Fig. 4.

PLK4 MTOCs can recruit STIL and γ-tubulin in Ca²⁺-released Xenopus extracts and are able to enhance centrosomal MT nucleation. (A) 3D-SIM images showing a ring-like structure of PLK4 MTOCs formed in Ca²⁺-released Xenopus extracts. α-tubulin and GFP–PLK4 are presented in red and green, respectively. Scale bar: 1 µm. (B) 3D reconstitution of PLK4 asters (Movie 3). Scale bars: 1 µm. (C) SIM images and profile across the arrow showing the colocalisation of STIL (red), γ-tubulin (magenta) and GFP–PLK4 (green) within PLK4 MTOCs. Scale bar: 1 µm. (Movie 4). (D) SIM images showing that GFP–PLK4 (green), STIL (red), α/β-tubulin (magenta) and γ-tubulin (blue) colocalise with PLK4 MTOCs. Scale bar: 1 µm. (E) Confocal images showing PLK4-induced MTOCs containing Rhodamine–tubulin in control extracts (Ctr) and STIL-depleted extract (ΔSTIL). Scale bar: 1 µm. (F) Western blots showing depletion of STIL in the extracts used in E. The total level of proteins in these extracts is shown using antibodies against XCep 192, γ-tubulin and PLK4. (G) PLK4 enhances MT nucleation. Confocal images showing MT nucleation using purified centrioles labelled with GFP–centrin incubated in Xenopus interphasic extract in the presence or absence of GFP–PLK4 (Rhodamine-labelled tubulin, red; centriole and PLK4, green). Images were taken after 30 min incubation (Movies 5 and 6). Scale bar: 5 µm. (H) Quantifications of MTs length (μm) visualised from the centrioles (GFP–centrin MTOCs) in the presence or absence of GFP–PLK4. MTs were measured from two independent experiments, where four different MTOCs were analysed (the total number of MTs measured in the presence of GFP–PLK4 was 225). The statistical data are presented as mean±s.d. ****P<0.0001, (Mann–Whitney U-test). (I) Representative schematic of PLK4 MTOC formation in Xenopus extracts.