Abstract
To elucidate the role of centriolar satellites in ciliogenesis, we deleted the gene encoding the PCM1 protein, an integral component of satellites. PCM1 null human cells show marked defects in ciliogenesis, precipitated by the loss of specific proteins from satellites and their relocation to centrioles. We find that an amino-terminal domain of PCM1 can restore ciliogenesis and satellite localization of certain proteins, but not others, pinpointing unique roles for PCM1 and a group of satellite proteins in cilium assembly. Remarkably, we find that PCM1 is essential for tethering the E3 ligase, Mindbomb1 (Mib1), to satellites. In the absence of PCM1, Mib1 destabilizes Talpid3 through poly-ubiquitylation and suppresses cilium assembly. Loss of PCM1 blocks ciliogenesis by abrogating recruitment of ciliary vesicles associated with the Talpid3-binding protein, Rab8, which can be reversed by inactivating Mib1. Thus, PCM1 promotes ciliogenesis by tethering a key E3 ligase to satellites and restricting it from centrioles.
Highlights
Primary cilia are antenna-like organelles that protrude from the surface of many differentiated cells and serve to orchestrate key signaling events required for development
We examined this cell line by immunofluorescence and transmission electron microscopy (TEM) and found that in contrast with previous studies, in which ciliation was reduced by 20–50% after knock-down, gene ablation resulted in a complete failure to ciliate when cells were made quiescent through serum deprivation (Figure 1A, Figure 1—figure supplement 1, and data not shown)(Lopes et al, 2011; Stowe et al, 2012)
We found that PCM1 knock-out cells completely lacked centriolar satellites, which were visualized through immunofluorescent detection with a panel of antibodies against known satellite proteins (Cep131, Cep290, Cep90, and Mib1), and this defect could be rescued by expression of Myc-PCM1 (Figure 1—figure supplement 1 and Figure 3)
Summary
Primary cilia are antenna-like organelles that protrude from the surface of many differentiated cells and serve to orchestrate key signaling events required for development (reviewed in Kobayashi and Dynlacht, 2011). An extensive array of centriolar and basal body proteins has been shown to play a critical role in ciliogenesis. Among these key regulators, Talpid is essential for ciliogenesis, and mutations in the gene encoding this protein lead to a panoply of developmental defects in model organisms and human ciliopathies, including Joubert Syndrome (Alby et al, 2015; Bachmann-Gagescu et al, 2015; Bangs et al, 2011; Davey et al, 2006; 2007; Malicdan et al, 2015; Roosing et al, 2015; Stephen et al, 2015). We have shown that Talpid associates with Rab, a component of ciliary vesicles, and silencing Talpid compromises the efficient recruitment of ciliary vesicles to basal bodies (Kobayashi et al, 2014)
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