Figure 6. MCPH1 interaction with condensin II is essential to prevent interphasic chromosome condensation.

(A) Co-immunoprecipitation of MCPH1 with NCAPH2-GFP. Nuclear extracts were prepared from wild type, Ncaph2^GFP/GFP and Ncaph2^GFP/GFP Mcph1^Δ/Δcells. Immunoprecipitation was performed using GFP-trap agarose beads and analysed by western blot using an anti-MCPH1 antibody (IP-GFP). 5 % of the lysate used for IP was loaded as INPUT control. Anti-Lamin B1 antibody was used as a loading control. (B) Deletion of the central domain of MCPH1. To address if the central domain of MCPH1 is necessary to mediate the interaction with condensin II, we first introduced a GFP-tag at the C-terminal end of MCPH1 in Ncaph2^Halo/Halo cells as the antibody against the protein was raised against the central domain. Then a second targeting was done to delete the central domain. As a result, the western blot represented in panel B using anti-MCPH1 antibody detects the wild-type protein or the GFP-tagged protein, homozygous Mcph1^GFP/GFP but does not detect anything after deletion of the central domain. Using anti-GFP antibody reveals that the protein deleted for the central domain is present in the cell at similar levels as the wild-type GFP-tagged protein. A slight decrease in size is observed due to the deletion of the central domain. Anti-Lamin B1 antibody was used as a loading control. (C) Co-immunoprecipitation of NCAPH2-Halo with MCPH1-GFP. Nuclear extracts were prepared from Ncaph2^Halo/Halo, Ncaph2^Halo/HaloMcph1^GFP/GFP and Ncaph2^Halo/HaloMcph1^{ΔcenGFP/ΔcenGFP} cells. Immunoprecipitation was performed using GFP-trap agarose beads and analysed by in-gel detection of NCAPH2-Halo using the Halo-ligand TMR or by western blot using an anti-NCAPD3 antibody (IP-GFP). 5 % of the lysate used for IP was loaded as input control. Anti-Lamin B1 antibody was used as a loading control. (D,E) Immunofluorescence analysis of the chromatin organisation in Ncaph2^Halo/HaloMcph1^GFP/GFP and Ncaph2^Halo/HaloMcph1^{ΔcenGFP/ΔcenGFP} cells. MCPH1-GFP is only detected in the cell nucleus enriched in dots colocalising with some γH2AX foci (D). The deletion of its central domain induces a similar condensation of interphasic chromosomes as the one observed after the complete loss of function of Mcph1 (E).