Figure 7.

The N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, whereas its central domain is additionally required for shaping metaphase chromosomes in patient cells. (A) MCPH1 patient cells were transduced with GFP-tagged full-length hMCPH1 by means of a lentivirus expression system. Mock-transduced cells and cells transduced with GFP alone were used as negative controls. Lysates were prepared from these cells and analyzed by immunoblotting with the indicated antibodies. (B) The cells described in A were fixed and stained with DAPI. The percentages of prophaselike cells (PLCs) in such populations were scored and plotted (*, P < 0.05; ***, P < 0.001 between each pair; PLCs were scored from three independently prepared coverslips [n = 3]). Representative examples of nuclear morphology in cells expressing GFP alone or GFP-hMCPH1 (wild type) are shown in Fig. S5 (A and B). By definition, the PLCs include not only G2 cells displaying PCC but also early G1 cells displaying delayed chromosome decondensation (Trimborn et al., 2006). In the current study, we follow this definition for the analysis of PLCs. However, when the percentages of the PLCs in patient cells were lowered to the basal level, we describe it as a “PCC rescue” for simplicity. (C) Chromosome spreads were prepared from the cells described in A and stained with DAPI and anti-Smc2. The morphology of chromosomes from 20 spreads was classified into three categories, and each sample was scored accordingly. (a) “Dumpy” is a category of short chromosomes with wavy axes that are commonly observed in the MCPH1 patient cells. (c) “Straight” is a category of long chromosomes with straight axes that are routinely observed in nonpatient cells. (b) “Intermediate” is a category belonging between dumpy and straight. Representative images of each category (stained with anti-Smc2) are shown at the top. Representative data from two independent experiments are shown. Bar, 5 µm. (D) MCPH1 patient cells were transduced with GFP alone or the GFP-tagged N-terminal domain of hMCPH1. Lysates were prepared from these cells and analyzed by immunoblotting as described in A. (E and F) The percentages of PLCs and the morphology of chromosome spreads were scored as described in B and C, respectively. For F, representative data from two independent experiments are shown. (G) A postulated change of condensin (Cond) II activity is shown by the red line. To prevent PCC in G2 phase, the N-terminal (N) domain of MCPH1 is sufficient. To properly shape metaphase chromosomes, both the N-terminal and central (cen) domains are required. Error bars indicate means ± SD.