Primary cilia are long (up to 9 μm long) narrow (less than 1 μm wide) microtubules based membrane protrusions that serve as key transducers of sensory stimuli. Cilia are dynamically regulated during the cell cycle and their formation involves centrosome remodeling, vesicle docking and coupled axoneme and membrane extension. Recently, the ESCRT-associated AAATPase, VPS4, was identified in Chlamydamonas at the transition zone, a region at the ciliary base that acts as a ciliary gate. Several studies showed that mutations affecting proteins located in this zone result in cilia-related diseases (ciliopathies) including retinal degeneration and kidney disease. In this study we investigate the role of VPS4 in primary cilia formation and maintenance. We find that in mammalian cells depletion of VPS4 or over expression of its dominant-negative version (VPS4 DN) impair cilia formation. Similarly, injection of zebrafish embryos with VPS4 DN reduced cilia formation in Kupffer’s vesicle and resulted in developmental delays and death. Using Structured Illumination Microscopy (SIM) we show that VPS4A DN localizes to the centrosome, specifically, to the transition zone. Mapping the composition of different centrosomal proteins by SIM reveals that several key ciliary components including γ-tubulin and Pericentriolar Material 1 (PCM1) exhibit altered organization while others (CEP164, CP110 and B9D2) are not affected. Moreover, using micropatterns, we demonstrate that over- expression of VPS4A DN alters centrosome positioning. Correlative EM analysis shows accumulation of ciliary vesicles at centrosomes in VPS4A DN expressing cells, suggesting a role for VPS4 in early stages of ciliogenesis. Taken together, our study suggests a role for VPS4 in cilia biogenesis and adds a new dimension to recent work that has defined several of the early steps in ciliogenesis.
