The primary cilium (plural cilia), which is a non-motile antenna-like organelle emerging from the surface of many eukaryotic cell types, plays a vital part in numerous cellular processes throughout the pre-natal human and animal development as well as in the adult organism. Throughout the years, we have significantly increased our understanding of the structure and functions of the primary cilium, and now this organelle has emerged to be essential for the proper functioning of several cellular and developmental signaling pathways. Consequently, mutations resulting in either abnormal formation or function of cilia underpin numerous diseases (ciliopathies) that can affect multiple organ systems. Therefore, it is vital to understand the molecular workings of cilia, not only from a fundamental science perspective but also for medical reasons.

This thesis is centered on two original manuscripts (manuscripts I and II), which explore the mechanisms involved in regulating ciliary composition, with a primary focus on ciliary targeting and homeostasis of PC2 in kidney epithelial cells. In manuscript I, we show that kinesin motor protein KIF13B regulates ciliary PC2 levels in a time-dependent manner. Previous studies in worms showed that PC2 is released into extracellular vesicles (EVs) to mediate cell-cell communication. Our findings build upon this knowledge, demonstrating how KIF13B controls the release and cargo content of EVs, including several known EV biogenesis regulators, such as HRS, TSG101, and ALIX, as well as PC2. We also provide evidence that KIF13B exhibits burst-like movement within the primary cilium of kidney epithelial cells and, together with PC2, is released in large EVs from such cells. In manuscript II, we focus on a potential new regulator of vesicular trafficking/targeting to the primary cilium. We show that scaffold protein DLG1, a core component of the Scribble polarity complex that regulates apicobasal establishment and maintenance in epithelial cells, is important for regulating ciliary length and composition in kidney epithelial cells, both in vitro and in vivo. Further studies showed that DLG1 interacts with PC2 trafficking regulators, SDCCAG3 and IFT20, and together mediate the targeting of PC2 to the primary cilium. Notably, a DLG1 missense variant mutated in patients with congenital anomalies of the kidney and urinary tract (CAKUT) was unable to confer ciliary targeting on SDCCAG3 and PC2. Finally, we identified several additional proteins that also rely on DLG1 for ciliary recruitment, including proteins that affect energy homeostasis, NFκB, and TGFβ signaling.

Overall, the findings presented in this thesis shed new light on the intricate mechanisms that regulate ciliary membrane composition and homeostasis in polarized kidney epithelial cells and implicate primary cilia dysfunction as a possible contributing factor to CAKUT.