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Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells

¹ Center for Evolutionary Genetics CNR, c/o Department of Genetics and Molecular Biology, University `La Sapienza', Via degli Apuli 4, 00185 Rome, Italy
² Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (55K): [in a new window]   Fig. 1. FISH staining on anaphase human primary fibroblasts (MRC-5 cells) recovering from a nocodazole-induced mitotic arrest. Alphoid-specific probes for chromosome 7 (FITC, yellow) and 11 (Rhodamine, red) were used. DNA was stained by DAPI (blue). (A) Normal distribution of chromosome 7 and 11 at spindle poles. (B) Loss of a single chromatid of chromosome 7. (C) Loss of both sister chromatids of chromosome 7. When both sisters are lost in anaphase cells, their kinetochores are frequently separated and only in a few cases are the two chromatids still connected at the kinetochore level as shown in D for chromosome 11. The graph below shows quantitative results of the in situ hybridization analysis on 6378 anaphases, of which 94 displayed lagging chromosomes, for a total of 130 chromosome loss events for the two chromosomes under study. The data show that single lagging chromatids are the great majority of loss events. Bar, 5 µm.

View larger version (47K): [in a new window]   Fig. 2. 3F3/2 immunostaining on mitotic human primary fibroblasts (MRC-5 cells). (A) MRC-5 cell arrested in mitosis by nocodazole treatment. All the kinetochores show 3F3/2 staining caused by the presence of the phosphoepitope recognized by the 3F3/2 antibody on unattached kinetochores. (B) Metaphase cell after release from a nocodazole-induced mitotic block. The 3F3/2 antibody does not stain kinetochores, which are aligned at the metaphase plate and under tension. The 3F3/2 antibody does stain spindle poles (arrows). (C,D) Anaphase cells with lagging chromosomes (arrows) after release from the mitotic block. There is no 3F3/2 staining on kinetochores of lagging chromosomes, nor on kinetochores of chromosomes correctly segregated to the spindle poles. DNA is pseudocolored in red; 3F3/2 antibody is shown in yellow. Bar, 5 µm.

View larger version (80K): [in a new window]   Fig. 3. Chromosome dynamics during late mitotic stages in an MRC-5 cell expressing an H2B-GFP fusion protein. H2B-GFP-transfected MRC-5 cells were arrested in mitosis by nocodazole treatment and released in fresh medium for 30 minutes. From this time, late prometaphase or metaphase cells were localized and single cells were observed by fluorescence (A) and phase contrast (B) microscopy at regular intervals. Two lagging chromosomes are visible in anaphase/telophase (60 minutes, 70 minutes) after chromosome alignment at the metaphase plate had occurred (20 minutes). Numbers at the bottom right corner indicate the time in minutes. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 4. Chromosome dynamics during late mitotic stages in an H2B-GFP-transfected PtK1 cell. H2B-GFP-transfected PtK1 cells were arrested in mitosis by nocodazole treatment and released in fresh medium for 30 minutes. From this time, late prometaphase or metaphase cells were localized and single cells were observed by fluorescence (A) and phase-contrast (B) microscopy at regular intervals. Numbers at the bottom right corner indicate the time in minutes. In the first frame (0 minutes), a PtK1 cell with all the chromosomes correctly aligned at the metaphase plate is shown; one lagging chromosome appears after anaphase onset (15 minutes) and persists as a lagging chromosome until late cytokinesis (55 minutes). Bar, 5 µm.

View larger version (68K): [in a new window]   Fig. 5. Analysis of lagging chromosome behavior during cell cleavage by time-lapse microscopy in a PtK1 cell released from a nocodazole-induced mitotic arrest. Numbers at the bottom right corner indicate the time in minutes from nocodazole washout. Two lagging chromosomes can be seen in the equatorial region of the dividing cell from cleavage furrow formation (60 minutes) until cytokinesis proceeds (85 minutes). In the last frame the position of the reforming membranes around the two daughter nuclei and the two micronuclei deriving from the two lagging chromosomes is highlighted by a white line for clarity. Bar, 5 µm.

View larger version (30K): [in a new window]   Fig. 6. Proposed events leading to the preferential loss of both sister chromatids at anaphase. Acquisition of a merotelic orientation of one sister kinetochore twists the position of the whole chromosome (A), promoting the merotelic orientation of the other sister kinetochore (B). In mono-oriented chromosomes, sister kinetochores face opposite poles (A), favoring chromosome bi-orientation (B).