Role of Titin in Nonmuscle Cytoskeletons

这项研究的长期目标是对肌动蛋白基细胞骨架的结构和功能有更深入的了解。该项目旨在研究t蛋白的作用，t蛋白是一种巨大的蛋白质，最近被发现是广泛表征的肠上皮细胞刷边细胞骨架的一个组成部分。基于多肽分子量、分子形态和免疫交叉反应性，刷状边界t蛋白似乎是肌蛋白titin的非肌同种异构体。肌凝素是肌节中与肌球蛋白粗丝相关的关键结构成分。同样，t蛋白与肌球蛋白在刷状边界共定位，t蛋白与刷状边界肌球蛋白在体外组装成超分子结构。本提案的具体目的是：1)确定t蛋白在肠上皮细胞刷状边界细胞骨架的不同结构域和其他类型细胞的细胞骨架结构（包括应力纤维）中的存在和结构组织；2)研究T-蛋白在肠上皮细胞胚胎发育过程中组织刷状缘细胞骨架组装中的作用；3)表征t蛋白与其他细胞骨架成分（包括肌球蛋白）之间的相互作用。这些研究的结果应该阐明非肌titin在控制细胞骨架组织及其为关键细胞过程产生力的能力中的重要性。在生物系统中最明显的运动形式是肌肉运动。在肌肉组织中，特别是骨骼肌中，收缩行为导致器官的剧烈运动，如肘部弯曲或头部转动。这些总的运动是蛋白质复合物相对于彼此的细胞内运动的综合结果。肌肉中主要的细胞内收缩蛋白是肌动蛋白和肌球蛋白，它们形成寡聚细丝，在atp驱动的受控反应中相互滑动，导致细胞缩短。整个肌肉缩短（收缩）是肌肉内细胞缩短的综合结果。然而，细胞和细胞内运动并不局限于肌肉组织；几乎所有类型的真核细胞都表现出各种基于肌动蛋白/肌球蛋白的运动行为，从细胞爬行到有丝分裂后子细胞的分离。虽然肌肉系统在分子细节上被很好地理解，但这些非肌肉肌动蛋白/肌球蛋白为基础的运动系统在分子组织方面与肌肉相似的程度尚不清楚。首席研究员现在已经确定了一种主要肌肉蛋白的非肌肉版本，它在肌肉肌动球蛋白的运动中起着重要作用。在这个项目中进行的实验将阐明t蛋白在非肌肉运动中所起的作用，从而将大大有助于我们对细胞运动的一般理解。

The long-term objective of this research is to gain a greater understanding of the structure and function of actin-based cytoskeletons. This project is designed to investigate the roles of T-protein, a huge protein that recently has been discovered as a component of the extensively characterized intestinal epithelial cell brush border cytoskeleton. Based on polypeptide molecular weight, molecular morphology, and immunocrossreactivity, brush border T-protein appears to be a nonmuscle isoform of the muscle protein titin. Muscle titin is a key structural component associated with the myosin thick filaments in sarcomeres. Likewise, T-protein is co-localized with myosin in brush borders, and T-protein and brush border myosin assemble into supramolecular structures in vitro. The specific aims of this proposal are to: 1) determine the existence and structural organization of T-protein in different domains of the intestinal epithelial cell brush border cytoskeleton and in cytoskeletal structures, including stress fibers, in other types of cells; 2) investigate the role of T- protein in organizing brush border cytoskeletal assembly during embryonic development of intestinal epithelial cells; and 3) characterize interactions between T-protein and other cytoskeletal components, including myosin. Results of these studies should elucidate the importance of nonmuscle titin in governing the organization of cytoskeletons and their ability to produce force for critical cellular processes. %%% The most obvious form of movement in biological systems is muscular motility. In muscle tissue, and particularly in skeletal muscle, contractile behavior results in gross movements of organs, such as the bending of an elbow or the turn of a head. These gross movements are the composite result of intracellular movements of protein complexes with respect to one another. The major intracellular contractile proteins in muscle are actin and myosin, which form oligomeric filaments that slide past each other in an ATP-fueled controlled reaction that results in cellular shortening. Whole muscles shorten (contract) as a composite result of the cells within the muscle shortening. However, cellular and intracellular movement is not limited to muscle tissue; virtually all types of eukaryotic cells display various actin / myosin based motility behaviors, ranging from cellular crawling to the separation of daughter cells after mitosis. While the muscle system is quite well understood in molecular detail, the extent to which these non- muscle actin /myosin based motility systems resemble muscle, in terms of molecular organization, is unclear. The Principal Investigator has now identified a non-muscle version of a major muscle protein which plays an important role in muscle actomyosin based motility. The experiments which will be performed in this project will elucidate the role played by the T-protein in non- muscle motility, and will thereby contribute greatly to our general understanding of cellular movement.