REGULATION OF FLAGELLAR ASSEMBLY IN CHLAMYDOMONAS

这项提案描述了一个项目，目的是了解细胞如何调节一个复杂的、含有微管的细胞器--鞭毛的组装。尽管对有丝分裂纺锤体中单线微管组装的调控已经进行了深入的研究，但关于细胞如何调节纤毛和鞭毛的更稳定的双线微管的组装，或者基体和中心粒的三线微管的组装，人们几乎一无所知。我们将使用不能正确控制其鞭毛长度的突变体作为切入点来剖析鞭毛组装的调节。已经证明，四个非连锁基因的突变会导致细胞失去对鞭毛长度的控制。已经证明，四个非连锁基因的突变会导致细胞失去鞭毛长度的控制。突变体的鞭毛最长可达正常长度的四倍。矛盾的是，这些突变体中的许多在截肢后鞭毛的再生方面显示出严重的缺陷。在之前的项目期间，我们克隆了这些基因中的三个，LF2、LF3和LF4。这项建议描述了使用广泛的技术来表征LF基因、其蛋白质产物以及与其相互作用以控制鞭毛长度的蛋白质的实验。LF1基因将被克隆，所有四个LF基因的基因和全长cDNA将被测序。这些蛋白质产物将在野生型和突变细胞中定位。强大的抑制子筛选将用于分离每个基因的新突变等位基因，其特定目标是识别错义突变，用于交互式抑制子筛选和结构/功能研究，分子和遗传技术将用于鉴定和表征与LF基因产物相互作用的蛋白质，以调节鞭毛长度。鉴于纤毛和鞭毛的结构和蛋白质组成的高度保守性，该项目应该为人类组装这些细胞器提供有用的见解。

This proposal describes a project to learn how cells regulate the assembly of a complex, microtubule-containing organelle, the flagellum. Although the regulation of assembly of singlet microtubules, such as those in the mitotic spindle, has been intensively studied, almost nothing is known about how cells regulate the assembly of the more stable doublet microtubules of cilia and flagella, or the triplet microtubules of the basal bodies and centrioles. We will use mutants which do not properly control their flagellar length as an entry point to dissect the regulation of flagellar assembly. Mutation in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutations in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutants grow flagella of up to four times normal length. A number of these mutants, paradoxically, show severe defects in the regrowth of flagella after amputation. During the preceding project period we cloned three of these genes, LF2, LF3 and LF4. This proposal describes experiments to use a broad range of techniques to characterize the LF genes, their protein products and the proteins with which they interact to control flagellar length. The LF1 gene will be cloned, and the gens and full length cDNAs for all four LF genes will be sequenced. The protein products will be localized in wild-type and mutant cells. Powerful suppressor screens will be used to isolate new mutant alleles of each of the genes, with a particular goal of identifying missense mutations for interactive suppressor screens and for structure/function studies Both molecular and genetic techniques will be used to identify and characterize proteins which interact with the LF gene products in the regulation of flagellar length. Given the high degree of conservation of both the structure and protein composition of cilia and flagella, this project should provide useful insights into the assembly of these organelles in humans.