Franck Z, Footer M, Bretscher A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Cell Biol. 1990 Dec;111(6 Pt 1):2475-85. doi: 10.1083/jcb.111.6.2475.
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 or 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.
绒毛蛋白是一种受Ca2+调节的F-肌动蛋白成束、切断、封端和成核蛋白，是肠刷状缘微绒毛核心的主要成分。其肌动蛋白结合特性、组织特异性以及在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，弗里德里希等人（Friederich, E., C. Huet, M. Arpin, and D. Louvard. 1989. Cell. 59:461 - 475）表明，在瞬时转染的成纤维细胞中表达绒毛蛋白会导致应力纤维消失，并且在一些细胞上出现大的细胞表面微绒毛。在这里，我们描述了将绒毛蛋白显微注射到正常缺乏该蛋白的细胞中的效果，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即时和长期影响。显微注射的细胞迅速失去其应力纤维，肌动蛋白重新组织成丰富的含有绒毛蛋白的皮质结构，包括微刺，并且在大约一半的细胞中形成大的表面微绒毛。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两到三次细胞分裂。显微注射缺乏绒毛蛋白成束活性但保留所有Ca2+依赖性特性的绒毛蛋白核心，破坏了应力纤维系统，并且对细胞表面形态没有影响。因此，绒毛蛋白的Ca2+依赖性活性负责应力纤维的破坏，而细胞表面结构的产生是其成束活性的结果。显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（通常与应力纤维相关的蛋白质）重新组织，而作为微绒毛核心丝成分的丝束蛋白和埃兹蛋白都很容易被招募到诱导的表面结构中。纽蛋白也从其在粘着斑中的正常位置重新分布。尽管肌动蛋白细胞骨架发生了这些变化，细胞仍能够分裂并进行胞质分裂，可以移动、在基质上铺展并形成褶皱。因此，我们表明单一的微丝相关蛋白可以重组细胞的整个微丝结构，而不会干扰基于微丝的一般功能，如胞质分裂、细胞运动和膜褶皱。
