Localization and dynamics of nonfilamentous actin in cultured cells.
Localization and dynamics of nonfilamentous actin in cultured cells.
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DOI：
10.1083/jcb.123.1.173
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发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
Wang YL
中科院分区：
文献类型：
--
作者：
Cao LG;Fishkind DJ;Wang YL
虽然丝状肌动蛋白在许多细胞类型中的分布已被很好地描述，但对非丝状肌动蛋白的分布仍知之甚少。为了确定丝状和非丝状肌动蛋白在培养的NRK细胞中的相对分布，我们使用了许多针对丝状和非丝状肌动蛋白的特异性不同的标记剂，包括单克隆和多克隆抗肌动蛋白抗体、维生素D结合蛋白(DBP)和荧光素。大量的斑点结构被鉴定出来，它们与鬼臼蛋白结合很差，但与一些抗肌动蛋白抗体呈阳性染色。这些珠状结构还被DBP染色，表明它们富含非丝状肌动蛋白。在将荧光标记的肌动蛋白显微注射到活细胞中后，也观察到了类似的点状结构，这使我们能够检查它们在活细胞中的动态。含有肌动蛋白的点状结构主要见于板脂细胞后的区域，尤其是在损伤融合单层诱导的铺展细胞中。注射了荧光肌动蛋白的细胞的时间推移记录表明，它们在前沿附近连续形成，并向着细胞核移动。我们的结果表明，至少部分未聚合的肌动蛋白分子定位在不连续的位置，可能是作为与单体隔离蛋白的复合体。这些结构可能代表G-肌动蛋白在细胞内的瞬时存储位置，在特定信号的刺激下，G-肌动蛋白可以迅速转化为肌动蛋白细丝。
Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.