MCPH1, mutated in primary microcephaly, is required for efficient chromosome alignment during mitosis

Abstract

MCPH1 gene, mutated in primary microcephaly, regulates cell progression into mitosis. While this role has been extensively investigated in the context of DNA damage, its function during unperturbed cell cycles has been given less attention. Here we have analyzed the dynamics of chromosome condensation and cell cycle progression in MCPH1 deficient cells under undamaging conditions. Our study demonstrates that chromosome condensation is uncoupled from cell cycle progression when MCPH1 function is lacking, resulting in cells that prematurely condense their chromosomes during mid G2-phase and delay decondensation at the completion of mitosis. However, mitosis onset occurs on schedule in MCPH1 deficient cells. We also revealed active Cdk1 to be mandatory for the premature onset of chromosome condensation during G2 and the maintenance of the condensed state thereafter. Interestingly, a novel cellular phenotype was observed while monitoring cell cycle progression in cells lacking MCPH1 function. Specifically, completion of chromosome alignment at the metaphase plate was significantly delayed. This deficiency reveals that MCPH1 is required for efficient chromosome biorientation during mitosis.

Figure 1

Cell cycle progression and dynamics of prophase like-condensation in control and MCPH1 patient cells. (a) Graphs comparing the cell cycle distribution, determined by FACS analyses, in untreated cell samples or cell samples incubated with nocodazole for 4 hours to induce M arrest. (b) For each sample from A, we determined in parallel the fraction of PLCs (prophase-like cells) by microscopic analyses of cytogenetic preparations. We also included cells incubated with nocodazole for 7 hours in these analyses. More than 500 cells were scored per sample. As expected, PLC frequency was negligible in control cells. Mean and S.D. data from at least 3 independent experiments are shown. Mean values are indicated. (c) Rate of H3PS10 positive cells in control and MCPH1 patient cells, determined by FACS after incubation with nocodazole for the indicated time points. Data from two independent experiments are presented. (d) Immunolocalization using antibodies against Cyclin B and Histone H3-PS10 proteins in proliferating cells from one MCPH1 patient. PLCs (“Prophase-like cells”) refers to cells with condensed chromatin inside a retained nuclear envelope, either as a consequence of premature onset of chromosome condensation or delayed decondensation at the end of mitosis. Note that G2-PLCs and real prophases are indistinguishable by DAPI staining but showed different IF patterns for both markers. G1-PLCs are also negative for both markers. Only a minor fraction of the observed cells with the typical morphology of prophase cells contained nuclear signals for both Cyclin B and Histone H3-PS10 proteins.

Figure 2

Analyses of mitosis progression by live-cell microscopy in cells depleted of MCPH1 function by siRNAs. (a) A brief description of the experimental procedure: HeLa cells stably expressing fluorescent histone H2B fused to Red1 were synchronized at the G1/S border by double thymidine block. MCPH1 depletion was achieved by transfection with siRNAs (oligo siRNA-MCPH1-3) during the release from the first thymidine block. Time-lapse images were collected using “Nikon Biostation IM Cell incubator” one hour after the release from the second thymidine block. (b) Dot-plots showing the time interval between different key mitotic events in minutes for untreated and MCPH1 depleted cells. The red line indicates the mean value. C.C. = chromosome condensation; NEB = nuclear envelope breakdown; C.D. = chromosome decondensation. At least 40 cells were analyzed in each case. Statistical comparisons for the mean and median data were done by T-student and Wilcoxon (W) tests respectively. **p < 0.01; N.S. not significant. (c) Selected frames showing the mitosis progression of representative HeLa H2B/Red1 cells from both untreated and MCPH1-depleted cell samples. Time from release is indicated in minutes. Arrow heads point to the first time point when chromosome condensation is clearly observable after release. Full movies are included in videos 1 and 2.

Figure 3

Analyses of prometaphase and metaphase progression in MCPH1 cells lacking MCPH1 function. (a) Live-cell microscopy analyses in HeLa H2B-Red1 cells depleted of MCPH1 by siRNAs (oligo siRNA-MCPH1-3). Graphs show the time required in individual control or MCPH1-depleted cells to i) align all the chromosomes at the metaphase plate (blue), ii) initiate chromosome segregation after metaphase alignment (red). Cells marked with an asterisk did not show the PLC phenotype before NEB. (b) Dot-plots from the data in A. The red line indicates the mean value. At least 40 cells were analyzed in each case. Statistical comparisons for the mean and median data were done by T-student and Wilcoxon (W) tests respectively. **p < 0.01; N.S. not significant. (c) Representative cells showing the progression through prometaphase and metaphase from both HeLa untreated and MCPH1-siRNA treated cell samples. Time from nuclear envelope breakdown (first frame) is indicated in minutes. Note that in the MCPH1-depleted cell most chromosomes align at the metaphase plate on time but some require more time to complete the process (pointed by arrows). Once metaphase alignment is fully achieved, cell progresses into anaphase without delay. (d) Dot-plots showing the duration of prometaphase in Hct-116 H2B-GFP and HeLa H2B-GFP cells depleted of MCPH1 by siRNAs. Analyses performed as described in a and b. (e) Percent of the corresponding mitotic stages observed in control and MCPH1 patient lymphoblasts. Analyses were performed by microscopic inspection of cytogenetic preparations obtained following a protocol that preserves the organization of chromosomes on the mitotic spindle. At least 100 mitotic cells were counted and classified according to the alignment stage of their chromosomes as follows: Prometaphase 1, if no signs of metaphase plate conformation is observed; Prometaphase 2, if most chromosomes are already aligned into a plate but few of them remain far; Metaphase, when all chromosomes are finally aligned (representative pictures in f; arrows point to chromosomes that remain far from the already formed plate). For better interpretation of our results, the data were adjusted considering the total amount of cells that were in either prometaphase or metaphase in control (1.7%) and patient (2.9%) cells (n = 1000). Representative data from analyses performed by two different persons are presented.

Figure 4

Analyses of cell cycle progression and dynamics of chromosome condensation in control and MCPH1 deficient cells treated with the Cdk1 inhibitor RO-3306. Fraction of M and G2 cells, determined by FACS analyses, in control (a) and MCPH1 patient cells (b) after either 4 h or 7 h of incubation with RO-3306 alone or combined with nocodazole (NOC), a spindle poison that causes M arrest. For each sample, we determined in parallel the fraction of PLCs by microscopic analyses of cytogenetic preparations. These data confirm that cells do not escape from G2 arrest. The fraction of PLCs is constantly reduced after prolonged incubation with RO-3306 in patient cells. In control cells, as expected, PLCs are nearly abseny. (c) Determination of the PLC frequency in U2OS cells depleted of MCPH1 by siRNAs and incubated with the Cdk1 inhibitor RO-3360 for 6 h. Mean and SD data are shown in all cases. Reduction of PLCs rate after RO-3306 treatment in b and c was statistically significant (p < 0.01, Χ² test).

Figure 5

Timing of PLC decondensation after incubation with the Cdk1 inhibitor RO-3360 in HeLa-H2B/Red1 cells. (a) Short description of the experimental procedure. RO-3306 was added 8 h after release from the second thymidine block to coincide with the occurrence of PLCs during G2 in the siRNA treated cells. (b) Selected frames showing the decondensation of a PLC (pointed by arrows) after incubation with RO-3360. Time after RO-3360 addition is indicated in minutes. (c) Combined dot- and box-plot showing the time (in minutes) that PLCs required to completely decondense their chromosomes after adding RO-3360. The broken line indicates the mean value. 30 PLCs were monitored.

Figure 6

Analyses of mitosis progression by live-cell microscopy in cells depleted of MCPH1 by siRNAs and incubated with the Cdk1 inhibitor RO-3360. (a) A brief description of the experimental procedure. HeLa H2B/Red1 cells were arrested in G2 by incubation with RO-3360 for 8 h and then released into normal fresh medium without the inhibitor. MCPH1 depletion was achieved by transfection with siRNAs during the previous 24 hours. Time-lapse recording started 30 minutes after the release from the RO-3360 incubation. Images were analyzed and processed using Image J software. (b) Dot-plots showing the time interval between different key mitotic events in minutes for untreated and MCPH1 depleted cells. The red line indicates the mean value. CC = chromosome condensation; NEB = nuclear envelope breakdown; CD = chromosome decondensation. At least 40 cells were analyzed in each case. Chromosome segregation and further decondensation were not analyzed in separate as both occur nearly simultaneously in control cells. Statistical comparisons for the mean and median data were done by T-student and Wilcoxon (W) tests respectively. **p < 0.01; N.S. not significant. (c) Selected frames showing the mitotic progression of representative HeLa cells from both control and MCPH1-depleted samples. Time from RO-3360 release is indicated in minutes. (d) Pairwise comparison of prometaphase timing (duration) in HeLa-H2B/Red1 cells after release from either double block with thymidine (Td) or RO-3360 incubation (RO).