Regulation of protein transport in cilia

纤毛中蛋白质运输的调节

• 批准号: 9213386
• 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/9213386
• 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; 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; high resolution imaging; human disease; in vivo imaging; insight; mutant; particle; predictive modeling; protein transport; public health relevance; 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）：纤毛是基于螺纹的微管细胞扩展，它们在细胞运动，流体传输和信号传导中起作用。许多发育障碍和疾病是由睫状功能和组装缺陷引起的。为了组装特定尺寸和组成的纤毛，细胞必须将数百种不同蛋白质从细胞体传输到细胞器中。假定蛋白质颗粒沿纤毛微管的双向运动，假定flagellar的转运（IFT）被认为是纤毛中蛋白质转运的主要途径。睫状组件，维护和信号传导需要IFT，但是，IFT仍未知哪些蛋白质是由IFT转运的。还不清楚从IFT中卸载纤毛货物中的位置以及IFT转运的蛋白质是否受到调节。由于纤毛蛋白可能以单分子或小簇的形式转运，因此对其运输的分析需要高度敏感的成像技术。使用总反射荧光（TIRF）显微镜，我们在纤毛中通过IFT对蛋白质转运的体内成像建立了。我们将使用Unicelluar Model Chlamydomonas Reinhardtii分析纤毛中的蛋白质转运，这使我们能够将纤毛中的高分辨率成像与遗传操纵和细胞器的生化分析相结合。我们对轴突蛋白DRC4的睫状运输进行了全面分析，并表明DRC4-GFP取决于IFT的纤毛进入和沿细胞器的分布。在特定目标1中，我们将成像从不同睫状室和子结构中选择的不同蛋白质，以确定它们与IFT的相互作用以进入纤毛的方式。我们将解决IFT颗粒如何用作许多不同蛋白质的载体以及IFT如何以正确比率将蛋白质传输到细胞器的问题。我们将测试将蛋白质负载到IFT颗粒上是否取决于细胞体内的蛋白质供应，以及从IFT中卸载货物的含量。我们的数据表明，当纤毛生长时，DRC4的传输频率大大增加，这表明可以调节IFT途径的能力。 IFT的调节是特定目标2的重点。我们将分析从生长和稳态纤毛中分离出的IFT颗粒在生化上是生化不同的，并且在粒子中较小缺陷的IFT突变体中如何影响货物运输。控制货物涌入的控制可能是建立特定纤毛长度的先决条件，这对于其机动和信号传导功能至关重要。我们不分析在具有睫状长度调节缺陷的突变体中的IFT和货物运输，例如长鞭毛2（LF2）。 LF2编码广泛保守的CDK样激酶，在疾病中具有新兴作用。 IFT在LF2纤毛中受到干扰；我们将检验以下假设：LF2激酶是一种调节剂FT，当有缺陷导致IFT颗粒过载时。我们注意到，IFT蛋白在纤毛中有结构缺陷的突变体中积累，这可能表明IFT途径上的反馈机制警告了错误组装的纤毛细胞。我们将测试细胞是否使用IFT途径来监视纤毛的正确尺寸和结构。