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- Fig. 1.
STIL is required for centriole duplication in U2OS cells. Asynchronously growing U2OS cells were transfected for 72 hours with control siRNA (GL2), three different STIL siRNAs (STIL1–STIL3) or PLK4 siRNA oligonucleotides. (A) Western blot analysis of STIL protein levels in control (GL2) and STIL-depleted U2OS cell lysates (STIL1–STIL3), using the STIL antibody ab89314 and an antibody against α-tubulin as a loading control. (B) After siRNA treatment, U2OS cells were fixed and stained with antibodies against CP110 (red) and Cep135 (green) for immunofluorescence microscopy. DNA was stained with DAPI (blue). Yellow rectangles illustrate probable orientation of centrioles. (C) Centriole numbers per cell in response to treatment with GL2, STIL or PLK4 siRNA, as in B. (n=3, 100 cells were analyzed in each experiment, error bars denote s.d.). Scale bars: 1 μm (magnifications) and 5 μm (overview; Merge + DAPI).
- Fig. 2.
STIL specifically localizes to the proximal end of daughter centrioles. (A) U2OS cells fixed and stained with antibodies against STIL (ca66, green) and CP110 (red); DNA (DAPI) is shown in gray. (B) STIL localizes closer to Cep164-negative centrioles. U2OS cells fixed and stained with antibodies against STIL (ca66, green), CP110 (red) and Cep164 (blue); DNA (DAPI) is shown in gray. The presumed orientation of the Cep164-positive mother centriole (M) is depicted with a yellow rectangle, the orientation of the respective Cep164-negative daughter centriole (D) with a white rectangle. The scheme to the left illustrates the relative localization of STIL (green), CP110 (red) and Cep164 (blue) within this centriole pair. (C) STIL colocalizes with SAS-6. U2OS cells were fixed and stained with antibodies against STIL (ca66, green), SAS-6 (red) and CP110 (blue); DNA (DAPI) is shown in gray. The presumed orientation of the SAS-6-negative mother centriole (M) is depicted with a yellow rectangle, the orientation of the respective SAS-6-positive daughter centriole (D) with a white rectangle. The scheme to the left illustrates the relative localization of STIL (green), SAS-6 (red) and CP110 (blue) within this centriole pair. (D) As shown by immunoelectron microscopy STIL localizes close to the interphase between mother and daughter centrioles (top row). U2OS cells were fixed and incubated with antibodies against STIL (ca66), followed by gold-labeled secondary antibodies. Schematic representations (bottom row) illustrate the orientation of mother (M) and daughter (D) centrioles; the localization of gold particles is denoted by black dots. Scale bars: 5 μm (A–C, Merge + DAPI) and 1 μm (A–C, all higher magnifications), 0.5 μm (D).
- Fig. 3.
Localization of STIL to centrioles is cell-cycle dependent. (A) Asynchronously growing U2OS cells fixed and stained for immunofluorescence microscopy with antibodies against STIL (ca66, green), CP110 (red) and Cep135 (blue); DNA (DAPI) is shown in gray. Representative images are shown for the different cell cycle stages (determined by the number of centrioles in interphase and DNA stain in mitosis). (B) Nocodazole (Noco)-arrested U2OS cells were released into mitosis in the presence or absence of MG132 and subjected to western blot analysis at different time points after release, using antibodies against STIL (ab89314) and cyclin B1. α-tubulin was monitored as loading control. (C) Same experiment as described in B except that Cdk1 inhibitor (RO-3306) was added to both MG132-treated and untreated cells. Anti-phospho-histone H3 (Ser10) antibodies were used to monitor mitotic progression. (D) Nocodazole (Noco)-arrested HeLa S3 cells previously treated with GL2 (control) or siRNA oligonucleotides targeting Cdc20 and/or Cdh1 were released into mitosis in the presence of Cdk1 inhibitor (RO-3306) and subjected to western blot analysis at different time points after release, using antibodies against STIL (ab89314), cyclin B1, Cdc20 and Cdh1. α-tubulin was monitored as a loading control. Scale bars: 5 μm (Merge + DAPI) and 1 μm (higher magnifications).
- Fig. 4.
Relationship between STIL, Ana2 and SAS-5. (A) Phylogenetic tree illustrating relationship between metazoan STIL and Drosophila Ana2 protein families. The tree was generated on phylogeny.fr (www.phylogeny.fr) using MUSCLE for alignment of amino acid sequences and PhyML (maximum likelihood method) for phylogeny. Number of amino acids (aa) for each sequence are indicated to the right. The scale bar indicates 0.5 substitutions per site. (B) Multiple protein sequence alignment (MUSCLE; colored according to the BLOSUM62 score, conservation visibility value was set to 30) of STIL/Ana2-related protein sequences. Regions encompassing highest sequence conservation (colored boxes on a scheme of human STIL) are shown. Alignment of three SAS-5 protein sequences of nematodes is shown below. STAN, STIL/ANA2; TIM, truncated in microcephaly.
- Fig. 5.
SAS-6 localizes to centrioles in the absence of STIL. (A) Asynchronously growing U2OS cells transfected for 72 hours with GL2, STIL or SAS-6 siRNA oligonucleotides. Cells were fixed and stained with antibodies against STIL (ca66, green), SAS-6 (red) and CP110 (blue) for immunofluorescence microscopy; DNA (DAPI) is depicted in gray. Representative images of prophase cells are shown (determined by DAPI staining). (B) Quantification of centriolar levels of STIL and SAS-6 fluorescence in cells treated as described in A. Only prophase cells (determined by DAPI staining) were considered. (n=3, 10 centriolar pairs were analyzed in each experiment, error bars denote s.d.). Note that in SAS-6-depleted cells, STIL is completely absent from centrioles, whereas in STIL-depleted cells, SAS-6 localizes to centrioles (albeit in reduced amounts). (C) Western blot analysis of STIL and SAS-6 protein levels in control- (GL2), STIL- (STIL 1-3) and SAS-6-depleted U2OS cell lysates, probed with antibodies against STIL (ab89314), SAS-6 and α-tubulin for loading control. Scale bars: 5 μm (Merge + DAPI) and 1 μm (higher magnifications).
- Fig. 6.
STIL overexpression results in centriole amplification. Asynchronously growing U2OS cells were transfected with plasmids encoding FLAG-tagged STIL, PLK4 or SAS-6 or empty vector. After 48 hours, cells were fixed and stained with antibodies against the FLAG tag (green), CP110 (red) and Cep135 (blue); DNA (DAPI) is shown in gray. Cells were then analyzed by fluorescence microscopy. (A) The number of centrioles per transfected cell was determined based on CP110 staining. The graph illustrates the percentage of cells in each indicated category for control-, STIL- PLK4- and SAS-6-transfected cells (n=3, 50 cells were analyzed in each experiment, error bars denote s.d.). (B) Representative images are shown for STIL-transfected and control cells. Note that in cells overexpressing STIL, multiple procentrioles form around a single centriole. (C) U2OS cells transiently expressing FLAG-tagged STIL were further stained with antibodies against CP110 (red), STIL (ca66, green) and Cep164 (blue). Representative images are shown and a scheme illustrates the localization of CP110 (red), STIL (green) and Cep164 (blue) to the flower-like structures. Note that Cep164, a marker for mature mother centrioles, localizes to the central centriole.(D) U2OS cells transiently expressing FLAG-tagged STIL were stained with antibodies against CP110 (red), STIL (ca66, green) and SAS-6 (blue). Note that both STIL and SAS-6 colocalize as a ring around the central centriole. Scale bars: 5 μm (Merge + DAPI) and 1 μm (all higher magnifications).
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