Control of Gene Activity and Organelle Biogenesis

描述(申请人提供)：微管(MT)在真核细胞中普遍存在，它们形成不同的细胞器，涉及细胞形状、细胞运动、细胞内运动、有丝分裂和减数分裂。在最精细的微管细胞器中，有基本体以及纤毛和鞭毛的轴丝，在人类的呼吸道、女性生殖道和癌症高发区的组织中都发现了这些细胞器。纤毛功能障碍会导致呼吸和生育问题。这项建议的长期目标是了解微管多样性的分子机制，特别是纤毛和基体的生物发生和功能。在确定了α-微管蛋白和β-微管蛋白的糖基化位点后，我们将确定和表征微管蛋白的其他初级序列和翻译后修饰(PTM)在确定MT细胞器的结构和功能方面的功能。以低等真核生物--嗜热四膜虫为模型。该系统的优点包括其相对较小的微管蛋白多基因超家族，以及最近发展的大量转化体细胞核和生发核的方法，使基因替换和基因破坏成为可能。这些方法将用于对α-微管蛋白、β-微管蛋白和伽马微管蛋白的功能及其二次修饰进行详细的突变分析。我们将分析一种新发现的、异常多样的β-微管蛋白作用于特定MT细胞器的机制。甘氨酸化影响纤毛MT功能的机制将通过遗传和生化方法进行研究，以鉴定和表征与甘氨酸化微管蛋白特异性相互作用的蛋白质。这些研究应该有助于阐明正常的纤毛生物发生和微管蛋白在产生MT多样性中的功能，这是理解细胞骨架功能所必需的，细胞骨架功能对正常发育至关重要，而且在肿瘤细胞中经常出现异常。

DESCRIPTION (provided by applicant): Microtubules (MTs) are ubiquitous in eukaryotic cells where they form diverse organelles involved in cell shape, cell motility, intracellular movements mitosis and meiosis. Among the most elaborate microtubular organelles are basal bodies and the axonemes of cilia and flagella, which, in humans, are found in the respiratory tract, the female reproductive tract, and in tissues with high incidences of cancer. Ciliary dysfunction can cause respiratory and fertility problems. The long-term goals of this proposal are to understand the molecular mechanisms underlying microtubule diversity with particular emphasis on the biogenesis and function of cilia and basal bodies. Having identified specific "axonemal sequences" as the sites of glyclylation in both alpha- and beta-tubulins, we will identify and characterize the function of other primary sequences and post-translational modifications (PTMs) of tubulins in specifying the structure and function of MT organelles. The lower eukaryote, Tetrahymena thermophila, is used as a model. Advantages of this system include its relatively small tubulin multigene superfamily and the recent development of methods for mass transformation of somatic and germinal nuclei, enabling gene replacement and gene disruption. These methods will be used for a detailed mutagenic analysis of the function of alpha-, beta-, and gamma-tubulin and of their secondary modifications. The mechanisms by which a newly discovered, unusually diverse beta-tubulin is targeted to specific MT organelles will be analyzed. The mechanism by which glycylation affects ciliary MT function will be studied by using genetic and biochemical approaches to identify and characterize proteins that interact specifically with glycylated tubulin. These studies should shed light on normal cilia biogenesis and the function of tubulins in generating MT diversity, that are required to understand cytoskeletal functions that are critical for normal development and that are frequently abnormal in neoplastic cells.