Stine Kjær Morthorst:
Regulation of vesicle trafficking at the primary cilium

Date: 26-04-2018    Supervisor: Lotte Bang Pedersen & Søren Tvorup Christensen



Primary cilia are microtubule-based antenna-like organelles emanating from the surface of most quiescent eukaryotic cells. The ciliary membrane is highly enriched in specific signaling receptors and ion channels, which enable the primary cilium to play an important role in the coordination of numerous different cellular and developmental signaling pathways. Regulated trafficking of ciliary membrane proteins to and from the ciliary compartment is crucial for the correct formation and signaling function of the primary cilium. Consequently, mutations in genes coding for components involved in the assembly or function of the primary cilium can lead to different disorders and diseases, collectively termed ciliopathies.

This thesis is based on three original articles (Article I, II and IV) and a review (Article III) focusing on mechanisms involved in regulating membrane protein trafficking to and from primary cilia and how such trafficking affects signaling.

In Article I we focus on components involved in the trafficking of proteins towards the ciliary base. First, we take a bioinformatics approach and identify transport protein particle complex 8 (TRAPPC8) and components of the TRAPPII complex as novel ASPM, SPD-2, Hydin (ASH) domain containing proteins, and further suggest that the ASH domain mediates their centrosomal localization. Additionally, we find TRAPPC8 to be involved in ciliogenesis, possibly through its involvement in RABIN8 recruitment to the ciliary base.

Our focus in Article II is on the mechanisms and specific proteins involved in the regulation of trafficking across the transition zone (TZ) barrier. We show that the kinesin-3 motor protein KIF13B interacts with NPHP4 and localizes to the ciliary-centrosome axis in a NPHP4- dependent manner. Further, depletion of either NPHP4 or KIF13B disrupts the accumulation of caveolin 1 (CAV1) at a specific TZ membrane region. Disruption of the CAV1 microdomain further inhibits Sonic Hedgehog (Shh)-induced accumulation of Smoothened in the ciliary compartment leading to reduced transcription of the downstream target gene, GLI1. The collective findings suggest that KIF13B and NPHP4 are required for the formation of a CAV1- enriched microdomain at the TZ membrane region necessary for the regulation of Shh signaling.

Finally, in Article IV the focus is on a potential new regulator of trafficking away from the ciliary compartment. We characterize the interaction between angiomotin isoform 2 (Ap80) and KIF13B and show that Ap80 localizes to the centrosome/cilia base in a KIF13B-independent manner. Additionally, FLAG-Ap80 co-localize with CAV1 and the early endosomal marker RAB5 at vesicles around the ciliary base and throughout the cytosol. Further, we find that depletion of Ap80 results in elongated cilia and from the collective results we suggest that Ap80 is involved in endocytosis at the ciliary base and thereby in regulating ciliary membrane homeostasis.

Collectively, the results in this thesis provide novel insight into the mechanisms regulating different steps in ciliary membrane protein trafficking important for proper cilia formation and signaling function. Future investigations will further elucidate these mechanisms and hopefully contribute to development of therapeutic approaches to combat ciliopathies.