Segregation and Positioning of Basal Bodies

基体和中心粒是微管细胞器，具有纵向和九重旋转不对称性。 除了它们在纤毛组装中的作用之外，基底体对于运动纤毛的旋转定位以正确地定向纤毛搏动的方向是重要的。 对于建立或识别不对称性以在特定位置将附属物组装到基体上的分子成分知之甚少。 使用模式生物莱茵衣藻，一个单细胞，双鞭毛绿色藻，PI已经分离出一系列的突变体与一个可变的鞭毛数（vfl）和缺陷的旋转定位的基体，在定向的probasal机构相对于成熟的基体，并在分离的基体在细胞分裂过程中。 已经克隆了两个VFL基因。 这个新项目继续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 4、VFL 5和VFL 6基因，并使用光学和电子显微镜定位VFL 5和VFL 6基因产物; 2）使用遗传方法包括插入诱变和化学诱变来鉴定参与基体分离和定位的其他基因; 3）利用遗传学方法和酵母双杂交技术研究vfl基因之间的相互作用，并鉴定相互作用蛋白。 实验方法将确定进化上保守的分子，指定旋转不对称的基体以及分子组成的纤维，结合到特定网站上的基体的墙壁。 这项研究将推进长期目标，即了解基底体结构的分子机制，附属物附着在基底体壁上的特定部位，以及在特定旋转位置启动基底体复制。 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.