Function of Myosins in Tetrahymena

9808301加文肌球蛋白是催化细胞内沿肌动蛋白微丝的线性空间位移的分子马达。基于肌球蛋白的运动的例子包括肌肉细胞的收缩和胞质分裂(细胞分裂时一个细胞分裂成两个)。尽管各种类型或类别的肌球蛋白的具体功能尚未完全阐明，但已发现了多种类型的非肌肉肌球蛋白。首席调查员的实验室最近证实，在纤毛原生动物四膜虫中存在肌球蛋白超家族的两个新成员。这是在纤毛原生动物中首次发现肌球蛋白。其中之一，Myo1p，是一种新的非传统肌球蛋白，可能是一个新的肌球蛋白类别的创始成员。另一个是P180，它显示了一个新的定位于运动纤毛底部的肌动蛋白微丝，这提出了一个有趣的可能性，即肌球蛋白可能整合了微管和微丝之间的相互作用。四膜虫易于进行遗传操作，是研究体内蛋白质功能的极佳实验生物。本项目的目标是完成MYO1基因的克隆和测序，克隆和测序P180基因，确定这些肌球蛋白的亚细胞位置(S)，并确定它们在四膜虫中的功能(S)。根据预测的氨基酸序列，将针对每种肌球蛋白产生抗体。这些抗体将用于免疫定位研究以及功能研究。功能也将通过基因组敲除进行评估。如果肌球蛋白参与微管-微丝的协同作用，例如，调节基底水平的纤毛活动，这项工作的结果可能不仅对纤毛原生动物的运动有影响，而且对脊椎动物细胞的纤毛活动也有意义。

9808301 Gavin Myosins are molecular motors that catalyze intracellular linear spatial displacement along actin microfilaments. Examples of myosin-based motility include the contraction of muscle cells and cytokinesis (the cleavage of a cell into two during cell division). A wide range of types of non-muscle myosin proteins have been discovered, although the specific functions of the various types or classes of myosins have not yet been fully elucidated. The Principal Investigator's laboratory recently demonstrated the existence of two new members of the myosin superfamily in the ciliated protozoan, Tetrahymena. These are the first myosins to be discovered in ciliated protozoa. One of these, Myo1p, is a novel unconventional myosin that may be the founding member of a new myosin class. The other, p180, exhibits a novel localization to actin microfilaments at the bases of the motile cilia, which raises the intriguing possibility that a myosin might integrate interactions between microtubules and microfilaments. Tetrahymena is amenable to genetic manipulation and is an excellent experimental organism for studies of protein function in vivo. The goals of this project are to complete the cloning and sequencing of MYO1, to clone and sequence the cDNA for p180, to identify the subcellular location(s) of these myosins, and to determine their function(s) in Tetrahymena. Antibodies will be raised against each myosin, based on predicted amino acid sequences. These antibodies will be used for immunolocalization studies as well as for functional studies. Function will also be assessed by genomic knockouts. If myosins are involved in microtubule-microfilament synergy, as for example, regulation of ciliary activity at the level of the basal body, the results of this work may well have implications not only for motility in ciliated protozoa but for ciliary activity in vertebrate cells as well.