INTRAFLAGELLAR TRANSPORT IN CHLAMYDOMONAS REINHARDTII

真核生物的纤毛和鞭毛是古老的细胞附属物，已经适应于运动和 感官功能。这些细胞器的活动形式能够推动某些细胞，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，使用协调的 许多纤毛的跳动，使细胞表面形成液体或粘液环境。不活动的纤毛 已经适应了对各种刺激的感知。经典的例子包括光感受器，它是 高度修饰的能够感知可见光的纤毛和气味丰富的嗅觉纤毛 感受器。由于它们在运动和感觉传导中的重要作用，纤毛和纤毛的缺陷 鞭毛与许多人类疾病密切相关，包括视网膜退化， 不动纤毛和Karagener综合征，男性和女性不孕症，脑积水和嗅觉障碍，Bardet- 贝德尔综合征是人类最常见的遗传病之一，多囊肾病。 着眼于细胞如何构建这些细胞器，我们研究了所需的鞭毛内运输(Ift)。 用于组装和维护这些结构。IFT的特征是蛋白质的运动 沿细胞器长轴的颗粒，既向顶端伸出(由Kinesin-2驱动的顺行IFT)和 回到细胞体(由胞浆动力蛋白1b/2驱动的逆行IFT)。IFT的主要功能是 将轴丝构筑块向远端运输，远端是细胞器的组装部位。 研究IFT的模式生物是单细胞双鞭毛绿藻衣藻 莱恩哈迪尔。在衣藻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结构。继续我们之前对IFT粒子的研究，我们的 具体目标是：(1)进一步表征IFT颗粒的结构；确定17种蛋白质如何 组装成复合体；(2)表征IFT颗粒与IFT马达蛋白的相互作用， 动蛋白-2和胞浆动力蛋白1b；(3)直接可视化假想的IFT货物的运输 包括微管蛋白和径向辐条复合体；以及(4)解决分离的IFT颗粒的功能 蛋白质。目标4的一个重要方面将涉及创建900个或更多的运动性突变体；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.