Sunday, May 11, 2025
5/11/2025
Trafficking and Sorting Mechanisms of Golgi Vesicles to Cilia - PD/PI: Takemaru, Ken-Ichi 7. PROJECT SUMMARY Cilia are evolutionarily conserved microtubule-based structures that protrude from the apical cell surface to perform diverse biological functions. Primary cilia are present on most cell types in the human body and play essential roles in embryonic and postnatal development of various organ systems as a dynamic chemo- and mechanosensing center. Dysfunctional primary cilia have been linked to numerous genetic disorders, such as organ laterality defects, polydactyly, and polycystic kidney disease (PKD), collectively known as ciliopathies. Therefore, a better understanding of ciliogenesis is crucial for the development of comprehensive strategies for the prevention and treatment of ciliopathies. The polarized vesicle trafficking of proteins and lipids from the trans-Golgi network (TGN) and recycling endosomes plays important roles in ciliogenesis. Upon fusion of vesicles at the ciliary base or the surrounding periciliary region, lipids and proteins are incorporated into the ciliary compartment. However, the molecular mechanisms for the formation, cargo sorting, and trafficking of cilium-bound vesicles are highly complex and remain poorly understood. Chibby 1 (Cby1) is a conserved small coiled-coil protein that localizes to the base of cilia and plays a crucial role in ciliogenesis. Cby1-knockout mice develop several ciliopathy features such as chronic airway infection, polydactyly, and PKD. Through affinity purification/mass spectrometry, we have identified coiled-coil domain-containing 186 (CCDC186) as a new Cby1-interacting protein. CCDC186 harbors molecular characteristics of golgins that function with small GTPases in budding, transport, tethering, and docking of Golgi-derived vesicles. We found that CCDC186 binds to and colocalizes with Cby1 at centrosomes and TGN. Depletion of CCDC186 in cultured cells results in a reduction in the recruitment of Cby1 to the ciliary base and impairs ciliogenesis. Consistent with its crucial role in ciliogenesis, CCDC186-knockout mice develop cystic kidneys, a hallmark of ciliary defects. Thus, our preliminary data suggest that CCDC186 facilitates the targeting of Cby1 vesicles to the ciliary base, thereby enhancing ciliogenesis. The overall goal of this application is to elucidate the molecular and functional mechanisms of Cby1- CCDC186 interactions in vesicle trafficking during ciliogenesis. In order to achieve this goal, we propose the following Specific Aims: Specific Aim 1. Investigate the physical and functional interactions of CCDC186 with Cby1 and small GTPases during ciliogenesis; Specific Aim 2. Investigate cystic kidney phenotypes and possible defects in renal cilia in CCDC186-knockout mice; Specific Aim 3. Isolate novel CCDC186 interactors using proximity labeling and affinity purification and characterize their functions during ciliogenesis. We anticipate that these experiments will contribute to a fundamental understanding of the molecular and cellular mechanisms of vesicle and cargo trafficking during ciliogenesis.
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