Mechanisms of regulated assembly and remodeling of Intraflagellar Transport trains

从胚胎发育到感觉知觉的各种细胞过程中，人体内几乎所有细胞的表面都有一根固定的感觉纤毛。纤毛的多方面运作范围与在真核生物门中建立和维持这些准细胞器的进化高度保守的机制形成鲜明对比。几乎所有的纤毛，无论是活动的还是固定的，都需要非膜结合的鞭毛内转运(IFT)来实现它们的结构和功能。然而，在分子水平上，IFT的潜在调控原则在很大程度上是未知的。所谓的多百万道尔顿的IFT-Trains是如何由20多个不同的小蛋白亚基组装而成的？这些列车是如何改装的，以执行由kinesin-2或dynein-2驱动的IFT列车的运输？到目前为止，很明显，IFT亚基的单独存在不足以组装这种多兆道尔顿复合体。在体内控制IFT序列组装和重塑的调控机制是什么？在过去的几年里，已经发现了几种纤毛定位的激酶。然而，这些激酶在分子水平上的具体作用仍不清楚。在一种假说驱动的机制方法中，我们发现纤毛特异的激酶直接磷酸化特定的IFT亚基。值得注意的是，磷酸化促进了一些亚基的寡聚化，而另一些亚基则被分解成单体。这是第一次证明了磷酸化调控的IFT亚基的结构重组。因此，我们的结果暗示可逆的磷酸化依赖的过程可以调节IFT序列的组装和重塑，这为我们在体外和体内提供了初步的证据。在我们拟议的工作中，我们将描述纤毛定位的激酶在体外和体内对各自IFT亚单位的复杂形成特性的调节影响。我们研究的一个主要目标是利用突变、SEC-MALS分析和定量磷质谱来定位IFT亚基的磷酸化调控的相互作用组。我们将通过化学交联和原子模拟进一步表征各个亚基的三维结构。正如第一个经历磷酸化的IFT亚基所证明的那样，我们的深入体外分析将用于设计特定的体内实验，以氨基酸精度揭示体内磷酸化调控过程对IFT的影响。我们的方法将提供新的调控视角，这些视角是以前组装的纤毛蛋白的“稳态”相互作用图中所缺少的。此前已在几个IFT亚基中描述了致病突变。鉴于磷酸化靶标是这些亚基中的一部分，我们的研究有可能为纤毛疾病及其治疗的分子理解做出贡献。

An immotile sensory cilium projects nearly from the surface of all cells in the human body that is essential for diverse cellular processes ranging from embryonic development to sensory perception. The multifaceted operational range of the cilium starkly contrasts the evolutionary highly conserved mechanism that builds and maintains these quasi-organelles across the eukaryotic phyla. Virtually all cilia, motile and immotile alike, require the non-membrane bound IntraFlagellar Transport (IFT) for their construction and function. Yet, underlying regulatory principles of IFT are largely unknown at molecular level. How are the so-called IFT-Trains of multi-mega Dalton in size are assembled from more than 20 different small protein subunits? How are these trains remodeled to enforce either kinesin-2- or dynein-2-driven transport of the IFT trains? It is so far clear that the sole presence of the IFT subunits are not sufficient to assemble such multi-mega Dalton complexes. What are the regulatory mechanisms that control the assembly and remodeling of IFT trains in vivo? Over the past years, several cilia-localized kinases haven discovered. However, the specific roles of these kinases at the molecular level remain unknown. In a hypothesis-driven mechanistic approach, we discovered that cilia-specific kinases directly phosphorylate specific IFT subunits. Strikingly, phosphorylation prompts oligomerization of some subunits while others are disassembled into monomers. This is the first demonstration of phosphorylation-regulated structural reorganization of IFT subunits. Our result thus implicate that reversible phosphorylation-dependent processes can regulate the assembly and remodeling of IFT trains, for which we provide preliminary evidence in vitro and in vivo. In our proposed work, we will delineate the regulatory impact of cilia-localized kinases on the complex formation properties of the respective IFT subunits in vitro and in vivo. One major goal of our studies is to map the phosphorylation-regulated interactome of the IFT subunits using mutagenesis, SEC-MALS analysis, and quantitative phospho-mass spectrometry. We will further characterize the 3-D structure of the respective subunits through chemical crosslinking and atomistic modeling. As demonstrated with the first IFT subunit that is subject to phosphorylation, our in-depth in vitro analysis will be used to design specific in vivo experiments to expose the impact of phosphorylation-regulated processes on IFT in vivo with amino acid-precision. Our approach will provide novel regulatory perspectives that are missing in previously assembled ‘steady-state’ interaction maps of ciliary proteins. Disease-causing mutations have previously been described in several IFT subunits. Given that phosphorylation targets are among those subunits, our studies bear the potential to contribute to the molecular understanding of ciliopathies and their treatment.