MICROVILLAR CYTOSKELETAL-MEMBRANE INTERACTIONS

对细胞骨架如何与细胞膜相互作用的研究将解释 几种微丝起关键作用的细胞现象， 包括趋化性、细胞运动、吞噬作用、胞质分裂， 细胞表面突起的形成。 为了执行许多这些细胞 由于肌动蛋白的功能，肌动蛋白丝必须与血浆密切相关， 膜的 确定肌动蛋白丝如何与膜相互作用， 例如，帮助阐明癌细胞如何转移。 的 肠微绒毛显示清晰可见的链接，连接核心 一束肌动蛋白丝到膜上，这个尖端的识别 成分完全未知。 螺旋布置的侧向链节 延伸到膜的微绒毛的长度是可见的 核心;它们由与钙调蛋白复合的110 kD多肽组成。 对110 K-钙调素复合物的研究可以提供对如何 肌动蛋白丝被锚定。 110 K-钙调素与F-肌动蛋白的结合是 ATP、钙和蛋白质浓度依赖性， 协同性 此外，110 K-钙调蛋白复合物类似于肌球蛋白 在其修饰肌动蛋白丝和水解ATP升高的能力中 推测它是非肌肉细胞中的运动分子。 的目标 本研究是为了表征细胞骨架与 膜的 特别是，我们将：（a）研究纯化的110 K- 钙调蛋白将直接与脂质相互作用，（B）使用交联 在完整的微绒毛上进行实验，以探测可能用于 将110 K-钙调素连接到膜上，然后对其进行表征 连接蛋白，（c）确定构成微绒毛的组分 （d）在细胞中寻找110 K-钙调素蛋白的存在， 其他组织类型，和（e）继续映射的功能结构域， 100 kD多肽，包括ATP结合位点、肌动蛋白- 结合位点和分子内的钙调蛋白结合位点。

The study of how the cytoskeleton interacts with the membrane will explain several cellular phenomena in which microfilaments play a critical role, including chemotaxis, cell locomotion, phagocytosis, cytokinesis, and the formation of cell surface projections. To perform many of these cellular functions, actin filaments must be intimately associated with the plasma membrane. Determining how actin filaments interact with the membrane will, for example, help to elucidate how cancer cells metastasize. The intestinal microvillus shows clearly visible links which connect a core bundle of actin filaments to the membrane, the identify of this tip component is completely unknown. Helically arranged lateral links extending to the membrane are visible down the length of the microvillar core; they are comprised of a 110 kD polypeptide complexed with calmodulin. Study of the 110K-calmodulin complex can provide needed insight into how actin filaments are anchored. Binding of 110K-calmodulin to F-actin is ATP, calcium, and protein concentration dependent and exhibits cooperativity. In addition, the 110K-calmodulin complex resembles myosin in its ability to decorate actin filaments and to hydrolyze ATP raising speculation that it is a motile molecule in non-muscle cells. The goal of this study is to characterize the interaction of the cytoskeleton with the membrane. In particular, we will: (a) investigate whether purified 110K- calmodulin will interact directly with lipid, (b) use cross-linking experiments on intact microvilli to probe for a protein which may serve to link 110K-calmodulin to the membrane, followed by characterization of this linker protein, (c) determine the components which comprise the microvillar tip region, (d) search for the presence of 110K-calmodulin proteins in other tissue types, and (e) continue to map the functional domains of the 100 kD polypeptide including identification of the ATP-binding site, actin- binding site, and calmodulin-binding site within the molecule.