Regulation of protein transport in cilia

纤毛中蛋白质运输的调节

• 批准号: 9023563
• 负责人: Karl Lechtreck
• 金额: $ 28.5万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-10 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9023563
• 关键词: Address; Affect; Affinity; Binding; Binding Sites; Biochemical; Blindness; Caenorhabditis elegans; Carrier Proteins; Cells; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Cluster Analysis; Complex; Data; Defect; Diffuse; Disease; Docking; Feedback; Flagella; Fluorescence Microscopy; Frequencies; Goals; Growth; Health; Image; Imaging Techniques; Kidney Diseases; Length; Liquid substance; Maintenance; Microtubules; Modeling; Molecular Target; Monitor; Mus; Obesity; Organelles; Pathway interactions; Pattern; Phenotype; Phosphorylation; Phosphotransferases; Positioning Attribute; Property; Proteins; Regulation; Research; Resolution; Role; Signal Transduction; Site; Structural defect; Structure; Testing; Travel; base; cell motility; developmental disease; equilibrium model; genetic manipulation; human disease; in vivo imaging; insight; mutant; particle; protein transport; repaired; single molecule; stoichiometry

DESCRIPTION (provided by applicant): Cilia are thread-like microtubule-based cell extensions which function in cell locomotion, fluid transport, and signaling. Many developmental disorders and diseases are caused by defects in ciliary function and assembly. To assemble cilia of a specific size and composition, cells have to transport hundreds of different proteins from the cell body into the organelle. Intraflagellar transport (IFT), a bidirectional motility of protein particles along ciliary microtubules, is assumed to be the major pathway for protein transport in cilia. IFT is required for ciliary assembly, maintenance, and signaling, however, it remains largely unknown which proteins are transported by IFT. It is also unclear where in the cilium cargoes are unloaded from IFT and whether the amount of protein transported by IFT is regulated. Because ciliary proteins are likely to be transported as single molecules or in small clusters, the analysis of their transport requires a highly sensitive imaging technique. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we have established in vivo imaging of protein transport by IFT in cilia. We will analyze protein transport in cilia using the unicelluar model Chlamydomonas reinhardtii, which allows us to combine high resolution imaging in cilia with genetic manipulation and biochemical analysis of the organelle. We performed a comprehensive analysis of ciliary transport of the axonemal protein DRC4 and showed that DRC4-GFP depends on IFT for ciliary entry and distribution along the organelle. In Specific Aim 1, we will image distinct proteins selected from different ciliary compartments and substructures to determine how they interact with IFT to move into cilia. We will address the question of how IFT particles serve as carriers for many distinct proteins and how IFT transports proteins in the correct ratio into the organelle. We will test whether protein loading onto IFT particles depends on protein supply in the cell body and to which extent unloading of cargoes from IFT is spatially controlled. Our data show that the transport frequency of DRC4 is greatly increased when cilia grow, suggesting that the capacity of the IFT pathway can be modulated. The regulation of IFT is the focus of Specific Aim 2. We will analyze whether IFT particles isolated from growing and steady-state cilia are biochemically distinct and how cargo transport is affected in IFT mutants with small defects in the particle. The control of cargo influx is likely to be a prerequisite to establish a specific length of cilia, which is critical for its motile and signaling functions. We ill analyze IFT and cargo transport in mutants with defects in ciliary length regulation such as long flagella 2 (lf2). LF2 encodes a widely conserved CDK-like kinase with an emerging role in disease. IFT is disturbed in lf2 cilia; we will test the hypothesis that LF2 kinase is a regulator f IFT, which when defective results in overloading of IFT particles. We noted that IFT proteins accumulate in mutants with structural defects in cilia, which might indicate a feedback mechanism on the IFT pathway which alerts the cell of incorrectly assembled cilia. We will test whether cells use the IFT pathway to monitor the correct size and structure of cilia.

描述（由申请人提供）：纤毛是基于微管的线状细胞延伸，其在细胞运动、液体运输和信号传导中发挥作用。许多发育障碍和疾病是由睫状体功能和组装缺陷引起的。为了组装特定大小和组成的纤毛，细胞必须将数百种不同的蛋白质从细胞体运输到细胞器中。鞭毛内运输（IFT）是蛋白质颗粒沿着纤毛微管的双向运动，被认为是纤毛中蛋白质运输的主要途径。 IFT 是纤毛组装、维护和信号传导所必需的，然而，目前仍不清楚 IFT 转运哪些蛋白质。目前还不清楚纤毛货物是从 IFT 中卸载的，以及 IFT 运输的蛋白质量是否受到调节。由于纤毛蛋白可能以单分子或小簇形式运输，因此对其运输的分析需要高度灵敏的成像技术。使用全内反射荧光 (TIRF) 显微镜，我们建立了纤毛中 IFT 蛋白质转运的体内成像。我们将使用单细胞模型莱茵衣藻分析纤毛中的蛋白质运输，这使我们能够将纤毛的高分辨率成像与细胞器的遗传操作和生化分析结合起来。我们对轴丝蛋白 DRC4 的纤毛运输进行了全面分析，结果表明 DRC4-GFP 依赖于 IFT 进入纤毛并沿细胞器分布。在具体目标 1 中，我们将对从不同纤毛区室和子结构中选择的不同蛋白质进行成像，以确定它们如何与 IFT 相互作用以进入纤毛。我们将解决 IFT 颗粒如何作为许多不同蛋白质的载体以及 IFT 如何将蛋白质以正确的比例运输到细胞器中的问题。我们将测试 IFT 颗粒上的蛋白质加载是否取决于细胞体内的蛋白质供应，以及从 IFT 卸载的货物在多大程度上受到空间控制。我们的数据表明，当纤毛生长时，DRC4 的运输频率大大增加，表明 IFT 途径的容量可以被调节。 IFT 的调节是具体目标 2 的重点。我们将分析从生长和稳态纤毛中分离的 IFT 颗粒是否在生化上不同，以及颗粒中存在小缺陷的 IFT 突变体中货物运输如何受到影响。控制货物流入可能是建立特定长度纤毛的先决条件，这对其运动和信号功能至关重要。我们将分析具有纤毛长度调节缺陷的突变体（例如长鞭毛 2 (lf2)）的 IFT 和货物运输。 LF2 编码一种广泛保守的 CDK 样激酶，在疾病中发挥着新兴作用。 lf2纤毛中的IFT受到干扰；我们将检验 LF2 激酶是 IFT 调节因子的假设，当其缺陷时，会导致 IFT 颗粒过载。我们注意到，IFT 蛋白在纤毛结构缺陷的突变体中积累，这可能表明 IFT 通路上存在反馈机制，可警告细胞错误组装的纤毛。我们将测试细胞是否使用 IFT 途径来监测纤毛的正确大小和结构。