CEP120 and SPICE1 Cooper

Current Biology 23, 1360–1366, July 22, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.06.002

Reportate

with CPAP in Centriole Elongation

David Comartin,1,2 Gagan D. Gupta,1 Eden Fussner,3

Etienne Coyaud,4 Monica Hasegan,1 Marco Archinti,6

Sally W.T. Cheung,1 Deborah Pinchev,1 Steffen Lawo,1

Brian Raught,4 David P. Bazett-Jones,3 Jens Luders,5

and Laurence Pelletier1,2,*1Samuel Lunenfeld Research Institute, Mount Sinai Hospital,600 University Avenue, Toronto, ON M5G 1X5, Canada2Department of Molecular Genetics, University of Toronto,Toronto, ON M5S 1A8, Canada3The Hospital for Sick Children and Department ofBiochemistry, University of Toronto, Toronto, ON M5G 1L7,Canada4Ontario Cancer Institute and Department of MedicalBiophysics, University of Toronto, MaRS TMDT 9-805,101 College Street, Toronto, ON M5G 1L7, Canada5Institute for Research in Biomedicine (IRB Barcelona),Carrer de Baldiri Reixac 10, 08028 Barcelona, Spain6ERA Biotec, Parc Cientıfic de Barcelona, Carrer de BaldiriReixac 15–21, 08028 Barcelona, Spain

Summary

Centrosomes organize microtubule (MT) arrays and arecomprised of centrioles surrounded by ordered pericentrio-

lar proteins [1–4]. Centrioles are barrel-shaped structurescomposed of MTs, and as basal bodies they template the for-

mation of cilia/flagella. Defects in centriole assembly can

lead to ciliopathies and genome instability [5]. The assemblyof procentrioles requires a set of conserved proteins [6–8]. It

is initiated at the G1-to-S transition by PLK4 and CEP152[9, 10], which help recruit SASS6 and STIL to the vicinity

of the mother centriole to organize the cartwheel [11–14].Subsequently, CPAP promotes centriolar MT assembly and

elongation in G2 [15–17]. While centriole integrity is main-tained by CEP135 and POC1 through MT stabilization

[18–20], centriole elongation requires POC5 and is restrictedby CP110 and CEP97 [21, 22]. How strict control of centriole

length is achieved remains unclear. Here, we show thatCEP120 and SPICE1 are required to localize CEP135 (but

not SASS6, STIL, or CPAP) to procentrioles. CEP120 asso-ciates with SPICE1 and CPAP, and depletion of any of

these proteins results in short procentrioles. Furthermore,CEP120 or CPAP overexpression results in excessive

centriole elongation, a process dependent on CEP120,SPICE1, and CPAP. Our findings identify a shared function

for these proteins in centriole length control.

Results

We originally identified CEP120 and SPICE1 as putative inter-acting proteins [23]. While both are required for centriole dupli-cation [23–25], CEP120 also participates in neuronal nuclearmigration [26], and its loss in planarians results in shorter cen-trioles [27]. Together, these data suggest that the association

*Correspondence: pelletier@lunenfeld.ca

between SPICE1 and CEP120 might reflect a shared functionin centriole biogenesis.To examine the role of CEP120 and SPICE1 in centriole

assembly, we exploited the PLK4-induced centriole overdupli-cation assay [28]. Here, CEP120 and SPICE1 knockdownreduced procentriole formation to levels comparable to thecentriole assembly proteins CPAP or CEP135 (Figures S1Eand S1F available online). We assessed the consequence ofdepleting the centriole assembly proteins CPAP and CEP135alongside CEP120 and SPICE1. We observed that with 48 hrofRNA interference (RNAi) prior toPLK4 induction, aproportionof mother centrioles were not recruiting SASS6 (Figure S1F)[29], suggesting that they may have been defective prior tooverduplication. We therefore allowed only 24 hr of RNAi priorto S phase arrest and PLK4 induction for 24 hr, followed bya 16 hr release into G2, which mitigated the loss of SASS6recruitment (Figures 1A and 1B). Importantly, proteinswere stillefficiently depleted by RNAi (Figure 1C), and despite SASS6recruitment around mother centrioles, procentriole formationwas inhibited as judged by lack of CETN2 labeling (Figures 1Aand 1B). Strikingly, CEP120, SPICE1, CEP135, or CPAP deple-tion coincided with the ring-like accumulation of SASS6aroundmother centrioles (Figures 1A and 1D), in stark contrastto the typical ‘‘rosette’’ distributions (Figure 1A).To follow up on this observation, we surveyed hundreds of

PLK4-induced centrioles labeled with SASS6, CETN2, andCP110 using three-dimensional structured-illumination micro-scopy (3D-SIM) (Figure 1E). We observed centriole proteinslocalized as rings aroundmother centrioles in some cases (Fig-ure 1E, left side), and in other cases as distinct procentriolesof increasing lengths as judged by radial distancing of CP110from mother centrioles (Figure 1E, right side). We alsoobserved a ring of PLK4 surrounding mother centrioles (Fig-ure 1F), whereas CEP120 and SPICE1 localized to the walls ofelongated procentrioles (Figure 1F), consistent with reportedCEP120 immunoelectron microscopy (immuno-EM) results[24]. The morphology of the structures observed implies asequential recruitment of downstreamproteins and, in parallel,radial expansion away from the mother centriole duringcentriole elongation (Figure 1E). In the earliest stages of thisprogression, the centriolar markers resemble rings. As elonga-tion proceeds, these rings resolve into ‘‘rosettes,’’ and eventu-ally into distinct, elongated centrioles. This suggests that whenCEP120 or SPICE1 is removed, the localization of procentriolecomponents remains ring like, as centrioles fail to elongate.Since 3D-SIM permitted us to unambiguously resolve

mother centrioles and PLK4-induced procentrioles, weproceeded to map the recruitment and localization depen-dency of known centriole assembly proteins with respect toCEP120 and SPICE1. First, CEP120 and SPICE1 were mutuallyrequired for their localization to procentrioles and werestrongly reduced upon CEP152, CPAP, or CEP135 depletion(Figures 2A and S2A). SASS6 and STIL depletion also causeda reduction in CEP120 and SPICE1 levels in the vicinity ofmother centrioles (Figure S2A). Second, CEP152 was localizedinside the ring of PLK4 (Figure S2B), consistent with theirreported interaction [9], and this localization was unaffectedby CEP120 or SPICE1 depletion (Figure S2B). Third, SASS6,

Figure 1. CEP120 and SPICE1 Are Required for Centriole Assembly

(A) Representative pseudocolor-merged immunofluorescence micrographs of PLK4-induced centriole assembly in U-2 OS cells depleted of the indicated

proteins and labeled with antibodies to SASS6 (green), CETN2 (red), and DAPI for DNA (blue). The bottom panels show magnified centrosomes (boxed in

white) for each depletion condition (large inset, merged image of SASS6 and CETN2; small insets, individual grayscale images).

(B) Bar graph showing frequency of overduplication (light gray), failed duplication without SASS6 recruitment (white), and failed duplication with SASS6

recruitment (dark gray) in cells processed as in (A) (n > 100, three replicates per condition, mean 6 SEM).

(C) Western blots showing depletion of proteins in this PLK4 assay.

(D) Bar graph showing frequency of SASS6 rings in this assay under the same conditions as in (A) and (B) (n > 100, three replicates, mean 6 SEM).

(E) 3D-SIM pseudocolor-merged (top panel) and individual micrographs (bottom panels) of centrosomes after PLK4 overexpression, labeled with antibodies

to CETN2 (blue), SASS6 (red), and CP110 (green). Centrosomes were staged from left to right based on the radial spread of the three centriole markers and

represent stages of procentriole elongation. CETN2 marks the mother centriole and distal tips of the procentrioles, CP110 marks the distal tips of procen-

trioles, and SASS6 marks the centriole lumen from the proximal end toward the distal end.

(F) Centrioles processed as in (E) and labeled with antibodies to Myc-PLK4 (red), CETN2 (blue), and either CEP120 (green; left panel) or SPICE1 (green; right

panel). The ring of Myc-PLK4 (dashes in white) delineates the region between the mother centriole (‘‘M,’’ marked by CETN2) and the proximal end of pro-

centrioles.

Scale bars in (A) represent 10 mm, with insets showing 83 and 43; scale bars in (E) and (F) represent 1 mm. See also Figure S1.

CEP120, SPICE1, and CPAP in Centriole Biogenesis1361

CPAP, and STIL were localized to individual procentrioles incontrol conditions, but as rings partially overlapping the outeredge of the PLK4 ring upon CEP120 or SPICE1 depletion (Fig-ure 2C). This observation is consistent with a previous reportshowing that SASS6 acts upstream of CEP120 [24].

Under control conditions, CEP135 localized to the mothercentriole and in a ring that partially overlapped the PLK4 ring(Figure 2B). We refer to this region as the procentriole assem-bly region (PCAR), since this is where the earliest procentrioleswere detected as labeled by SASS6 (Figure 1E). Qualitatively,we observed that CEP120, SPICE1, or CPAP depletion in thePLK4 assay blocked the localization of CEP135 to the PCAR(Figure 2B). We verified this with quantitative image analysisof CEP135 levels within the PCAR in a random population ofcentrosomes (Figure S2C and the Supplemental ExperimentalProcedures) and validated this effect in cycling and S-phase-arrested HeLa cells (Figures S2D–S2F). We note that whencentriole duplication is blocked in CEP120, SPICE1, CPAP,

and CEP135 RNAi-treated cells, a marked increase in thePLK4 signal surrounding themother centriole is observed (Fig-ure 2 and S2). This implies a procentriole-dependent feedbackregulation of PLK4 levels that will warrant further investigation.We also examined the recruitment of regulators of centriole

elongation. While POC5 localization was largely unaffected byCEP120 or SPICE1 depletion, the capping proteins CP110 andCEP97 did not localize to procentrioles (Figures S2G–S2K).Upon CEP97 or CP110 depletion, CEP120 and SPICE1 formedelongated structures that extended beyond CETN2 at thedistal end of the centrioles, consistent with their establishedrole in restricting centriole length (Figures S2H–S2K) [22].When POC1A or POC5 were depleted, CEP120 localized toapparent procentrioles that lacked or had reduced localiza-tion of CETN2, whereas SPICE1 only sparsely localized tothe procentrioles (Figure S2D). Taken together, these resultssuggest that POC5 recruitment to the PCAR does not requireCEP120 and SPICE1 activity or the recruitment of CEP97 and

Figure 2. CEP120 and SPICE1 Are Required for CEP135 Localization to the Procentriole Assembly Region

3D-SIM pseudocolor-merged (all left panels) and individual grayscale (all others) micrographs of centrosomes depleted of the indicated proteins and

induced to overduplicate by PLK4 overexpression, followed by labeling with antibodies to CETN2 (blue), Myc-PLK4 (red), and SPICE1 or CEP120

(green; A), CEP135 (green; B), or SASS6, STIL, or CPAP (green; C). PLK4 micrographs in control conditions are scaled for visibility. All other micrographs

are quantitatively scaled so that their relative intensities can be compared. Scale bars represent 1 mm. See also Figure S2.

Current Biology Vol 23 No 141362

CP110. Additionally, the recruitment of CEP120 and SPICE1 toelongated centrioles is independent of CEP97 and CP110recruitment.

Next, we confirmed the CEP120-SPICE1 interaction bycoimmunoprecipitation (coIP). Their association required full-length CEP120 and both SPICE1 coiled-coil domains (FiguresS3C and S3D) [23]. To identify centriole assembly proteins thatinteract with CEP120, we expressed FLAG-tagged CEP120 asa BirA* fusion protein (BirA-R118G mutant, hereafter FLAG-BirA*-CEP120) to facilitate biotinylation of nearby proteins andperformed affinity purification mass spectrometry (AP-MS)using streptavidin-Sepharose beads [30]. In the BioIDapproach, biotinylation occurs before lysis, so weak or tran-sient interactions can be detected [30]. Importantly, FLAG-BirA*-CEP120 was functional and localized to centrosomes(Figures S3A, S3B, S1C, and S1D). Among the most prevalentAP-MS hits were CPAP and SPICE1 (Table S1). To validatethese interactions, we performed coIP with FLAG-CEP120,and we detected interactions with GFP-tagged SPICE1 andGFP-tagged CPAP (Figure 3A). Together, these data suggestthat CEP120 associates with both SPICE1 and CPAP. Interest-ingly, we noted that CP110 and CEP97 were also detected inour BioID experiment (Table S1) [22].

To test the possibility that CEP120 and SPICE1 function withCPAP, we looked for overexpression effects in cycling cells[15–17]. CEP120-GFP or CPAP-GFP, but not SPICE1-GFP,caused abnormal centriole elongation without affecting cell-cycle progression (Figures 3B, 3C, S3E, and S3F). As withCPAP, CEP120-induced centriole elongation was specific tocells in G2 (Figures S3G and S3H) [15–17]. Furthermore, lossof CEP120, SPICE1, or CPAP significantly inhibited centrioleelongation after CPAP or CEP120 overexpression in G2-arrested cells (Figures 3D–3I). This was not due to the loss ofcentrioles, since SASS6-RNAi had little effect on the frequencyof elongated centrioles, and mother centriole elongation wasalso blocked by CEP120, SPICE1, or CPAP depletion. (Figures3D–3I). Similar results were obtained when CEP135 was usedas a marker of elongation (data not shown). Thus our dataindicate that CEP120, SPICE1, and CPAP cooperate duringcentriole elongation.In serial EM sections of centrioles after CEP120, SPICE1, or

CPAP depletion in the PLK4 assay, we primarily observedeither short procentrioles or electron dense material aroundmother centrioles (Figure S4A). Therefore, we measuredprocentriole length and width in reconstructed tomograms ofcontrol and CEP120-, SPICE1-, and CPAP-depleted cells

Figure 3. CEP120 Functionally Interacts with CPAP and SPICE1

(A)Western blots from IPs in which FLAG-CEP120 was coexpressed with GFP fusions to USP37, SPICE1, or CPAP. Blots showGFP (top) and FLAG (middle)

blots after coIP, and GFP blots of inputs (bottom).

(B andC) Representativemicrographs (B) and elongation frequency bar graph (C) of U-2OS cells overexpressing SPICE1-GFP, CEP120-GFP, or CPAP-GFP,

labeled with antibodies against GFP (green) and acetylated a-tubulin (red; Ac.Tub.). White boxes are magnified insets shown in pseudocolor merge and

grayscale.

(D–I) Representativemicrographs (D andG) and elongation frequency bar graphs (E, F, H, and I) of U-2 OS cells overexpressing GFP tagged CEP120 (D–F) or

GFP taggedCPAP (G–I) in combinationwith RNAi depletion of SASS6, SPICE1, andCPAP or CEP120 (see the Supplemental Experimental Procedures). Cells

were labeled with antibodies against CEP164 (blue in insets), GFP (green), and glutamylated a-tubulin (red; Gl.Tub.). Insets are as in (B).

All bar graphs show mean6 SEM of three replicates in which at least 100 cells were examined visually for centrioles longer thanw2 mm per replicate (C, E,

and H), and for the presence of CEP164 (F and I). Scale bars in (B), (D), and (G) represent 10 mm, with all insets showing 23. See also Figure S3 and Table S1.

CEP120, SPICE1, and CPAP in Centriole Biogenesis1363

(Figures 4A–4C). The average cross-sectional (barrel) width ofprocentrioles was unchanged (Figure 4C), but microtubuleswere shorter in all three RNAi conditions (Figure 4B). We alsoobserved a decrease in acetylated and glutamylated tubulinin the PCAR region in CEP120- and SPICE1-depleted cells(Figure 4D). These results are consistent with a role forCEP120, SPICE1, and CPAP in centriole elongation. Next, weused taxol to artificially elongate centrioles in G2-arrested cells[31]. Depletion of CEP120, SPICE1, or CPAP blocked centrioleelongation after taxol treatment (Figures S4B andS4C). Finally,we used colchicine to shorten procentrioles in the PLK4 assay[32]. Similar to CEP120, SPICE1, or CPAP depletion, we

observed decreased CEP135, but not SASS6, recruitment(Figures 4E and 4F). Under these conditions, CEP120, SPICE1,CPAP, and CP110 still localized to the PCAR but with limitedradial extension, consistent with shorter procentrioles (Fig-ure S4D). Interestingly, PLK4 amounts were increased in thePCAR region of colchicine-treated cells, as seen previously(see Figures 2 and S4D).

Discussion

Using PLK4-induced centriole overduplication assays and 3D-SIM, we reveal a role for CEP120 and SPICE1 in procentriole

Figure 4. CEP120, SPICE1, and CPAP Are Required for Procentriole Elongation

(A–C) Tomogram projections (average projections of 20–30 nm central volume sections) of mother and procentrioles from PLK4-induced cells depleted of

CPAP, CEP120, or SPICE1 or with nontargeting small interfering RNA (A). Bar graphs show mean lengths of individual procentriole microtubules (B) and

cross-sectional widths of procentrioles (C) measured from multiple tomograms from each condition with IMOD software (measurements in control,

CEP120-, SPICE1-, and CPAP-depleted conditions respectively were n = 16, 12, 16, and 14 microtubules (MTs) for B and n = 6, 5, 6, and 3 procentrioles

for C). Widths of procentrioles were measured at the widest detectable point in tomograms under all conditions. Representative microtubules are high-

lighted in green in (A).

(D) Representative pseudocolor merge (left-most panels) or grayscale micrographs of U-2 OS centrosomes that were PLK4-induced and depleted of

CEP120 or SPICE1 and labeled with antibodies to glutamylated (top panels) or acetylated (bottom panels) a-tubulin (red) and CEP135 (green).

(E) Representative micrographs of PLK4-induced U-2 OS centrioles in the presence (right column) or absence (left column; control) of colchicine (10 mM;

see the Supplemental Experimental Procedures) prior to fixation and labeling with antibodies to glutamylated tubulin (Gl.Tub.; blue), CEP135 (green),

and SASS6 (red).

(F) Box-and-whisker plot of the total integrated intensity of each of the three labels (wholemasks) in every centrosome, taken from (E), andmeasured from at

least 50 cells in each condition. The red bar shows the mean, light gray the 95% confidence interval, and dark gray 1 SD.

(G) Model depicting where within the centriole biogenesis pathway CEP120 and SPICE1 operate.

Scale bars in (A) represent 100 nm; scale bars in (D) and (E) represent 1 mm. See also Figure S4.

Current Biology Vol 23 No 141364

elongation. We show that CEP120 interacts with SPICE1 andCPAP, providing new insight into how these proteins togethercontribute to centriole elongation. The Drosophila ortholog ofCPAP, SAS-4, provides a scaffold to recruit multiple extralu-menal centriolar proteins [33]. Here, we find that CPAP isrequired for the recruitment of CEP120 and SPICE1 to PLK4-induced procentrioles (see model in Figure 4G). At later stagesof centriole assembly, CEP120 and SPICE1 appear to be more

peripheral to CPAP on the procentriole barrel. This is similar toEM data showing that CPAP localization is luminal and distinctfrom CEP120 [1, 24, 28]. By 3D-SIM, all three proteins similarlyoccupy the PCAR when procentrioles are artificially short-ened by colchicine treatment. Therefore, the interactions weobserve may be transient and/or cytoplasmic.We have shown that CEP120, SPICE1, and CPAP function

downstream of SASS6, consistent with previous studies in

CEP120, SPICE1, and CPAP in Centriole Biogenesis1365

CEP120-depleted cells [24]. Our localization data also indicatethat CEP135 recruitment is reduced after CEP120, SPICE1, orCPAP depletion. This conclusion is supported by workshowing that CEP135 and CPAP interact and that loss ofCEP135 leads to short centrioles [34]. A corollary is that theDrosophila ortholog of CEP135 (BLD10) is a MT-binding pro-tein that stabilizes basal bodies and that the human CEP135localizes to the MT barrel of centrioles by immuno-EM[18, 19, 28]. Consistent with this, we also show that chemicalinhibition of centriole elongation reduces CEP135 recruitmentto procentrioles. Drosophila CEP135 is dispensable for cart-wheel formation but is required to maintain robust centriolearchitecture [35]. It is therefore tempting to speculate thatthe decrease in the number of centrioles after CEP120 orSPICE1 depletion may be due to the assembly of abnormal/short procentrioles [23, 25]. This would be consistent with areport demonstrating that CEP135-deficient cells have per-turbed centriolar triplet MT organization [34]. Our proteinrecruitment assays further indicate that CPAP can localizeto the PCAR independently of CEP120 and SPICE1 (Figure2C). Although CPAP depletion prevents the recruitment ofCEP120, SPICE1, and CEP135 to the PCAR, short procen-trioles do form, but with apparently less-robust centriolarMTs. This contrasts with work in C. elegans in which centriolarMTs are completely lost in the absence of the CPAP orthologSAS-4 [6, 8, 36]. This discrepancy could be explained by adifferential requirement in human cells for CPAP in centriolarMT formation or residual CPAP activity. Another possibility isthat CPAP is important for the initial formation/anchoring ofcentriolar microtubules and that CEP120, SPICE1, CEP135,and CPAP are then required for subsequent elongation/stabilization of procentrioles.

Overexpression of CEP120 or CPAP induces the elongationof centrioles, requiring each other and SPICE1. CPAP can binda- and b-tubulin dimers through its PN2-3 domain [37] and canregulate the nucleotide status of tubulin [33]. SPICE1 occupiesthe mitotic spindle, but whether CEP120 or SPICE1 also bindtubulin directly remains unknown [25]. One possibility is thatCEP120, CEP135, and CPAP recruit SPICE1 to centrioles,where SPICE1 regulates microtubule stability through an un-known mechanism. Accordingly, CEP120 or SPICE1 depletionleads to a decrease in the levels of modified tubulin in thePCAR region. It will be of interest to assess the tubulin-stabilizing activity of CEP120 and SPICE1 and interactionswith known MT-stabilizing proteins.

Taken together, our data suggest that CEP120 and SPICE1act with CPAP to promote the elongation of procentrioles,being similarly required for the recruitment of distal MTcapping proteins and CEP135 to procentrioles. The resultsdescribed here further our understanding of the intricate path-ways that cooperate to ensure robust, timely, and measuredelongation of centrioles.

Supplemental Information

Supplemental Information includes Supplemental Experimental Proce-

dures, four figures, and four tables and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2013.06.002.

Acknowledgments

We thank Doug Holmyard and Mike Strauss for assistance with EM and

tomography and Alex Bird, Andrew Fry, Chad Pearson, Guowei Fang,

Stephane Angers, and Anne-Claude Gingras for reagents. We thank E.

Nigg for the PLK4 line and Li-Huei Tsai for CEP120 antibody. L.P. and

D.B.-J. hold Canada Research Chairs. This work was funded by grants

from the NSERC (RGPIN-355644-2008), CIHR (MOP-123468), and Krembil

Foundation to L.P. and the Plan Nacional (BFU2009-08522 and BFU2012-

33960) and IRB Barcelona to J.L.

Received: February 12, 2013

Revised: May 6, 2013

Accepted: June 3, 2013

Published: June 27, 2013

References

1. Lawo, S., Hasegan, M., Gupta, G.D., and Pelletier, L. (2012).

Subdiffraction imaging of centrosomes reveals higher-order organiza-

tional features of pericentriolar material. Nat. Cell Biol. 14, 1148–1158.

2. Fu, J., and Glover, D.M. (2012). Structured illumination of the interface

between centriole and peri-centriolar material. Open Biol 2, 120104.

3. Mennella, V., Keszthelyi, B., McDonald, K.L., Chhun, B., Kan, F., Rogers,

G.C., Huang, B., and Agard, D.A. (2012). Subdiffraction-resolution

fluorescence microscopy reveals a domain of the centrosome critical

for pericentriolar material organization. Nat. Cell Biol. 14, 1159–1168.

4. Sonnen, K.F., Schermelleh, L., Leonhardt, H., and Nigg, E.A. (2012).

3D-structured illumination microscopy provides novel insight into

architecture of human centrosomes. Biol Open 1, 965–976.

5. Nigg, E.A., and Raff, J.W. (2009). Centrioles, centrosomes, and cilia in

health and disease. Cell 139, 663–678.

6. Pelletier, L., O’Toole, E., Schwager, A., Hyman, A.A., and Muller-

Reichert, T. (2006). Centriole assembly in Caenorhabditis elegans.

Nature 444, 619–623.

7. Gonczy, P. (2012). Towards amolecular architecture of centriole assem-

bly. Nat. Rev. Mol. Cell Biol. 13, 425–435.

8. Delattre, M., Canard, C., and Gonczy, P. (2006). Sequential protein

recruitment in C. elegans centriole formation. Curr. Biol. 16, 1844–1849.

9. Dzhindzhev, N.S., Yu, Q.D., Weiskopf, K., Tzolovsky, G., Cunha-

Ferreira, I., Riparbelli, M., Rodrigues-Martins, A., Bettencourt-Dias,

M., Callaini, G., and Glover, D.M. (2010). Asterless is a scaffold for the

onset of centriole assembly. Nature 467, 714–718.

10. Habedanck, R., Stierhof, Y.D.,Wilkinson, C.J., andNigg, E.A. (2005). The

Polo kinase Plk4 functions in centriole duplication. Nat. Cell Biol. 7,

1140–1146.

11. Tang, C.J., Lin, S.Y., Hsu, W.B., Lin, Y.N., Wu, C.T., Lin, Y.C., Chang,

C.W., Wu, K.S., and Tang, T.K. (2011). The human microcephaly protein

STIL interacts with CPAP and is required for procentriole formation.

EMBO J. 30, 4790–4804.

12. Kitagawa, D., Kohlmaier, G., Keller, D., Strnad, P., Balestra, F.R.,

Fluckiger, I., and Gonczy, P. (2011). Spindle positioning in human cells

relies on proper centriole formation and on the microcephaly proteins

CPAP and STIL. J. Cell Sci. 124, 3884–3893.

13. van Breugel, M., Hirono, M., Andreeva, A., Yanagisawa, H.A.,

Yamaguchi, S., Nakazawa, Y., Morgner, N., Petrovich, M., Ebong, I.O.,

Robinson, C.V., et al. (2011). Structures of SAS-6 suggest its organiza-

tion in centrioles. Science 331, 1196–1199.

14. Kitagawa, D., Vakonakis, I., Olieric, N., Hilbert, M., Keller, D., Olieric, V.,

Bortfeld, M., Erat, M.C., Fluckiger, I., Gonczy, P., and Steinmetz, M.O.

(2011). Structural basis of the 9-fold symmetry of centrioles. Cell 144,

364–375.

15. Tang, C.J., Fu, R.H., Wu, K.S., Hsu, W.B., and Tang, T.K. (2009). CPAP is

a cell-cycle regulated protein that controls centriole length. Nat. Cell

Biol. 11, 825–831.

16. Schmidt, T.I., Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Lavoie,

S.B., Stierhof, Y.D., and Nigg, E.A. (2009). Control of centriole length

by CPAP and CP110. Curr. Biol. 19, 1005–1011.

17. Kohlmaier, G., Loncarek, J., Meng, X., McEwen, B.F., Mogensen, M.M.,

Spektor, A., Dynlacht, B.D., Khodjakov, A., and Gonczy, P. (2009).

Overly long centrioles and defective cell division upon excess of the

SAS-4-related protein CPAP. Curr. Biol. 19, 1012–1018.

18. Bayless, B.A., Giddings, T.H., Jr., Winey, M., and Pearson, C.G. (2012).

Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces.

Mol. Biol. Cell 23, 4820–4832.

19. Carvalho-Santos, Z., Machado, P., Alvarez-Martins, I., Gouveia, S.M.,

Jana, S.C., Duarte, P., Amado, T., Branco, P., Freitas, M.C., Silva, S.T.,

et al. (2012). BLD10/CEP135 is a microtubule-associated protein that

controls the formation of the flagellum central microtubule pair. Dev.

Cell 23, 412–424.

Current Biology Vol 23 No 141366

20. Pearson, C.G., Osborn, D.P., Giddings, T.H., Jr., Beales, P.L., and

Winey, M. (2009). Basal body stability and ciliogenesis requires the

conserved component Poc1. J. Cell Biol. 187, 905–920.

21. Azimzadeh, J., Hergert, P., Delouvee, A., Euteneuer, U., Formstecher,

E., Khodjakov, A., and Bornens, M. (2009). hPOC5 is a centrin-binding

protein required for assembly of full-length centrioles. J. Cell Biol.

185, 101–114.

22. Spektor, A., Tsang, W.Y., Khoo, D., and Dynlacht, B.D. (2007). Cep97

and CP110 suppress a cilia assembly program. Cell 130, 678–690.

23. Hutchins, J.R., Toyoda, Y., Hegemann, B., Poser, I., Heriche, J.K.,

Sykora, M.M., Augsburg, M., Hudecz, O., Buschhorn, B.A.,

Bulkescher, J., et al. (2010). Systematic analysis of human protein

complexes identifies chromosome segregation proteins. Science 328,

593–599.

24. Mahjoub, M.R., Xie, Z., and Stearns, T. (2010). Cep120 is asymmetrically

localized to the daughter centriole and is essential for centriole assem-

bly. J. Cell Biol. 191, 331–346.

25. Archinti, M., Lacasa, C., Teixido-Travesa, N., and Luders, J. (2010).

SPICE—a previously uncharacterized protein required for centriole

duplication and mitotic chromosome congression. J. Cell Sci. 123,

3039–3046.

26. Xie, Z., Moy, L.Y., Sanada, K., Zhou, Y., Buchman, J.J., and Tsai, L.H.

(2007). Cep120 and TACCs control interkinetic nuclear migration and

the neural progenitor pool. Neuron 56, 79–93.

27. Azimzadeh, J., Wong, M.L., Downhour, D.M., Sanchez Alvarado, A., and

Marshall, W.F. (2012). Centrosome loss in the evolution of planarians.

Science 335, 461–463.

28. Kleylein-Sohn, J., Westendorf, J., Le Clech,M., Habedanck, R., Stierhof,

Y.D., and Nigg, E.A. (2007). Plk4-induced centriole biogenesis in human

cells. Dev. Cell 13, 190–202.

29. Dammermann, A., Muller-Reichert, T., Pelletier, L., Habermann, B.,

Desai, A., and Oegema, K. (2004). Centriole assembly requires both

centriolar and pericentriolar material proteins. Dev. Cell 7, 815–829.

30. Roux, K.J., Kim, D.I., Raida, M., and Burke, B. (2012). A promiscuous

biotin ligase fusion protein identifies proximal and interacting proteins

in mammalian cells. J. Cell Biol. 196, 801–810.

31. Kuriyama, R., Dasgupta, S., and Borisy, G.G. (1986). Independence of

centriole formation and initiation of DNA synthesis in Chinese hamster

ovary cells. Cell Motil. Cytoskeleton 6, 355–362.

32. Kuriyama, R. (1982). Effect of colcemid on the centriole cycle in Chinese

hamster ovary cells. J. Cell Sci. 53, 155–171.

33. Gopalakrishnan, J., Chim, Y.C., Ha, A., Basiri, M.L., Lerit, D.A., Rusan,

N.M., and Avidor-Reiss, T. (2012). Tubulin nucleotide status controls

Sas-4-dependent pericentriolar material recruitment. Nat. Cell Biol.

14, 865–873.

34. Lin, Y.C., Chang, C.W., Hsu, W.B., Tang, C.J., Lin, Y.N., Chou, E.J., Wu,

C.T., and Tang, T.K. (2013). Human microcephaly protein CEP135 binds

to hSAS-6 and CPAP, and is required for centriole assembly. EMBO J.

32, 1141–1154.

35. Roque, H., Wainman, A., Richens, J., Kozyrska, K., Franz, A., and Raff,

J.W. (2012). Drosophila Cep135/Bld10 maintains proper centriole struc-

ture but is dispensable for cartwheel formation. J. Cell Sci. 125, 5881–

5886.

36. Dammermann, A., Maddox, P.S., Desai, A., and Oegema, K. (2008).

SAS-4 is recruited to a dynamic structure in newly forming centrioles

that is stabilized by the gamma-tubulin-mediated addition of centriolar

microtubules. J. Cell Biol. 180, 771–785.

37. Cormier, A., Clement, M.J., Knossow, M., Lachkar, S., Savarin, P.,

Toma, F., Sobel, A., Gigant, B., and Curmi, P.A. (2009). The PN2-3

domain of centrosomal P4.1-associated protein implements a novel

mechanism for tubulin sequestration. J. Biol. Chem. 284, 6909–6917.