Figure 4. Mis18α associates with centromeres in a Mis18β-independent manner but requires Mis18β for efficient CENP-A loading.

(A) Left panel shows SDS-PAGE analysis of cobalt and amylose pull-down of His-MBP-Mis18β_{188–229 WT} and mutants with His-SUMO-Mis18α_191–233. SDS-PAGE shows protein bound to nickel resin as input (I) and protein bound to amylose resin to assess interaction (P). Right panel shows Western blot analysis of co-immunoprecipitation (Co-IP) experiments using Mis18α antibody to test interaction of Mis18α−mCherry and Mis18β-GFP with and without mutations in the C-terminal α-helices or GFP as a control. Top panel shows blot against mCherry, middle panel shows blot against GFP, and bottom panel shows blot against tubulin as loading control. (B) Representative fluorescence images (left panel) and quantification (right panel) used to evaluate the ability of Mis18β_WT-GFP (n = 963, 927) and Mis18β_L199D/I203D-GFP (n = 1312, 1221) to co-localise with mCherry-Mis18α at endogenous centromeres. Middle panel, quantification of Mis18β signal. Right panel, quantification of Mis18α signal (Mann–Whitney U test; ****P ≤ 0.0001). Cells were co-transfected with either control (n = 1131, 935) or Mis18β siRNA (with no transfected Mis18β-GFP n = 1170), in three independent experiments shown in black, blue and red. Error bars show mean ± SD. (C) Representative fluorescence images (left panel) and quantification (right panel) used to evaluate the ability of Mis18β_WT-GFP (n = 1036) and Mis18β_L199D/I203D GFP (n = 947) to deposit new CENP-A-SNAP at endogenous centromeres (Mann–Whitney U test; ****P ≤ 0.0001). Cells were co-transfected with either control (n = 840) or Mis18β siRNA (with no transfected Mis18β-GFP n = 824), as stated, in three independent experiments shown in black, blue and red. Error bars show mean ± SD. Scale bars, 10 μm. All conditions have been normalised to control conditions: cells transfected with control siRNA and Mis18β_WT-GFP. Source data are available online for this figure.