Regulation of protein transport in cilia

描述(申请人提供)：纤毛是基于微管的线状细胞延伸，在细胞运动、液体运输和信号传递中起作用。许多发育障碍和疾病是由睫毛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输送到细胞器中。鞭毛内转运(IFT)是纤毛中蛋白质颗粒沿纤毛微管的双向运动，被认为是纤毛中蛋白质转运的主要途径。IFT是纤毛组装、维持和信号传递所必需的，然而，目前还不清楚IFT运输哪些蛋白质。也不清楚纤毛中的货物是从IFT的哪里卸货的，也不清楚IFT运输的蛋白质数量是否受到监管。由于纤毛蛋白可能以单分子或小簇的形式运输，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，我们已经建立了体内成像的蛋白质转运的IFT纤毛。我们将使用单细胞模型莱茵衣藻来分析纤毛中的蛋白质运输，这使得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相结合。我们对轴丝蛋白DRC4的纤毛运输进行了全面的分析，结果表明DRC4-GFP依赖IFT进行纤毛进入和沿细胞器的分布。在具体目标1中，我们将成像从不同纤毛间隔和亚结构中选择的不同蛋白质，以确定它们如何与IFT相互作用进入纤毛。我们将讨论IFT颗粒如何作为许多不同蛋白质的载体，以及IFT如何以正确的比例将蛋白质运输到细胞器中。我们将测试是否将蛋白质装载到IFT颗粒上取决于细胞体中的蛋白质供应，以及从IFT卸货的空间控制程度。我们的数据显示，随着纤毛的生长，DRC4的运输频率大大增加，这表明IFT途径的容量是可以调节的。IFT的调控是特定目标2的重点。我们将分析从生长和稳定状态的纤毛中分离的IFT颗粒是否在生化上是不同的，以及具有微小缺陷的IFT突变体中货物运输是如何受到影响的。控制货物的流入可能是建立特定长度纤毛的先决条件，纤毛长度对其运动和信号功能至关重要。我们将分析纤毛长度调节缺陷的突变体，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，在疾病中具有新的作用。IFT在LF2纤毛中受到干扰；我们将测试LF2激酶是IFT调节因子的假设，当缺陷时会导致IFT颗粒超载。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途径上的反馈机制，提醒细胞纤毛组装不正确。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。

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.