INTRAFLAGELLAR TRANSPORT IN CHLAMYDOMONAS REINHARDTII

真核生物的纤毛和鞭毛是古老的细胞附属物， 感官功能这些细胞器的运动形式能够推动一些细胞，如精子， 原生动物通过液体环境，而其他细胞，如人的纤毛气管， 许多纤毛的跳动以在细胞表面推动液体或粘液环境。非运动纤毛 已经适应了感知各种刺激。经典的例子包括光感受器， 高度修饰的纤毛可以感知可见光，嗅觉纤毛高度富集气味剂 受体。由于它们在运动和感觉传导中的重要作用，纤毛和 鞭毛与许多人类疾病包括视网膜变性密切相关， 不动纤毛和Kartagener综合征，男性和女性不育，脑积水和嗅觉丧失，Bardet- Beidl综合征和人类最常见的遗传性疾病之一多囊肾病。 着眼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 用于这些结构的组装和维护。IFT以蛋白质的运动为特征 颗粒沿着细胞器的长轴，既向外到尖端（由驱动蛋白-2驱动的顺行IFT）， 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作为细胞器组装位点的远端。 IFT研究的模式生物是单细胞双鞭毛绿色藻、衣原体 莱因哈德氏菌对衣原体IFT颗粒中发现的17种蛋白质的生化分析已经开始， 揭示了它们复杂的寡聚体结构。继续我们以前对IFT粒子的研究， 具体的目的是：（1）进一步表征IFT颗粒的结构;确定17种蛋白质是如何与IFT颗粒结合的。 组装成复合物;（2）表征IFT颗粒与IFT马达蛋白的相互作用， 驱动蛋白-2和细胞质动力蛋白1b;（3）直接显示假设的IFT货物的运输 包括微管蛋白和辐射辐复合物;和（4）解决分离IFT颗粒的功能 proteins.目标4的一个重要方面将涉及创造900个或更多的运动突变体;目前，我们 在超过1/2的IFT颗粒蛋白中缺乏突变体。

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 one of the most commongenetic 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.