Mechanisms of regulated assembly and remodeling of Intraflagellar Transport trains

不动的感觉纤毛几乎从人体所有细胞的表面突出，这对于从胚胎发育到感觉知觉的多种细胞过程至关重要。纤毛的多方面操作范围与在真核门中构建和维持这些准细胞器的进化高度保守机制形成鲜明对比。事实上，所有纤毛，无论是运动纤毛还是不动纤毛，都需要非膜结合的鞭毛内运输 (IFT) 来实现其构建和功能。然而，IFT 的基本监管原则在分子水平上很大程度上是未知的。所谓的数兆道尔顿大小的 IFT 链是如何由 20 多个不同的小蛋白质亚基组装而成的？ 如何改造这些列车以强制执行 IFT 列车的驱动蛋白 2 或动力蛋白 2 驱动的运输？到目前为止，很清楚的是，仅存在 IFT 亚基不足以组装这种大型道尔顿复合物。控制IFT列车在体内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激酶。然而，这些激酶在分子水平上的具体作用仍然未知。在假设驱动的机制方法中，我们发现纤毛特异性激酶直接磷酸化特定的 IFT 亚基。引人注目的是，磷酸化促使一些亚基寡聚化，而另一些亚基则分解成单体。这是 IFT 亚基磷酸化调节结构重组的首次演示。因此，我们的结果表明可逆磷酸化依赖性过程可以调节 IFT 序列的组装和重塑，为此我们在体外和体内提供了初步证据。在我们提出的工作中，我们将描述纤毛定位激酶对体外和体内各个 IFT 亚基的复合物形成特性的调节影响。我们研究的一个主要目标是利用诱变、SEC-MALS 分析和定量磷酸化质谱来绘制 IFT 亚基磷酸化调节的相互作用组图。我们将通过化学交联和原子建模进一步表征各个亚基的 3-D 结构。正如第一个进行磷酸化的 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，以氨基酸精度揭示磷酸化调节过程对体内 IFT 的影响。我们的方法将提供先前组装的纤毛蛋白“稳态”相互作用图谱中所缺少的新颖的监管视角。先前已在几个 IFT 亚基中描述了致病突变。鉴于磷酸化靶点就在这些亚基中，我们的研究有可能有助于对纤毛病及其治疗的分子理解。

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.