MICROINJECTION OF VILLIN INTO CULTURED-CELLS INDUCES RAPID AND LONG-LASTING CHANGES IN CELL MORPHOLOGY BUT DOES NOT INHIBIT CYTOKINESIS, CELL MOTILITY, OR MEMBRANE RUFFLING
MICROINJECTION OF VILLIN INTO CULTURED-CELLS INDUCES RAPID AND LONG-LASTING CHANGES IN CELL MORPHOLOGY BUT DOES NOT INHIBIT CYTOKINESIS, CELL MOTILITY, OR MEMBRANE RUFFLING
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DOI：
10.1083/jcb.111.6.2475
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发表时间：
1990-12-01
影响因子：
7.8
通讯作者：
BRETSCHER, A
中科院分区：
文献类型：
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作者：
FRANCK, Z;FOOTER, M;BRETSCHER, A
绒毛蛋白是一种钙离子调控的F-肌动蛋白成束、切断、加帽和成核蛋白，是小肠刷状缘微绒毛核心的主要成分。其肌动蛋白结合特性、组织特异性和细胞分化过程中的表达表明，它可能参与肠上皮细胞中微丝的组织形成刷状缘。最近，Friederich等人，（Friederich，E.，C. Huet，M. Arpin和D.卢瓦德1989. Cell. 59：461-475）显示，瞬时转染的成纤维细胞中的绒毛蛋白表达导致应力纤维的损失和一些细胞上大细胞表面微绒毛的出现。在这里，我们描述了绒毛蛋白显微注射到通常缺乏这种蛋白质的细胞中的效果，这使我们能够研究引入不同浓度的绒毛蛋白对微丝组织和功能的直接和长期影响。显微注射的细胞迅速失去了它们的应力纤维和肌动蛋白重组成丰富的绒毛含有皮质结构，包括microspikes，并在约一半的细胞，大表面微绒毛。肌动蛋白组织的这种变化在细胞中持续至少24小时，在此期间，它们经历了三次细胞分裂中的两次。微注射绒毛核心，缺乏成束活性的绒毛，但保留所有的Ca 2+依赖的属性，破坏了应力纤维系统，并没有影响细胞表面形态。因此，绒毛蛋白的Ca 2+依赖性活动负责应力纤维的破坏，而细胞表面结构的产生是其集束活动的结果。微注射绒毛蛋白导致肌球蛋白、原肌球蛋白和α-辅肌动蛋白的重组，这些蛋白通常与应力纤维相关，而fimarin和ezrin，它们也是微绒毛核心丝的组分，很容易被吸收到诱导的表面结构中。在局灶性粘连中，骨钙素也从其正常位置重新分布。尽管肌动蛋白细胞骨架发生了这些变化，但细胞仍能分裂并进行胞质分裂、移动、在基质上扩散和起皱。因此，我们表明，一个单一的microdelicent相关蛋白可以重组整个微丝结构的细胞，而不干扰一般microdelicent为基础的功能，如胞质分裂，细胞运动，膜皱褶。
Villin, a Ca2+-regulated F-actin bundling, severing, capping, and nucleating protein, is a major component of the core of microvilli of the intestinal brush border. Its actin binding properties, tissue specificity, and expression during cell differentiation suggest that it might be involved in the organization of the microfilaments in intestinal epithelial cells to form a brush border. Recently, Friederich et al., (Friederich, E., C. Huet, M. Arpin, and D. Louvard. 1989. Cell. 59:461-475) showed that villin expression in transiently transfected fibroblasts resulted in the loss of stress fibers and the appearance of large cell surface microvilli on some cells. Here, we describe the effect of villin microinjection into cells that normally lack this protein, which has allowed us to examine the immediate and long-term effects of introducing different concentrations of villin on microfilament organization and function. Microinjected cells rapidly lost their stress fibers and the actin was reorganized into abundant villin containing cortical structures, including microspikes and, in about half the cells, large surface microvilli. This change in actin organization persisted in cells for at least 24 h, during which time they had gone through two of three cell divisions. Microinjection of villin core, that lacks the bundling activity of villin but retains all the Ca2+-dependent properties, disrupted the stress fiber system and had no effect on cell surface morphology. Thus, the Ca2+-dependent activities of villin are responsible for stress fiber disruption, and the generation of cell surface structures is a consequence of its bundling activity.Microinjection of villin led to the reorganization of myosin, tropomyosin, and alpha-actinin, proteins normally associated with stress fibers, whereas both fimbrin and ezrin, which are also components of microvillar core filaments, were readily recruited into the induced surface structures. Vinculin was also redistributed from its normal location in focal adhesions. Despite these changes in the actin cytoskeleton, cells were able to divide and undergo cytokinesis, move, spread on a substratum, and ruffle. Thus, we show that a single microfilament-associated protein can reorganize the entire microfilament structure of a cell, without interfering with general microfilament-based functions like cytokinesis, cell locomotion, and membrane ruffling.