Function of Myosins in Tetrahymena

Gavin Myosins是沿肌动蛋白微丝催化细胞内线性空间位移的分子马达。肌球蛋白为基础的运动包括肌肉细胞的收缩和细胞质分裂（细胞分裂时一个细胞分裂成两个细胞）。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，但已经发现了各种类型的非肌肉肌球蛋白蛋白。首席研究员的实验室最近在纤毛原生动物四膜虫中证明了肌球蛋白超家族的两个新成员的存在。这是在纤毛原生动物中首次发现的肌凝蛋白。其中，Myo1p是一种新颖的、非常规的肌凝蛋白，可能是一个新的肌凝蛋白类的创始成员。另一个是p180，它在运动纤毛基部的肌动蛋白微丝上表现出一种新的定位，这提出了一种有趣的可能性，即肌凝蛋白可能整合了微管和微丝之间的相互作用。四膜虫易于遗传操作，是研究体内蛋白质功能的优良实验生物。本项目的目标是完成MYO1的克隆和测序，p180的cDNA克隆和测序，确定这些肌球蛋白的亚细胞位置，并确定它们在四膜虫中的功能。根据预测的氨基酸序列，将产生针对每种肌球蛋白的抗体。这些抗体将用于免疫定位研究以及功能研究。功能也将通过基因敲除来评估。如果肌凝蛋白参与微管-微丝协同作用，例如，在基底体水平上调节纤毛活性，那么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性也有影响。

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.