KINESIN RELATED MICROTUBULE MOTORS IN MYOFIBRILLOGENESIS

这项提议的主要目标是测试微管是否 主动或被动参与肌原纤维组装， 小鼠成肌细胞。间接证据表明，微管可能 参与至少四个在时间和/或空间上分离 横纹肌细胞的分化过程：细胞伸长; 肌原纤维组装;肌浆网（和T-小管）加工;和 成肌细胞融合如果肌原纤维的组装需要微管， 独立于其他三个过程，那么这支持了 微管积极参与肌原纤维组装假说。 我们的建议旨在区分四种微管- 通过识别微管， 参与这些过程的依赖分子。的 一个广泛、多样和多功能的驱动蛋白相关微管家族 马达是最有可能寻找分子的地方， 不同的微管依赖过程。在此期间，A 将从分化的细胞中制备消减cDNA表达文库 C2 C12成肌细胞。驱动蛋白相关蛋白， 在成肌细胞分化过程中上调的细胞将通过以下方法鉴定： 利用抗驱动蛋白肽从消减文库中克隆表达 抗血清将分析回收的推定的肌源性驱动蛋白， 使用柱与肌原纤维蛋白缔合能力 层析和沉淀分析。基因工程表型， 反义RNA和DNA将与异位表达联合使用， 运动性测定、缺失构建体和实时动力学观察 通过视频显微镜将肌源性驱动蛋白分配到四个 上述功能类别。一种肌源性驱动蛋白的鉴定 其与新生肌原纤维相关，并且是肌原纤维 组装将是微管积极参与的直接证据 在肌原纤维形成中。此外，这样的发现将构成 第一个直接证据表明，在细胞质 重组。到供资期结束时， 参与肌肉发育早期阶段的蛋白质， 将在理解上取得显著进展， 微管在肌原纤维发生中的作用。该提案针对的是 基本的，但被忽视的，肌肉发展的领域，这将有助于我们 更好地了解临床肌病和亚细胞模式 在所有生物系统中。

The broad goal of this proposal is to test whether microtubules are actively or passively involved in myofibril assembly in differentiating mouse myoblasts. Circumstantial evidence suggests that microtubules may participate in at least four temporally and/or spatially separate processes in differentiating striated muscle cells: cell elongation; myofibril assembly; sarcoplasmic reticulum (and T-tubule) elaboration; and myoblast fusion. If microtubules are required for myofibril assembly, independently of the other three processes then this supports the hypothesis that microtubules participate actively in myofibril assembly. Our proposal is designed to differentiate between the four microtubule- dependent events in myogenic differentiation by identifying microtubule- dependent molecules that participate in each of these processes. The broad, diverse and multifunctional family of kinesin-related microtubule motors is the most likely place to look for molecules that mediate distinct microtubule-dependent processes. During this funding period a subtractive cDNA expression library will be prepared from differentiating C2C12 myoblasts. Kinesin-related proteins that are specifically upregulated during myoblast differentiation will be identified by expression cloning from the subtractive library using anti-kinesin peptide antisera. The recovered putative myogenic kinesins will be analyzed for the ability to associate with myofibrillar proteins using column chromatography and pelleting assays. Null phenotypes engineered using antisense RNA and DNA will be used in combination with ectopic expression, motility assays, deletion constructs and observation of real-time dynamics by video microscopy to assign the myogenic kinesin(s) to one of the four aforementioned functional categories. Identification of a myogenic kinesin which associates with nascent myofibrils and is required for myofibril assembly will be direct evidence for active participation of microtubules in myofibrillogenesis. Furthermore, such a discovery would constitute the first direct evidence for cytoskeletal crosstalk during cytoplasmic reorganization. By the completion of the funding period, a new class of proteins involved in early stages of muscle development will be characterized and significant progress will be made toward understanding the role of microtubules in myofibrillogenesis. This proposal addresses a fundamental, yet neglected, area of muscle development which will help us better understand both clinical myopathies and also subcellular pattern formation in all biological systems.