Mechanisms of regulated assembly and remodeling of Intraflagellar Transport trains

鞭毛内运输列车的规范组装和改造机制

• 批准号: 449713185
• 负责人: Dr. Zeynep Ökten
• 金额: --
• 依托单位: Lehrstuhl für Molekulare Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/449713185?language=en
• 关键词: Mechanisms; regulated; assembly; remodeling; Intraflagellar

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.

不动的感觉纤毛几乎从人体内所有细胞的表面伸出，这对于从胚胎发育到感觉感知的各种细胞过程是必不可少的。纤毛的多方面的操作范围形成鲜明对比的进化高度保守的机制，建立和维持这些准细胞器在真核生物门。事实上，所有的纤毛，运动和不运动的一样，需要非膜结合的鞭毛内运输（IFT）的建设和功能。然而，IFT的潜在调控原理在分子水平上很大程度上是未知的。所谓的多兆道尔顿大小的IFT-火车是如何从20多个不同的小蛋白质亚基组装而成的？ 这些火车是如何改造，以加强驱动蛋白-2或动力蛋白-2驱动的运输的IFT列车？目前清楚的是，IFT亚基的单独存在不足以组装这种多兆道尔顿复合物。体内控制IFT序列组装和重塑的调控机制是什么？在过去的几年中，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。在一个假设驱动的机制的方法，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，而另一些亚基分解成单体。这是IFT亚基磷酸化调节的结构重组的第一个证明。因此，我们的研究结果表明，可逆的磷酸化依赖的过程可以调节IFT列车的组装和重塑，我们提供了初步的证据，在体外和体内。在我们提出的工作中，我们将描绘纤毛定位激酶的调控影响，在体外和体内的各个IFT亚基的复合物形成特性。我们的研究的一个主要目标是使用诱变、SEC-MALS分析和定量磷酸化质谱来绘制IFT亚基的磷酸化调节的相互作用组。我们将通过化学交联和原子模型进一步表征各个亚基的3-D结构。正如第一个受到磷酸化的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，以暴露磷酸化调节过程对体内IFT的影响，并具有氨基酸精度。我们的方法将提供新的监管角度，在以前组装的纤毛蛋白的“稳态”相互作用地图失踪。致病突变先前已在几个IFT亚基中描述。鉴于磷酸化的目标是在这些亚基，我们的研究承担的潜力，有助于纤毛病变及其治疗的分子理解。