INTRAFLAGELLAR TRANSPORT IN CHLAMYDOMONAS REINHARDTII

描述（由申请人提供）：真核纤毛和鞭毛是古老的细胞附属物，已经适应了运动和感觉功能。这些细胞器的运动形式能够推动一些细胞如精子和原生动物通过液体环境，而其他细胞如人的纤毛气管使用许多纤毛的协调跳动来推动细胞表面上的液体或粘液环境。非运动性纤毛已经适应于感知广泛的刺激。经典的例子包括光感受器，其是可以感知可见光的高度修饰的纤毛，以及高度富集气味感受器的嗅觉纤毛。由于纤毛和鞭毛在运动和感觉传导中的重要作用，它们的缺陷与许多人类疾病密切相关，包括视网膜变性、不动纤毛和Kartagener综合征、男性和女性不育症、脑积水和嗅觉丧失、Bardet-Beidl综合征和人类最常见的遗传疾病之一多囊肾病。专注于细胞如何建立这些细胞器，我们研究鞭毛内运输（IFT），这是必要的组装和维护这些结构。IFT的特征在于蛋白质颗粒沿着细胞器的长轴运动，既向外运动到尖端（由驱动蛋白-2驱动的顺行IFT）又返回到细胞体（由细胞质动力蛋白1b/2驱动的逆行IFT）。IFT的主要功能是将轴丝构建块运输到远端尖端，该远端尖端用作细胞器的组装位点。IFT研究的模式生物是单细胞双鞭毛绿色衣藻，莱茵衣藻。对衣原体IFT颗粒中发现的17种蛋白质的生化分析已经开始揭示它们复杂的寡聚体组织。我们的目标是：（1）进一步研究IFT颗粒的结构，确定17种蛋白质是如何组装成复合物的，（2）研究IFT颗粒与IFT马达蛋白、驱动蛋白2和细胞质动力蛋白1b的相互作用，（3）进一步研究IFT颗粒的结构和功能。（3）直接可视化包括微管蛋白和辐射辐复合物的假设IFT货物的运输;和（4）解决单独IFT颗粒蛋白的功能。Aim 4的一个重要方面将涉及900个或更多运动突变体的产生;目前我们在超过1/2的IFT颗粒蛋白中缺乏突变体。

DESCRIPTION (provided by applicant): Eukaryotic cilia and flagella are ancient cellular appendages that have been adapted for motile and sensory functions. Motile forms of these organelles are capable of propelling some cells like sperm and protozoa through a liquid environment while other cells like the ciliated trachea of man use the coordinated beating of many cilia to propel a liquid or mucous environment over the surface of the cells. Nonmotile cilia have been adapted to sense a wide range of stimuli. Classic examples include the photoreceptors which are highly modified cilia that can sense visible light and the olfactory cilia which are highly enriched in odorant receptors. Because of their important roles in both motility and sensory transduction, defects in cilia and flagella have been intimately linked with a number of human diseases including retinal degeneration, immotilie cilia and Kartagener's syndromes, male and female infertility, hydrocephalus and anosmia, Bardet-Beidl syndrome and 1 of the most common genetic diseases in man, polycystic kidney disease. Focusing on how cells build these organelles, we study intraflagellar transport (IFT) which is required for the assembly and maintenance of these structures. IFT is characterized by the movement of protein particles along the long axis of the organelle, both out to the tip (anterograde IFT driven by kinesin-2) and back to the cell body (retrograde IFT driven by cytoplasmic dynein 1b/2). A primary function of IFT is to transport axonemal building blocks out to the distal tip which serves as the site of assembly for the organelle. The model organism for the study of IFT is the unicellular biflagellate green alga, Chlamydomonas reinhardtii. Biochemical analysis of the 17 proteins found in the Chlamydomonas IFT particles has begun to reveal their complex oligomeric organization. Continuing on our previous study of the IFT particles, our specific aims are: (1) to further characterize the architecture of IFT particles; to identify how the 17 proteins assemble into complexes; (2) to characterize the interaction of the IFT particles with the IFT motor proteins, kinesin-2 and cytoplasmic dynein 1b; (3) to directly visualize the transport of hypothesized IFT cargo including tubulin and radial spoke complexes; and (4) to address the function of separate IFT particle proteins. An important aspect of Aim 4 will involve the creation of 900 or more motility mutants; currently we lack mutants in more than 1/2 of the IFT particle proteins.