The Role of an Intraflagellar Transport Protein in Cell-Cycle Control

鞭毛内转运蛋白在细胞周期控制中的作用

• 批准号: 7907649
• 负责人: Christopher R Wood
• 金额: $ 2.61万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907649
• 关键词: Antibodies; Back; Bardet-Biedl Syndrome; Binding; Carrier Proteins; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cell Proliferation; Cell division; Cells; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Complex; Cytokinesis; Cytoplasmic Protein; Defect; Disease; Distal; Excision; Family; Green Algae; Guanosine; Guanosine Triphosphate Phosphohydrolases; Health; Human; Immunofluorescence Microscopy; Immunoprecipitation; Kidney Diseases; Link; Maintenance; Mass Spectrum Analysis; Mediating; Mitosis; Molecular; Organism; Pattern; Process; Protein Binding; Proteins; RNA Interference; Role; Sequence Analysis; Sucrose; Testing; Wood material; Yeasts; cell growth; density; gel electrophoresis; human disease; knock-down; neuronal cell body; particle; protein complex; yeast two hybrid system

DESCRIPTION (provided by applicant): Most vertebrate cells are ciliated and the genesis and resorption of these cilia are coordinated with progression through the cell cycle. However, no direct causal relationship between the two processes has been demonstrated. The assembly and maintenance of cilia are mediated by intraflagellar transport (IFT), a process by which multimeric protein complexes, called IFT particles, move bidirectionally along the cilium to shuttle axonemal precursors to the distal tip for assembly and turnover products back to the cell body for removal. Recently, RNAi knockdown of a core IFT particle protein, IFT27, resulted in cell-cycle defects in addition to defects of flagellar assembly in the green alga, Chlamydomonas reinhardtii. Because sequence analysis of IFT27 reveals significant similarity to proteins of the Rab family of guanosine triphosphatases (GTPases), it is hypothesized that IFT27 may be functioning as a regulator of the cell cycle in a way similar to that observed for Rabs in higher organisms. To investigate this hypothesis, the cellular localization of IFT27 will be examined by immunofluorescence microscopy in order to determine if the distribution of IFT27 changes in concert with the cell cycle. Next, an IFT27-containing sucrose gradient fraction will be obtained from cytoplasmic protein extracts prepared during the cell cycle stage at which IFT27 is observed to localize most prominently to the cell body interior. To identify proteins that bind to IFT27 during the cell cycle, the cytoplasmic IFT27-containing fraction will be subjected to immunoprecipitation with IFT27 antibodies and the resulting coimmunoprecipitates will be identified by gel electrophoresis and mass spectrometry. To assess the function of these cytoplasmic binding partners of IFT27, each will be knocked down by RNAi. Knockdowns will be examined for defects in cell cycle progression, cell division and flagellar assembly. The relevance of these studies to human health is supported by the rapidly emerging body of evidence linking defects in the assembly and maintenance of cilia to a growing list of human diseases including Bardet-Biedl syndrome, and polycysitic kidney disease (PKD). The fact that a decrease in IFT27 causes loss of the cilium, taken together with the fact that the presence of IFT27 is required for completion of mitosis and cytokinesis provides a possible molecular link between the cilium and diseases of cell proliferation.

描述（由申请人提供）：大多数脊椎动物细胞都是纤毛的，这些纤毛的起源和吸收与整个细胞周期的进展相协调。但是，尚未证明这两个过程之间的直接因果关系。纤毛的组装和维护是由氟法内转运（IFT）介导的，该过程是多聚体蛋白络合物（称为IFT颗粒）沿着纤毛的双向移动，从纤毛上移动，以穿梭轴突前体向远端尖端，以使组装和转移产物回到细胞体中以进行移植。最近，除了在绿色藻类（绿色藻类，reinhardtii）中，RNAi敲低IFT颗粒蛋白IFT27，还导致细胞周期缺陷。由于对IFT27的序列分析表明，与Rab的三磷酸酶（GTPases）的Rab家族的蛋白质显着相似，因此假设IFT27可能以类似于较高生物体的RAB的方式作为细胞周期的调节剂。为了研究这一假设，将通过免疫荧光显微镜检查IFT27的细胞定位，以确定IFT27的分布是否与细胞周期共同变化。接下来，将从在细胞周期阶段制备的细胞质蛋白提取物获得含有IFT27的蔗糖梯度分数，在该细胞周期阶段观察到IFT27最突出地定位于细胞体内的内部。为了鉴定在细胞周期中与IFT27结合的蛋白质，将对IFT27抗体进行免疫沉淀，并通过凝胶电泳和质谱法确定所得共免疫沉淀。为了评估IFT27的这些细胞质结合伴侣的功能，每个人都将被RNAi击倒。将检查敲低的细胞周期进程，细胞分裂和鞭毛组件的缺陷。这些研究与人类健康的相关性得到了迅速新兴的证据体系，这些证据将纤毛中的缺陷和维持纤毛的缺陷与包括Bardet-Biedl综合征和多性肾脏疾病（PKD）在内的越来越多的人类疾病列表联系起来。 IFT27减少会导致纤毛的损失，这一事实与以下事实：完成有丝分裂和细胞因子所必需的IFT27的存在提供了纤毛和细胞增殖疾病之间的可能分子联系。