Shining light on motor regulation in the cilium

Chapter 1: General introduction All living organisms must sense their surroundings to find food and detect threats. Cilia, which took hundreds of millions of years to evolve, help eukaryotes perceive their environment by detecting extracellular chemicals like hormones or environmental signals. This thesis explores cilia, a complex biological structure, making it essential to first understand evolution by natural selection. Evolution by natural selection follows three basic principles: (I) Genetic mutations occur randomly and can be beneficial, harmful, or neutral. (II) These mutations are heritable. (III) Since more individuals are born than the environment can support, competition arises. Those with advantageous traits are more likely to survive and reproduce, making these traits more common in future generations. This is evolution by natural selection in simple terms. Cilium The cilium consists of a microtubule backbone, the axoneme, surrounded by a membrane enriched with transmembrane receptors to detect extracellular signals. At the cilium’s base is the transition zone, which controls protein entry and exit. Protein transport is driven by intraflagellar transport (IFT), using IFT-A and IFT-B complexes. Two kinesin-2 motors (OSM-3 and kinesin-II in C. elegans) drive anterograde transport, while retrograde transport is powered by IFT dynein. C. elegans The model organism C. elegans is a transparent, hermaphroditic roundworm used extensively in biological research. It has a simple structure, is easy to maintain, and was the first animal to have its complete genome sequenced in 1998. Chapter 2: Mechanisms of Regulation in IFT This chapter explores IFT regulation from three perspectives: (i) the track (axoneme), (ii) motor proteins, and (iii) regulatory proteins. The axoneme uses different tubulin proteins to regulate IFT, and in Chlamydomonas, anterograde trains use the B-tubule while retrograde trains use the A-tubule. C. elegans uses both homodimeric and heterotrimeric kinesin-2 motors, with dynein inactivated at the ciliary tip to prevent a tug of war. Chapter 3: DYF-5 Affects IFT Train Turnaround DYF-5, a kinase related to CILK1, regulates IFT train turnaround. In the absence of DYF-5, C. elegans cilia become disorganized, kinesin-II is no longer restricted to the cilium’s proximal segment, and retrograde transport is severely reduced. This leads to an accumulation of IFT components at the ciliary tip and increased variation in IFT velocities, affecting ciliary function. Chapter 4: Sorting at the Ciliary Base and Ciliary Entry of IFT Components This chapter investigates how IFT components enter the cilium. We observed using single-molecule imaging that IFT-A and IFT-B arrive in vesicles, while the BBSome diffuses to the ciliary base. The components assemble sequentially: first IFT-B, then IFT-A, followed by the BBSome binding to the IFT train. Chapter 5: Outlook In the final chapter, I discuss potential follow-up experiments, including identifying DYF-5 targets and investigating the link between ciliary sensing and IFT regulation. Preliminary results on DYF-18’s effect on retrograde transport are also mentioned. W