Control of Gene Activity and Organelle Biogenesis

描述（由申请人提供）：微管（MT）在真核细胞中普遍存在，它们形成不同的细胞器，参与细胞形状、细胞运动、细胞内运动、有丝分裂和减数分裂。 最复杂的微管细胞器是纤毛和鞭毛的基体和轴丝，在人类中，发现于呼吸道、女性生殖道和癌症高发组织中。 纤毛功能障碍会导致呼吸和生育问题。 这项提案的长期目标是了解微管多样性的分子机制，特别强调纤毛和基体的生物起源和功能。 在确定了特定的“轴丝序列”的α-和β-微管蛋白的糖基化位点，我们将确定和表征其他主要序列和翻译后修饰（PTM）的微管蛋白的功能，在指定的MT细胞器的结构和功能。 低等真核生物，嗜热四膜虫，被用作模型。 该系统的优点包括其相对较小的微管蛋白多基因超家族和最近开发的用于体细胞和胚细胞核的大规模转化的方法，使得基因置换和基因破坏成为可能。 这些方法将用于α-、β-和γ-微管蛋白功能及其二级修饰的详细致突变分析。 将分析一种新发现的、异常多样的β-微管蛋白靶向特定MT细胞器的机制。 通过使用遗传和生物化学的方法来识别和表征与甘氨酰化微管蛋白特异性相互作用的蛋白质，将研究甘氨酰化影响纤毛MT功能的机制。 这些研究应阐明正常纤毛的生物发生和微管蛋白在产生MT多样性的功能，这是需要了解细胞骨架功能，这是正常发育的关键，往往是异常的肿瘤细胞。

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.