1. Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number
Midori Ohta, Koki Watanabe, Tomoko Ashikawa, Yuka Nozaki, Satoko Yoshiba, Akatsuki Kimura, Daiju Kitagawa 
Cell Reports 
Volume 23, Issue 11, Pages e4 (June 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 3160-3169.e4DOI: (10.1016/j.celrep.2018.05.030)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
The Autophosphorylation of Plk4 Is Critical for Limiting a Single Site for Procentriole Assembly
(A) U2OS cells stained with indicated antibodies were observed using super resolution microscopy. White broken line, outline of the Nuclei. Insets, magnified views around the centrosome. A red arrowhead, an intense focus in a Plk4 ring of the mother centriole. Scale bar, 0.5 (inset) and 5 μm (whole nuclei). Note that we judged centrioles to be “before” or “after” duplication based on the staining patterns of Cep152 and Cep192.
(B) (Top) Top view of the centriole duplication process and distribution of Plk4 (blue) and Cep152 (red). A blue arrowhead, an intense focus of the Plk4 ring. (Middle) How to quantify the signal intensities of Plk4 along with the mother centriole wall. (Bottom) Representative panels of centriolar Plk4 and Cep152. Scale bar, 0.5 μm. See also STAR Methods.
(C) Signal intensity of the Plk4 around the mother centriole wall according to the method in (B). n = 18 or n = 13 for before or after duplication respectively. The values are ±SEM.
(D) U2OS cells were stained with indicated antibodies. The graph shows total intensities of p-S305 Plk4 signal at centrosomes before (–) and after (+) HsSAS-6 loading (∗∗p < 0.01). The values are ±SEM. Scale bar, 0.5 μm. An arrowhead shows a mother centriole.
(E) U2OS cells were treated with aphidicoline for 12 hr and arrested after procentriole formation, followed by addition of 100 nM centrinone for 12 hr. (Left) Representative panels of the centrioles and percentages of the centrosomes with Plk4 ring and STIL. (Right) Quantifications of signal intensities of centriolar Plk4 and STIL. (N > 30, ∗∗p < 0.01). The values are ±SEM. An arrowhead, a mother centriole. Scale bar, 0.5 μm. See also Figures S1 and S2.
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4. Figure 2
The C-Terminal Region of STIL, Rather Than the Coiled Coil, Is Essential for Stimulating Plk4 Kinase Activity
(A) HEK293T cells expressing Plk4-3Flag together with HA, HA-STIL, HsSAS-6-GFP, or HA-Cep152 were analyzed by western blotting using indicated antibodies. Tubulin was used for loading control. The values at the bottom indicate the average ratios (relative to the HA co-expression) of Plk4-3Flag protein level from two independent experiments.
(B) U2OS cells were treated with siRNA targeting control, STIL, HsSAS-6, or Cep152, followed by transfection of Plk4-3FLAG vector. The cells were analyzed by western blotting using indicated antibodies. The values at the bottom indicate the average ratios (relative to control siRNA) of Plk4-3Flag level from two independent experiments.
(C) Diagrams of the Plk4 and STIL proteins. Each fragment used for the experiment is shown in below. Degron: aa 272–311; PB: polo-box domain. CC (coiled-coil): aa 721–746; STAN: aa 1051–1147; TIM: aa The position of ND (S285A, T289A) is shown as a red line in the degron motif.
(D) HEK293T cells expressing Plk4-3Flag together with HA vector (HA), HA-STIL FL, aa 1–1210, aa 1–1147, and N were analyzed by western blotting using indicated antibodies. An asterisk shows non-specific band, which was used for internal loading control. The histograms depict the average ratios (relative to the HA) of Plk4-3Flag level from three independent experiments. The values are ±SEM. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant compared to the HA co-expression in this figure.
(E and F) HEK293T cells expressing Plk4ND-3Flag together with HA vector (HA), HA-STIL FL, N, N3C (E), and FL, N3C, C (F) were analyzed by western blotting using anti-FLAG, STIL, HA, or tubulin antibodies. In (F), the histograms depict the average ratios of phosphorylated Plk4ND (p-Plk4ND) to the total of Plk4ND from three independent experiments.
(G) Recombinant STIL fragments N1, N2, N3, and C tagged with HA were incubated with recombinant GST-Plk4 full length for in vitro kinase assay. The incorporation of [γ-32P] ATP to the substrates was visualized by autoradiography, and the loaded proteins were monitored by blue staining.
(H) Recombinant STIL N3, C and GST proteins were incubated with recombinant GST-Plk4 KL1 in the presence of ATP for in vitro kinase assay. The incorporation of [γ-32P] ATP to the substrates and the loaded proteins were analyzed in the same way as in Figure 2G. See also Figure S3.
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5. Figure 3
Direct Interaction between the STIL STAN and the First Linker Region of Plk4
(A and B) GST pull-down assay was performed using recombinant GST-Plk4 FL wild-type (WT) or kinase-dead (KD) with HA-STIL[N3] (A) or HA-STIL[C] (B). Precipitant materials were analyzed by western blotting using indicated antibodies.
(C) Diagrams of the Plk4 and STIL proteins. Each fragment used for the GST pull-down assay is shown in below. The minimum regions of Plk4 and STIL for their interaction are shown in red. The position of ND is shown as a red line in the degron motif.
(D and E) GST pull-down assay was performed using recombinant GST-Plk4 FL WT, kinase-dead, or fragments (L1PB3 and PB1-3 in D, L1 in E) with HA-STIL[C]. Precipitant materials were analyzed by western blotting using HA antibodies or by blue staining (BS).
(F) GST pull-down assay was performed using recombinant GST-Plk4[L1PB3] with STIL fragments (C, CS, and CSC). Precipitant materials were analyzed by western blotting using STIL antibody or by BS.
(G) (Top) Schematic diagram of the experimental procedure. HEK293T cells expressing HA-STIL[C] alone or together with Plk4ND-3Flag WT or KD were treated with 10 mM MG132 for 6 hr. (Bottom) The lysates were subjected to co-immunoprecipitation assay and analyzed by western blotting using indicated antibodies. See also Figure S4.
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6. Figure 4
The Conserved TIM Domain of STIL Limits the Localization of Plk4 at Centrioles and Procentriole Number
(A) Diagrams of STIL domains and mutants used in this figure. Replacements of amino acids were shown in red.
(B) Alignment of the TIM domain within human, mouse, Xenopus and zebrafish STIL, and Drosophila Ana2. Identical residues are colored in yellow; similar residues are in gray. Asterisks: the residues identical in all aligned sequences; colons: conserved substitutions; periods: semi-conserved substitutions. Red asterisks indicate the residues, which are changed in the LR-AC mutant.
(C) HEK293T cells expressing Plk4ND-3Flag together with HA vector, HA-STIL FL, ΔTIM (aaΔ1268–1287), LR-AC (L1280A, R1281C), or 5A (S1061A, S1116A, S1181A, T1238A, and T1250A) were analyzed by western blotting using anti-Flag, STIL, or tubulin antibodies. The histograms depict the average ratios (relative to the HA co-expression) of phosphorylated Plk4ND (p-Plk4ND) to the total Plk4ND protein level. The values are mean ±SEM from three independent experiments. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant relative to the HA co-expression.
(D) In vitro kinase assay was performed using recombinant GST-Plk4[KL1] and HA-STIL[C], [CLRAC], [CΔTIM], and [TIM: aa 1268–1287]. The incorporation of [γ-32P] ATP to the substrates and the loaded proteins were analyzed in the same way as in Figure 2G. Protein amount of TIM fragment was detected by western blotting using anti-STIL antibody.
(E and F) U2OS cells were treated with control siRNA (siCnt) or siRNA targeting 3′ UTR of endogenous STIL (siSTIL), followed by transfection with an HA vector (HA), HA-STIL full-length WT, ΔTIM, or LR-AC mutant. The cells were immunostained with antibodies against HA and Plk4 (E) or centrin (F).
(E) Signal intensities of centriolar localization of endogenous Plk4 and HA-STIL proteins were shown in the box-and-whisker plot (N ≥ 33).
(F) Quantification of fraction of centrosomes with the indicated number of daughter centrioles from three independent experiments (N ≥ 30, the values are mean ± SEM). ∗p < 0.05, ∗∗p < 0.01, n.s., not significant. Scale bar, 0.5 μm. See also Figure S4.
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