PMC

(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.

(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.

(A) Schematic outlining the generation of G1 cells with engaged centrioles as described previously (; ). 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).

(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 .