Segregation and Positioning of Basal Bodies

基底体和中心粒是微管细胞器，具有纵向和九倍旋转不对称性。除了在纤毛组装中的作用外，基底体对于活动纤毛的旋转定位以正确地定位纤毛搏动的方向也是重要的。人们对建立或识别这种不对称性的分子成分知之甚少，这种不对称性使附属物在特定位置组装到基底上。利用模式生物莱茵衣藻，一种单细胞、双鞭毛的绿色藻类，PI分离出一系列鞭毛数(VFL)可变的突变株，这些突变株在基体旋转定位、原基体相对于成熟基体的取向以及细胞分裂过程中基体分离方面存在缺陷。目前已克隆出两个VFL基因。这个新项目继续了PI对VFL突变体的研究。该项目的具体目标是1)克隆和表征VFL基因，并使用光学和电子显微镜对VFL5和VFL6基因产物进行定位；2)通过插入突变和化学诱变等遗传方法确定更多参与基础机体分离和定位的基因；3)利用遗传方法和酵母双杂交技术检测VFL基因之间的相互作用并确定相互作用的蛋白质。这种实验方法将识别进化上保守的分子，这些分子规定了基底体内的旋转不对称，以及结合到基底体壁上特定位置的纤维的分子成分。这项研究将推进理解基底体结构、附属物附着到基底体壁上的特定位置以及在特定旋转位置启动基底体复制的分子机制的长期目标。该项目将提供三个层次的研究培训：本科生、研究生和博士后。

Basal bodies and centrioles are microtubule organelles with both longitudinal and nine-fold rotational asymmetry. In addition to their role in ciliary assembly, basal bodies are important for the rotational positioning of motile cilia to properly orient the direction of ciliary beat. Little is known about the molecular components that establish or recognize the asymmetry to assemble appendages onto basal bodies at specific locations. Using the model organism Chlamydomonas reinhardtii, a unicellular, biflagellate green alga, the PI has isolated a series of mutants with a variable flagellar number (vfl) and with defects in rotational positioning of basal bodies, in orientation of probasal bodies with respect to mature basal bodies, and in segregation of basal bodies during cell division. Two VFL genes have been cloned. This new project continues the PI's study of the vfl mutants. The specific aims of the project are to 1) Clone and characterize the VFL4, VFL5, and VFL6 genes and localize the VFL5 and VFL6 gene products using light and electron microscopy; 2) Identify additional genes involved in basal body segregation and positioning using genetic approaches including insertional mutagenesis and chemical mutagenesis; 3) Examine interactions among vfl genes and identify interacting proteins using genetic approaches and yeast two-hybrid technology. The experimental approach will identify evolutionarily conserved molecules that specify rotational asymmetry in basal bodies as well as molecular components of fibers that bind to specific sites on the walls of basal bodies. The study will advance the long-term goal of understanding the molecular mechanisms that underlie the structure of basal bodies, the attachment of appendages to specific sites on the wall of the basal body, and the initiation of basal body duplication at a specified rotational position. The project will provide research training at three levels: undergraduate, graduate and postdoctoral.