CYTOSKELETON/MEMBRANE INTERACTIONS

肌动蛋白相关膜骨架参与细胞粘附、运动和细胞存活调控。这项应用提出了继续研究一种新型的膜骨架，其特征是超级绒毛蛋白的存在，超级绒毛蛋白是一种最近发现的肌动蛋白结合外周膜蛋白。超级绒毛蛋白是从牛中性粒细胞质膜中分离出来的，它与稳定的肌动蛋白丝、fodrin、磷脂酰肌醇-4-激酶α、Rho激酶和其他几种蛋白质有关。Supervillin包含几个F-actin结合位点、一个黏附靶向序列、预测的核定位信号、潜在的核输出信号和可能的螺旋结构域。根据细胞周期的生长状态和/或阶段，超绒毛蛋白定位于细胞核内或与f -肌动蛋白一起定位于细胞-细胞和细胞-底物粘附部位。超绒毛蛋白序列的过度表达导致局灶黏附的破坏和肌动蛋白细胞骨架在质膜和/或细胞核中重组成束。在许多癌细胞系中观察到超绒毛蛋白信息和/或蛋白水平升高，这表明超绒毛蛋白过表达可能发生在肿瘤发生过程中。初步的反义实验表明，超级反派蛋白可能对细胞存活至关重要。我们提出，超级绒毛蛋白相关的膜骨架有助于运动细胞的结构完整性，并且超级绒毛蛋白可能参与控制肌动蛋白和肌球蛋白在质膜上的组装。我们进一步假设超级绒毛蛋白和相关蛋白参与核结构和/或功能的各个方面，并且这种类型的膜骨架有助于调节细胞的粘附、生长、存活和/或运动。为了验证这些假设，我们提出：(1)完成中性粒细胞质膜中超绒毛蛋白相关膜骨架的生化和形态学表征；(2)确定超绒毛蛋白相关膜骨架在上皮细胞和癌细胞的生长、粘附、运动和机械应力中的作用。该项目的最终目标是了解肌动蛋白相关膜骨架在细胞运动、粘附和脱离底物和其他细胞过程中的功能。这些信息将增加我们对正常细胞行为和病理状况的理解，如发育异常和癌细胞转移，甚至可能产生一些有用的肿瘤分期诊断工具。

The actin-associated membrane skeleton is involved in cell adhesion, motility, and the regulation of cell survival. This application proposes the continued study of a new type of membrane skeleton characterized by the presence of supervillin, a recently-discovered actin-binding peripheral membrane protein. Supervillin is isolated from bovine neutrophil plasma membranes in association with stabilized actin filaments, fodrin, phosphatidyl-inositol-4-kinase alpha, Rho kinase, and several other proteins. Supervillin contains several F-actin binding sites, a focal adhesion targeting sequence, predicted nuclear localization signals, a potential nuclear export signal, and possible coiled-coil domains. Depending upon growth state and/or stage in the cell cycle, supervillin localizes within nuclei or with F-actin at sites of cell-cell and cell-substrate adhesion. Overexpression of supervillin sequences leads to disruption of focal adhesions and re-structuring of the actin cytoskeleton into bundles at the plasma membrane and/or in cell nuclei. Increased levels of supervillin message and/or protein are observed in many carcinoma cell lines, suggesting that supervillin overexpression may occur during tumorigenesis. Preliminary antisense experiments suggest that supervillin may be essential for cell survival. We propose that the supervillin-associated membrane skeleton contributes to the structural integrity of motile cells and that supervillin may be involved in the control of actin and myosin assembly at the plasma membrane. We further hypothesize that supervillin and associated proteins participate in aspects of nuclear architecture and/or function and that this type of membrane skeleton contributes to the regulation of cell adhesion, growth, survival, and/or motility. To test these hypotheses, we propose: (1) to complete the biochemical and morphological characterization of the supervillin-associated membrane skeleton in neutrophil plasma membranes and (2) to determine the role(s) of the supervillin-associated membrane skeleton during growth, adhesion, motility, and mechanical stress of epithelial and carcinoma cells. The ultimate goal of this project is to understand how the actin-associated membrane skeleton functions during cell motility, adhesion, and detachment from substrates and other cells. This information will increase our understanding of both normal cellular behaviors and pathological conditions, such as developmental abnormalities and cancer cell metastasis, and may even produce some useful diagnostic tools for tumor staging.