Sanders M C, Wang Y L
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
J Cell Biol. 1990 Feb;110(2):359-65. doi: 10.1083/jcb.110.2.359.
Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.
已知大多数非肌肉细胞维持着相对较高浓度的未聚合肌动蛋白。为了确定肌动蛋白的聚合是如何被调控的，通过超声处理荧光素鬼笔环肽标记的肌动蛋白丝制备的外源性成核位点被显微注射到活的瑞士3T3细胞和NRK细胞中。显微注射后，成核位点以簇状形式存在超过一小时，并且未引起细胞相形态的可检测变化。通过针对内源性肌动蛋白的免疫荧光以及用罗丹明鬼笔环肽对细胞进行染色确定，显微注射既未诱导内源性肌动蛋白从成核位点广泛聚合，也未引起肌动蛋白丝分布的变化。此外，通过整合结合的罗丹明鬼笔环肽的荧光强度估计的肌动蛋白聚合程度似乎未受影响。为了确定显微注射后成核位点是否仍然活跃，首先向细胞注射成核位点，在孵育20分钟后，再向细胞显微注射单体罗丹明标记的肌动蛋白。罗丹明标记的肌动蛋白与成核位点广泛结合，表明至少一些成核活性得以维持，并且内源性肌动蛋白的行为与外源性肌动蛋白亚基不同。同样，当含有成核位点的细胞被提取并用罗丹明标记的肌动蛋白孵育时，罗丹明标记的肌动蛋白以细胞松弛素敏感的方式与成核位点结合。这些观察结果表明，封端和成核抑制不能解释活细胞中肌动蛋白聚合的调控。然而，单体的隔离可能起着关键作用。
