live cells and determined that the pHi in pseudopods is same as that of general cytoplasm. During cytokinesis, the relative concentration of actin in the cleavage furrow was significantly higher than in the general cytoplasm. In migrating cells, actin was recruited surprisingly rapidly, particularly in the pseudopod. We found that the region of high actin concentration moves relative to the leading edge when a pseudopod projects or retracts. When the pseudopod retracts, the actin density dissipates within 5 seconds. We have also found that actin accumulates in developing pseudopods in an oscillatory manner, and this timing coordinates with advancement of the centroid. This is the first study to reveal the dynamic changes in relative concentration of actin in live cells and to quantitatively correlate these changes with the locomotive behavior of the amoeba.