Segregation and Positioning of Basal Bodies

基体和中心粒是微管细胞器，具有纵向和九倍旋转不对称。除了在纤毛组装中起作用外，基底体对运动纤毛的旋转定位也很重要，以正确地定向纤毛跳动的方向。关于建立或识别在特定位置将附属物组装到基体上的不对称性的分子成分知之甚少。利用模式生物莱因哈蒂衣藻（Chlamydomonas reinhardtii，一种单细胞双鞭毛绿藻），PI分离出一系列具有可变鞭毛数（vfl）的突变体，这些突变体在基体的旋转定位、前基体相对于成熟基体的朝向以及细胞分裂过程中基体的分离方面存在缺陷。两个VFL基因已被克隆。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VFL4、VFL5和VFL6基因，并利用光镜和电镜对VFL5和VFL6基因产物进行定位；2)利用插入诱变和化学诱变等遗传方法确定参与基底体分离和定位的其他基因；3)利用遗传方法和酵母双杂交技术研究vfl基因间的相互作用，鉴定相互作用蛋白。实验方法将识别进化上保守的分子，这些分子指定基底体内的旋转不对称性，以及结合在基底壁上特定位置的纤维分子成分。该研究将促进了解基底结构的分子机制，附属物附着在基底壁上的特定位点，以及在特定旋转位置开始基底复制的长期目标。该项目将提供本科生、研究生和博士后三个层次的研究培训。

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.