Mechanisms of regulated assembly and remodeling of Intraflagellar Transport trains

• Applicant: Dr. Zeynep Ökten
• Subject Area: Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449713185

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.

• DFG Programme: Research Grants