Verkhovsky A B, Borisy G G
A. N. Belozersky Laboratory of Molecular Biology, Moscow State University, Russia.
J Cell Biol. 1993 Nov;123(3):637-52. doi: 10.1083/jcb.123.3.637.
The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.
在采用一种揭示肌球蛋白潜在形态的新方法后，通过相关荧光显微镜和电子显微镜研究了成纤维细胞片层中肌球蛋白的组织情况。使用一种与传统平滑肌肌球蛋白（肌球蛋白II）的X-罗丹明类似物，在显微注射后与内源性肌球蛋白共定位，以追踪活成纤维细胞中肌球蛋白的分布。然后，通过铂复型电子显微镜检查相同的细胞。为了可视化肌球蛋白的结构排列，必须去除其他细胞骨架纤维结构：在注射肌球蛋白之前，用诺考达唑处理活细胞使微管解聚；持续的诺考达唑处理还诱导中间丝聚集在细胞核附近，从而将它们从片层区域去除；通过用重组凝溶胶蛋白孵育细胞骨架使细胞裂解后去除肌动蛋白丝。通过荧光显微镜检查这种处理引起的肌球蛋白组织的可能变化。在诺考达唑处理的细胞和对照细胞之间未观察到肌球蛋白分布模式的显著差异。细胞裂解和肌动蛋白的耗尽也未诱导肌球蛋白的重组，这通过直接比较活细胞状态下和凝溶胶蛋白处理后相同细胞中肌球蛋白的分布得以证明。对肌动蛋白耗尽的细胞骨架的良好铺展的周边区域进行电子显微镜检查，发现了一个双极肌球蛋白微丝网络，它们在其末端球状区域相互收缩。该网络的形态与通过荧光显微镜观察到的肌球蛋白分布非常吻合。提出了一种通过肌球蛋白丝网络折叠进行细胞收缩的新机制。
