Fig 1: Hypothetical model: self-organization of Plk4 directs a single daughter centriole formation per mother centriole. Schematic illustration of hypothetical model. Increasing centriolar Plk4 concentration after mitosis drives condensation and autonomous activation of Plk4 prior to STIL-HsSAS6 loading. Self-organization property of Plk4 driven by the regulated condensation induces spatial pattern formation of autophosphorylated Plk4, which results in a bias formation of activated Plk4. The single focus is a possible target site for STIL-HsSAS6 loading. Model 1: active and inactive Plk4 species that have different diffusion coefficients interact with and generate biased distribution of Plk4 around the mother centriole through trans-autophosphorylation. Inactive Plk4 stably self-assembles onto centrioles, while high concentration of Plk4 promotes autonomous activation of Plk4 through trans-autophosphorylation14. Activated Plk4 trans-autophosphorylates inactive Plk4 to become active state14 and suppresses stable self-assembly of inactive Plk4. The interaction mode of these two components is similar to Turing’s reaction-diffusion model or its analog, the lateral inhibition model. Model 2: active Plk4 proteins are phase-separated into liquid-like droplets around mother centrioles and they eventually form a single and large droplet of active Plk4 through coalescence or Ostwald ripening. Surface tension of Plk4 droplet may work as a key force shaping asymmetric structure of Plk4 around mother centrioles. These two models may cooperatively work for spatial pattern formation of Plk4
Fig 2: Phosphorylation of STIL by Plk4 triggers centriole duplication.(A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ-32P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P<0.001, two-tailed t-test). Representative images are shown for control, Flag-STIL wt and 5A-transfected cells. Scale bars: 10 µm (merge), 2 µm (magnifications). Western blotting using antibodies against Flag and α-tubulin was performed to visualize expression of STIL constructs as indicated.
Fig 3: Knockdown of STIL suppressed cell proliferation and enhanced cell apoptosis. (A) STIL protein levels were determined by Western blot; cell viability, proliferation, and apoptosis were examined by CCK-8 (B), EdU (C), and flow cytometry (D), respectively. Compared to the si-NC group, *p < 0.05, **p < 0.01.
Fig 4: Cdc6 restrains centrosome duplication via inhibiting Sas-6 in a Plk4 phosphorylation regulated way.(a) Endogenous Cdc6 interacts with Sas-6 in cells. HEK293 total cell extract was immunoprecipitated with a Cdc6 antibody, and probed with Cdc6, Sas-6, Cep164, CPAP and STIL antibodies. (b) Cdc6 directly binds Sas-6 in vitro. In vitro transcribed and translated His-tagged Sas-6 was incubated with or without in vitro transcribed and translated His-tagged Cdc6. His-Cdc6 was then immunoprecipitated with a Cdc6 antibody. The bound proteins were analysed with Cdc6 and Sas-6 antibodies. (c) Simultaneous depletion of Sas-6 eliminates the Cdc6 depletion-induced centriole amplification. U2OS cells were transfected with control siRNA, Cdc6 siRNA or Sas-6 siRNA; or co-transfected with Cdc6 siRNA and Sas-6 siRNA. The cells were then stained with centrin1 (red) and Sas-6 (green) antibodies to identify the cells with efficient depletion of Sas-6. (d) Quantitation of cells with indicated centrin1-positive dots number in c. Approximately 100 cells were counted per sample, and three independent experiments were conducted. (e) Cdc6 WT or Cdc6 2A mutant, but not Cdc6 2D mutant, suppresses the Sas-6-induced centriole amplification. Myc-tagged Sas-6 was co-transfected with GFP, GFP-tagged Cdc6 WT or GFP-tagged Cdc6 2A or 2D mutant for 28 h. The cells were then stained with Myc and centrin1 antibodies. (f) Quantitation of cells with >4 centrioles in e. Approximately 100 cells were counted per sample, and three independent experiments were conducted. The statistical data in d and f are presented as means±s.d. **P<0.01, and ***P<0.001; N.S., no significant difference (Student's t-test). DNA was stained with DAPI. Scale bars, 10 μm. Insets in c and e are high-magnification views of the regions indicated in the low-magnification images.
Fig 5: Cdc6 colocalizes with Plk4 and cartwheel proteins Sas-6 and STIL during S phase.(a) Cdc6 colocalizes with Plk4. U2OS cells were co-transfected with mCherry-tagged Cdc6 (upper panel) or Myc-tagged Cdc6 (lower panel) with GFP-tagged Plk4. Myc-tagged Cdc6 was stained with a Myc antibody. (b) Cdc6 colocalizes with Plk4 and Sas-6 viewed by 3D-SIM. 3D-SIM images of U2OS cells transfected with GFP-tagged Plk4 (upper panel) or GFP-tagged Sas-6 (lower panel) and stained for endogenous Cdc6 and CP110. (c–f) Cdc6 colocalizes with Plk4, STIL and Sas-6 viewed by STED. Immunofluorescence of endogenous Cdc6 (green) and Plk4 (red), or Cdc6 (green) and STIL (red) in U2OS cells (c,e). Immunofluorescence of endogenous Cdc6 (red) in U2OS cells transfected with GFP-tagged Plk4 or GFP-tagged Sas-6 (d,f). (g) Schematic diagrams illustrating the localization of proteins at the centrosome in b–f. Different proteins are indicated by the colours of staining in b–f. DNA was stained with DAPI. Scale bars in a,c–f, 10 μm. Scale bar in b, 0.2 μm. Insets in a,c–f are high-magnification views of the regions indicated in the low-magnification images.
Supplier Page from Abcam for Anti-STIL/SIL antibody