Flagellar Motility and Assemlby

鞭毛运动和组装

• 批准号: 8914625
• 负责人: George B Witman
• 金额: $ 70.42万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8914625
• 关键词: Affect; Animal Model; Antibodies; Bardet-Biedl Syndrome; Binding; Biochemical; Biochemical Genetics; Biological; Blindness; Cells; Chlamydomonas; Cilia; Color; Complex; Coupling; Cytoplasm; Defect; Disease; Dynein ATPase; Flagella; Future; Genes; Genetic; Genetic study; Genomic DNA; Goals; Hand; Health; Homologous Gene; Human; Individual; Instruction; Investigation; Learning; Life; Location; Mammals; Methods; Modeling; Motor; Movement; Mus; Mutate; Organism; Partner in relationship; Phenotype; Photoreceptors; Primary Ciliary Dyskinesias; Process; Protein Export Pathway; Proteins; Research; Role; Structure; Techniques; Testing; Total Internal Reflection Fluorescent; base; cell motility; cell type; embryonic stem cell; gene induction; genetic regulatory protein; human disease; interest; knock out mouse project; link protein; mutant; novel; particle; protein B; repository; research study; tool

The long-term goals of this research are to understand the structure, assembly, and function of cilia and flagella. The studies will utilize Chlamydomonas and mice as model organisms, and will focus on processes and proteins that are highly conserved among ciliated organisms. A combination of genetic, biochemical, and cell biological approaches will be taken. Intraflagellar transport (IFT), which is necessary for assembly of cilia, will be investigated. IFT involves the movement of particles and cargo within the cilium. Studies are proposed to learn more about the motors that transport the IFT particles, the particles themselves, and the BBSome -- an IFT cargo adapter. Experiments will use existing Chlamydomonas mutants to determine the specific function of the IFT-particle protein IFT74, and of FAP133 -- the intermediate chain for the retrograde IFT motor, dynein 1B. Studies will Investigate the specific roles of several BBSome proteins and establish the basis for BBSome-IFT particle interaction. The hypothesis that the BBSome exports proteins from the flagella will be tested. The relationship between the BBSome and phosphollpase D, a putative cargo of the BBSome, will be investigated. Studies will explore how the transition zone protein CEP290 regulates entry of IFT particles and BBSomes into the flagellum. Studies will define the domains of CEP290 necessary for assembly into the transition zone, assign specific functions to the domains, and Identify interacting proteins. An existing mutant defective In an uncharacterized motility regulating protein will be studied to determine the location of the protein In the axoneme and to Identify Its interacting partners. To discover novel proteins involved in flagellar assembly, in flagellar motility, and In the induction of genes encoding flagellar proteins, new insertional mutants will be made by a method that allows rapid identification of the genomic DNA flanking both ends of the insert, so that the mutated gene is quickly identified. Characterization of selected mutants will be facilitated by rescuing the mutants with constructs expressing HA- or GFP-tagged proteins so that the proteins can be quickly localized, observed by TIRF microscopy in living flagella, and followed in biochemical experiments.

这项研究的长期目标是了解纤毛和鞭毛的结构，组装和功能。这些研究将利用衣原体和小鼠作为模型生物，并将专注于在纤毛生物中高度保守的过程和蛋白质。将采用遗传，生化和细胞生物学方法的结合。将研究纤毛组装所必需的flagellar内运输（IFT）。 IFT涉及粒子和货物在纤毛内的运动。提出研究以了解有关运输IFT颗粒，颗粒本身和BBSOME的电动机的更多信息 - IFT货物适配器。实验将使用现有的衣原体突变体来确定IFT颗粒蛋白IFT74和FAP133的特定功能 - 逆行IFT运动的中间链，Dynein 1B。研究将研究几种BBSOME蛋白的特定作用，并为BBSOME-IFT颗粒相互作用建立基础。将测试BBSOME出口蛋白的假设。将研究BBSOME和Phospholpase D（BBSOME的推定货物）之间的关系。研究将探索过渡区蛋白CEP290如何调节IFT颗粒和BBSOMES进入鞭毛的进入。研究将定义组装到过渡区，为域分配特定功能并识别相互作用蛋白的CEP290的域。 将研究一个未表征的调节蛋白质中的现有突变体有缺陷，以确定蛋白质在轴突中的位置并鉴定其相互作用的伴侣。为了发现与鞭毛组装有关的新型蛋白质，鞭毛运动和诱导编码鞭毛蛋白的基因时，将通过一种允许快速鉴定插入片段两端的基因组DNA的方法制造新的插入突变体，从而快速识别突变基因。通过用表达HA-或GFP标记的蛋白的构建体拯救突变体的突变体的表征将促进，以便可以快速定位蛋白质，通过TIRF显微镜在活的鞭毛中观察到，然后进行生物化学实验。