SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication

Abstract

Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. Here, we found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number.

Figure 1. SAS-6 is transiently recruited to the proximal lumen of mother centrioles in early S-phase

(A) U2OS cells in S phase were processed for immunofluorescence to visualize centriole (centrin-2) and SAS-6. (B) Quantification of cells with weak SAS-6 foci in S phase. Error bars, standard deviation. n>80, N=3. (C) Quantification of weak SAS-6 foci in late S phase. n>100, N=3. (D) Weak SAS-6 localizes to the proximal end of mother centrioles. Linear alignment of centrin-2 (distal-end component) and C-Nap1 (proximal end) is marked by a white line. Here, rabbit antibodies recognizing the C-terminus of C-Nap1 were used with a mouse anti-SAS-6 antibody. m, mother; d, daughter. (E) 3D-SIM image of luminal components CPAP and centrin-2 in comparison to non-luminal component C-Nap1. Here, a mouse antibody recognizing the N-terminus of C-Nap1 was used with a rabbit anti-CPAP antibody. Imaris 3D rendering is presented as indicated. (F) 3D-SIM images of SAS-6, C-Nap1, and centrin in S-phase centrioles stained with the antibodies indicated. m, mother. All S-phase cells in (A), (B), (C), (D) and (F) were identified by BrdU labeling (see Experimental Procedures), not shown.

Figure 2. G1 centrioles can recruit and organize SAS-6 into an ordered structure similar to the cartwheel

(A) RPE-1 cells arrested in G1 for 48 hours followed by expression of HA-tagged SAS-6^ND were imaged by 3D-SIM. n>45, N=3. (B) G1-arrested RPE-1 cells expressing SAS-6^ND as described in (A) were released into S phase by serum reintroduction and stained with the indicated antibodies. S-phase cells were identified by BrdU labeling, not shown. n>21, N=3. (C) Schematic diagram of domain organization of SAS-6 and the epitopes recognized by the antibodies used in STED analyses. (D) Representative STED images of SAS-6 staining with anti-C-term antibody on normal S-phase centrioles were shown. Graph presents STED measurements of ring/focus diameter by averaging the long and short axes. Actual size of the structure is estimated by subtracting STED resolution from STED measurements as indicated. Error bar, standard deviation. n=40. (E) Frequency of observing C-Nap1 ring under 3D-SIM. n=46. (F) STED imaging of SAS-6^ND stained with anti-C-term and anti-N-term antibodies. Actual size of the structure is estimated as indicated. Error bars, standard deviation. n as indicated. (G) SAS-6^F131E localizes to the proximal lumen of G1 centrioles. 3D-SIM image of G1-arrested RPE-1 cells inducibly expressing HA-tagged SAS-6^F131E as described in (A). n>39, N=3. (H) STED imaging of SAS-6^F131E stained with anti-C-term and anti-N-term antibodies. Actual size of the structure is estimated as indicated. Error bars, standard deviation. n as indicated.

Figure 3. Molecular requirements for SAS-6 recruitment to and release from centriole lumen

(A) Collections of various SAS-6 deletion mutants tagged with HA (red). Centriolar localizations of these mutants are summarized. (B and C) Localization and expression of SAS-6 mutants. Indicated SAS-6 mutants were inducibly expressed in G1-arrested RPE-1 cells, and their luminal localizations were examined. (D) HeLa cells depleted of CPAP (two sequential RNAi for 6 days) were induced to express SAS-6^ND. Centrioles in G1 or S-phase cells were examined for SAS-6 localization as indicated. Similar results were obtained with three independent siRNA oligos (see Experimental Procedures). Error bars, standard deviation. n>38, N=3. The significance (two-tailed t-test) is indicated, *** P < 0.0001. (E) U2OS cells depleted of the indicated protein were examined for the localization of the endogenous SAS-6 during S phase. n>31, N=2. (F) U2OS cells depleted of PLK4 or STIL were examined for the localization of the endogenous SAS-6 during S phase by 3D-SIM. Error bars, standard deviation. n>26, N=3. (G) U2OS cells depleted of PLK4 were induced to express SAS-6^ND. The duplication status and localization of the elevated SAS-6^ND were examined and quantified in S-phase cells by 3D-SIM with indicated antibodies. Note that duplication was not rescued, and S-phase cells containing unduplicated centrioles (centrin singlet) were counted. n>25, N=3. (H) Wild-type, early S-phase U2OS cells containing one bright and one weak SAS-6 foci were examined for the localization of STIL. Note that STIL only associates with the bright SAS-6 focus. (I) STIL localization in control or PLK4-depleted U2OS cells expressing SAS-6^ND was examined. n>29, N=3. (J) Immunoblots showing STIL level is unaffected by PLK4 knockdown. (K and L) SAS-6^F131E was inducibly expressed in U2OS cells depleted of the endogenous SAS-6. Localizations of SAS-6^F131E (K) and STIL (L) were then examined in S-phase cells by 3D-SIM and regular microscopy, respectively as indicated. n>24, N=3. S-phase cells in (D), (E), (F), (G), (I), (K) and (L) were identified by BrdU labeling, not shown. See also Figure S1.

Figure 4. Engaged centrioles block luminal recruitment of SAS-6

(A) Schematic outlining the generation of G1 cells with engaged centrioles as described previously (Tsou et al., 2009; Wang et al., 2011). SAS-6^ND expression was induced in proliferating cells for 16 hours before adding BI-2536 to inhibit Plk1. Mitotic arrested cells were forced to exit mitosis 3 hours after Plk1 inhibition by Cdk1 inhibitor RO-3306 for 5 hours. Multinuclei cells, which were cells that had gone through mitosis in the absence of Plk1 activity, were examined for the centriolar localization of SAS-6 with indicated antibodies. n>26, N=3. (B) SAS-6 recruitment to mother centriole lumen is inhibited by engaged centrioles. Immunofluorescence image of a G1 cell containing engaged centrioles with indicated antibodies. (C) Model for a template-based mechanism of centriole duplication. SAS-6 dimers are individually recruited to the proximal lumen of mother centrioles, where they are organized by the surrounding geometry to undergo 9-fold symmetrical assembly. The assembled SAS-6 oligomer is then released from the lumen and forms the cartwheel that drives daughter centriole formation. Centriole engagement blocks re-recruitment of SAS-6 to the mother centriole lumen and thus prevents centriole re-duplication. Centriole disengagement and cartwheel removal at the end of mitosis allow the centrioles to be used as a template for their own biogenesis. Mother centriole (grey), daughter centriole (green), CPAP (blue), SAS-6 (red).