Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly

Abstract

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominantnegative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage

“…Although several centriolar proteins such as STIL, SAS-6, CEP120, SPICE1, and CEP135 can interact with CPAP and positively regulate the centriole duplication and elongation process (30,32,45,46), this is the first study that uncovers the mechanism by which CPAP levels are regulated in the cell to restrict the centriole length. Earlier we showed that centrobin binds to CPAP directly, and this interaction is essential for maintaining CPAP levels on centrioles (48).…”

“…The duplication process involves three stages: initiation, elongation, and maturation. Initiation process includes the recruitment of essential centriolar proteins such as CEP152, PLK4, hSAS-6, STIL, CPAP 2 , CEP135, CP110, ␥-tubulin, and centrobin to the proximal end of preexisting mother centrioles (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Once this pro-centriolar protein complex is formed, centrioles are elongated to achieve a maximum length of ϳ500 nm and width of ϳ200 nm by the addition of ␣/␤-tubulin heterodimers onto it (37).…”

The Centrosome and Its Duplication Cycle

“…Similarly, when Cep135 was disrupted in the chicken DT40 cell line, there was only a small decrease in the centriole number with no major defects in centrosome composition or structure (Inanc et al 2013). However, human Cep135 is required for excessive centriole duplication following Plk4 overexpression (Kleylein-Sohn et al 2007), and a recent report showed that depletion of Cep135 reduces the centrosome number (Lin et al 2013a). Thus, although a common function of Cep135 seems to ensure the intact structure of centrioles, in most cases, its loss does not have a devastating effect (Roque et al 2012;Inanc et al 2013;Lin et al 2013a).…”

“…A tubulin-binding domain is critical for this function (Hsu et al 2008;Cormier et al 2009), and its stable incorporation into centrioles is dependent on gtubulin and microtubule assembly (Dammermann et al 2008). CPAP is reported to interact with STIL and another centriole protein, Cep135 (Tang et al 2011;Cottee et al 2013;Hatzopoulos et al 2013;Lin et al 2013a), but how these molecules cooperate to regulate procentriole assembly requires further analysis. The functional importance of Cep135 was first shown by the requirement for its ortholog, Bld10p, for cartwheel formation in Chlamydomonas and Paramecium Jerka-Dziadosz et al 2010).…”

Small organelle, big responsibility: the role of centrosomes in development and disease

“…Strikingly, four of these carry MCPH-linked mutations [26][27][28]. CPAP/ MCPH6 appears to be a key node of the MCPH protein network, because it binds STIL/MCPH7, CEP152/MCPH9 and CEP135/MCPH8, although probably not simultaneously [29][30][31][32]. A missense MCPH mutation weakens the ability of CPAP to bind STIL and impairs centriole production [32][33][34][35].…”