Control of Centriole Length by CPAP and CP110

Abstract

Centrioles function as the major components of centrosomes, which organize microtubule (MT) arrays in proliferating cells, and as basal bodies for primary cilia formation in quiescent cells. Centrioles and basal bodies are structurally similar, barrel-shaped organelles composed of MTs. In proliferating cells, two new centrioles, termed procentrioles, form during the S phase of the cell cycle in close proximity to the proximal ends of the two preexisting parental centrioles, often at a near-orthogonal angle. Considerable progress has been made toward understanding the biogenesis of centrioles, but the mechanisms that determine their lengths remain unknown. Here we show that overexpression of the centriolar protein CPAP in human cells enhances the accumulation of centriolar tubulin, leading to centrioles of strikingly increased length. Consistent with earlier work, we also find that elongated MT structures can be induced by depletion of the distal end-capping protein CP110 from centrioles. Importantly, though, these structures differ from genuine primary cilia. We thus propose that CPAP and CP110 play antagonistic roles in determining the extent of tubulin addition during centriole elongation, thereby controlling the length of newly formed centrioles.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…Although this observation may appear surprising, considering that Sas‐6 is a key structural component of the centriolar cartwheel (Gonczy, 2012; Hirono, 2014), it supports the view that recruitment of Sas‐6 to nascent centrioles is extensively controlled by posttranslational mechanisms (Dzhindzhev et al , 2014; Keller et al , 2014; Ohta et al , 2014; Arquint et al , 2015; Kratz et al , 2015). Similarly, although CPAP and CP110 have both been implicated in controlling the length of centrioles (Kohlmaier et al , 2009; Schmidt et al , 2009; Tang et al , 2009), their abundance in different cell lines shows no correlation, arguing that their functions at centrioles do not simply reflect protein concentration. Finally, we note that most centrosomal proteins were least abundant in the KE37 T‐lymphoblastoid line, which is commonly used for centrosome purification (Gosti‐Testu et al , 1986; Andersen et al , 2003).…”

“…Proteins were then resolved by SDS–PAGE and transferred onto a nitrocellulose membrane. Primary antibodies were directed against α‐tubulin (clone DM1A, 1:5,000; Sigma‐Aldrich, St. Louis, MO, USA), γ‐tubulin (clone GTU‐88, 1:5,000; Sigma‐Aldrich, St. Louis, MO, USA), GFP (ab290; 1:5,000; Abcam, Cambridge, UK), STIL (ab89314, 1:2,000; Abcam, Cambridge, UK), Sas‐6 (Kleylein‐Sohn et al , 2007), CP110 (1:2,000, Schmidt et al , 2009), cyclin A (1:2,000, Maridor et al , 1993), cyclin B1 (05‐373, 1:2,000, Merck Millipore, Darmstadt, Germany), and phospho‐histone H3/serine 10 (3377, 1:1,000, Cell Signaling Technology, Danvers, MA, USA); secondary antibodies were HRP‐conjugated anti‐mouse immunoglobulin (170‐6516, 1:3,000, Bio‐Rad, Hercules, CA, USA) or anti‐rabbit immunoglobulin (170‐6515, 1:3,000, Bio‐Rad, Hercules, CA, USA).…”

“…The protein CPAP gets recruited to promote the assembly of microtubules onto the cartwheel (Tang et al , 2011a; Cottee et al , 2013; Sharma et al , 2016), and this step is likely assisted by Cep135 (the human homolog of Chlamydomonas Bld10) (Hirono, 2014). CPAP also cooperates with additional proteins, including CP110, in determining the length of nascent centrioles (Kohlmaier et al , 2009; Schmidt et al , 2009; Tang et al , 2009; Comartin et al , 2013; Lin et al , 2013; Sharma et al , 2016). Finally, centriole maturation is completed by the acquisition of subdistal and distal appendages (Paintrand et al , 1992; Tateishi et al , 2013).…”

Tankyrase 1 regulates centrosome function by controlling CPAP stability

“…Centrosomal P4.1-associated protein (CPAP), a human SAS4-related protein, was found to be required for centrosome function in human cells [6,7]. Depletion of CPAP in human cells prevented centrosome duplication, whereas overexpression of CPAP led to aberrant centriole elongation, supernumerary centrioles and spindle multipolarity [8][9][10][11]. A crucial role for CPAP and centriole maturation in humans is evidenced by its mutation in autosomal recessive primary microcephaly (MCPH) [12][13][14].…”

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

“…Recently, CPAP (also referred to as CENPJ) overexpression was reported to cause the centriolar microtubules to elongate beyond the predetermined length of 0.5 m and led to accumulation of multipolar spindles (35,39,40). Depletion of cellular CPAP also resulted in loss of centrosome integrity and led to a higher number of cells with mono-and multipolar (35) as well as abnormal, misoriented spindles (4).…”