VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

Abstract

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can’t hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer’s vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics

“…In addition, we assessed spindle orientation in cells depleted of the ESCRT components Hrs (ESCRT‐0) or CHMP4B (ESCRT‐III; Fig EV2F and G), since ALIX often influences biological processes in concert with the ESCRT machinery (Christ et al , ). However, neither depletion of Hrs nor CHMP4B significantly affected the spindle tilting (Fig EV2F), consistent with the reported lack of ESCRT‐III/CHMP4B at centrosomes (Ott et al , ). This suggests that whereas regulation of MS orientation depends on ALIX, it is independent of core ESCRT proteins.…”

“…Source data are available online for this figure. at centrosomes (Ott et al, 2018). This suggests that whereas regulation of MS orientation depends on ALIX, it is independent of core ESCRT proteins.…”

The regulation of cilium assembly and disassembly in development and disease

“…Apart from the Golgi-centriole pathway, components of the multi-subunit endosomal sorting complex required for transport (ESCRT) are essential for sustained docking of PCVs, supporting the involvement of the endosome trafficking pathway in CV growth (Ott et al, 2018). After PCV docking, the membrane-shaping protein EHD1 is recruited to PCVs to facilitate their fusion into a large CV, which caps the entire distal ends of MCs (Fig.…”

ESCRT subunit CHMP4B localizes to primary cilia and is required for the structural integrity of the ciliary membrane

“…A previous proteomic analysis of isolated ciliary transition zones of Chlamydomonas identified endosomal sorting complexes required for transport (ESCRT) proteins, which constitute a multicomponent molecular machinery required for the generation and stabilization of membrane curvature. 15 Expression of a dominant negative form of VPS4 reduces centrosomal γ-Tubulin levels and also interferes with microtubule growth and ciliogenesis. 12 The upstream ESCRT components (ESCRTs-0, I, and II) assemble into heteromultimers on the endosomal surface to initiates the assembly of the ESCRT-III complex, which together with vacuolar protein sorting-associated protein 4 (VPS4) is responsible for bending endosomal membranes.…”

“…10,11 ESCRT proteins are involved in a diverse membrane remodeling events, including intraluminal vesicle formation at the late endosome, the final abscission stage of cytokinesis, and the budding of HIV-1 and other lentiviruses. 15 The ESCRT-0 subunits STAM and Hrs drive internalization of polycystins at the ciliary base of Caenorhabditis elegans and thus downregulate polycystin abundance in the ciliary membrane. 13 ESCRT-III proteins deform a membrane by forming curved oligomeric filaments on the membrane.…”