Stability of the small -tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

JCS035238 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. T-Coffee alignment of protein sequences of human HCA66 (Hs, NP_060898) with mouse HCA66 (Mm, AAO15383), Drosophila CG7246 (Dm, AAF46437) and the protein UTP6p from Saccharomyces cerevisiae (Sc, NP_010737). Grey boxes show semi-conserved residues, black boxes indicate identical residues. The predicted HAT repeats are underlined and numbered 1-7.

• Supplemental Figure S2 -

  Fig. S2. Overexpression of GFP:HCA66 constructs. (A) Upper panel: U-2 OS cells were transfected with either GFP, GFP:HCA66, GFP:HCA66_151-597 or GFP:HCA66_1-86, and after 48 hours processed for immunofluorescence with anti-PCM-1, anti-centrin or anti-γ-tubulin antibodies (red). Lower panel: quantification of the number of cells displaying centrosomal localization of γ-tubulin, GCP2, NEDD1, PCM-1 and centrin (mean from three experiments, error bars indicate s.d., ∼250 cells were scored per condition). (B) U-2 OS cells transfected with GFP:HCA66 or GFP:HCA66_1-86, processed for flow cytometry after 48 hours. Top panels indicate signal distribution of GFP intensity. The following panels show flow cytometry of the total cell population, as well as of cells with low and high expression of the GFP-tagged proteins, as marked in the top panels. (C) U-2 OS cells were transfected with GFP:HCA66_1-86. After 48 hours, the untransfected and GFP-positive cells were sorted. Lysates from these cells were analysed by immunoblotting with anti γ-tubulin. α-Tubulin is shown as a loading control. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Silencing of HCA66 affects bipolar spindle formation, but does not affect transcription of gamma-TuSC RNAs. (A) U-2 OS cells were transfected for 48 hours with either control or HCA66-4 siRNA. Microtubules were depolymerised in the cold for 1.5 hours and allowed to regrow for 1 minute, fixed and processed for immunofluorescence of α-tubulin. (B) U-2 OS cells were transfected with HCA66 siRNA, and processed for immunofluorescence of centrin, Plk1 and TPX2 after 48 hours, as indicated. (C) Reverse transcription of RNA extracted from U-2 OS cells, treated for 48 hours with either control (C) or HCA-4 (H) siRNA, followed by PCR using specific primers for actin, HCA66 (HCA), γ-tubublin (γ-tub), GCP2 and GCP3. DNA size markers are shown on the left. Non-adjacent wells on the same gel have been juxtaposed to compose this figure. (D) Table summarizing the effects of various concentrations of the translation inhibitor cycloheximide or the proteasome inhibitor MG132 in U-2 OS cells on the mitotic index, on γ-tubulin localization to the centrosome, and on the percentage of mitotic cells containing monopolar spindles (n=500 cells for the calculation of the mitotic index; n=minimum of 100 cells for the detection of γ-tubulin or for the spindle phenotype). For comparison, the effects of siRNA against HCA66 are listed, as described in the main text. Scale bar: 10 µm.