Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - Overexpression of cyclin A increases Mps1 levels at centrosomes. (A) Four-fold magnified images of centrosomes from four representative pairs of adjacent GFP-cyclin A transfected (GFP+) and untransfected (GFP-) S-phase arrested HeLa cells from the experiment described in Figure 3A. The anti-Mps1 signal is shown in pseudocolor intensity scale to highlight the difference in intensity between transfected and untransfected cells. Green, GFP-cyclin A (GFP); red, γ-Tubulin ( γ-Tub); pseudocolor, Mps1; scale bar = 2µm. (B) Background corrected Fluorescence Intensities (F_M) of the Mps1 signal for the centrosomes to the left in A; green bars, F_M of the GFP+ centrosome; black bar, F_M of the GFP- centrosome. F_M was calculated using the formula described by Howell et al. (2000), F_M = (F_L - F_S) x (A_S (A_L - A_S)), where F_S and F_L are the integrated fluorescence intensities of the Mps1 signal in a 15 x 15 pixel box surrounding each centrosome and a 20 x 20 pixel box surrounding the first box, and A_S and A_L are the area of the two boxes.
• Supplemental Figure 2 - Cdk2 and proteasome activities regulate the accumulation of Mps1 tethered to centrosomes with the PACT domain. (A) A representative S-phase arrested HeLa cell expressing GFP-Mps1-PACT. Green, GFP-Mps1-PACT; red, γ-Tubulin; scale bar = 5 µm. Insets show four-fold magnified images of centrosomes, scale bar = 2 µm. (B) Proteasome inhibition reverses the loss of GFP-Mps1-PACT from centrosomes in the absence of Cdk2. Bar graph represents the apparent transfection efficiency of GFP-Mps1-PACT in S-phase arrested HeLa cells transfected with Cdk2-specific siRNAs after treatment with DMSO or MG115, normalized to that of DMSO treatment. White bar, DMSO; black bar, MG115.
• Supplemental Figure 3 - Overexpression of Mps1 accelerates the over production of centrioles in U2OS cells, and like cyclin A causes the over production of centrioles in HeLa cells. (A) Bar graph showing the percentage of U2OS cells with more than four centrioles (as judged with the 20H5 antibody against centrin) after expressing GFP or GFP-Mps1, during the first 24 hr of S-phase arrest. White bar, GFP; black bar, GFP-Mps1. (B) Bar graph showing the percentage of HeLa cells with more than four centrioles (as judged with the 20H5 antibody against centrin) after expressing GFP, GFP-cyclin A, or GFP-Mps1Δ12/13 during 48 hr of S-phase arrest. White bar, GFP; black bar, GFP-cyclin A; gray bars, GFP-Mps1Δ12/13. (C) Centriole staining in representative GFP, GFP-cyclin A, or GFP-Mps1Δ12/13 expressing HeLa cells from the experiment in B. Red, centrin; green, GFP; blue, DNA; scale bar = 5 µm. Insets show four-fold magnified images of centrosomes, scale bar = 2 µm.
• Supplemental Figure 4 - GFP-Mps1^STA does not accumulate at centrosomes in the presence of Cdk2 activity. HeLa cells were sequentially transfected with control siRNAs and GFP-Mps1^STA, then analyzed as described in Figure 5. Red, γ-Tubulin; green, GFP-Mps1^STA; blue, DNA; scale bars = 5 µm. Insets show four-fold magnified images of centrosomes, scale bars = 2 µm. (A) GFP-Mps1^STA does not accumulate at centrosomes in HeLa cells transfected with control siRNAs. Red, γ-Tubulin; green, GFP-Mps1^STA; blue, DNA; scale bar = 5 µm. Insets show four-fold magnified images of centrosomes, scale bar = 2 µm. (B) Normalized GFP and γ-Tubulin signals; lines, brackets and arrowheads are as in Figure 5.
• Supplemental Figure 5 - Mps1^Δ12/13 cause centrosome re-duplication when tethered to centrosomes with the PACT domain. (A) Representative HeLa cell expressing GFP-Mps1^Δ12/13-PACT that executed centrosome re-duplication after 48 hr of S-phase arrest. Red, γ-Tubulin; green, GFP; blue, DNA; scale bars = 5 µm. Insets show four-fold magnified images of centrosomes, scale bars = 2 µm. (B) Bar graph showing the percentage of HeLa cells expressing GFP-PACT (white), GFP-Mps1-PACT (black), or GFP-Mps1^Δ12/13-PACT (gray) that have three or more centrosomes after 48 hr of S-phase arrest.
• Supplemental Figure 6 - GFP-Mps1^STD is not removed from centrosomes in the absence of Cdk2 activity. HeLa cells were sequentially transfected with Cdk2-specific siRNAs and GFP-Mps1^STD, then analyzed as described in Figure 5. (A) GFP-Mps1^STD remains at centrosomes in cells transfected with Cdk2-specific siRNAs. Red, γ-Tubulin; green, GFP-Mps1^STD; blue, DNA; scale bar = 5 µm. Insets show four-fold magnified images of centrosomes, scale bar = 2 µm. (B) Normalized GFP and γ-Tubulin signals; lines, brackets and arrowheads are as in Figure 5.