Plk4-Induced Centriole Biogenesis in Human Cells

Abstract

We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.

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

“…2C). CP110, which stains the distal ends of centrioles (27), was not significantly different in the control and centrobin-overexpressing cells (Fig. 2D).…”

“…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).…”

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

“…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).…”

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].…”