Cao L G, Fishkind D J, Wang Y L
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
J Cell Biol. 1993 Oct;123(1):173-81. doi: 10.1083/jcb.123.1.173.
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.
尽管丝状肌动蛋白在许多细胞类型中的分布已得到充分表征，但非丝状肌动蛋白的分布仍知之甚少。为了确定培养的NRK细胞中丝状和非丝状肌动蛋白的相对分布，我们使用了多种标记剂，这些标记剂对丝状或非丝状形式的特异性不同，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）和荧光鬼笔环肽。我们发现了许多点状结构，它们与鬼笔环肽结合不佳，但用几种抗肌动蛋白抗体染色呈阳性。这些珠状结构也能用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后，也观察到了类似的点状结构，这使我们能够研究它们在活细胞中的动态变化。含肌动蛋白的点状结构主要在片状伪足后面的区域观察到，特别是在由汇合单层细胞损伤诱导的铺展细胞中。对注射了荧光肌动蛋白的细胞进行延时记录表明，它们在前沿附近不断形成，并向细胞核向心移动。我们的结果表明，至少部分未聚合的肌动蛋白分子定位于离散位点，可能是以与单体隔离蛋白形成复合物的形式存在。这些结构可能代表细胞内G-肌动蛋白的瞬时储存位点，在特定信号刺激下可迅速转化为肌动蛋白丝。
