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First published online 2 September 2003
doi: 10.1242/jcs.00716

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Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms

Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (167K): [in a new window]   Fig. 1. (A-D) Effect of microinjection of dominant negative mitotic checkpoint proteins on the presence of lagging chromosomes in anaphase in PtK1 cells under different experimental conditions. Arrows point at lagging chromosomes in anaphase. In microinjected cells, the tip of the needle is indicated by an asterisk. Scale bar: 10 µm. A QuickTime movie (Movie 1) of the cell shown in D is available at http://jcs.biologists.org/supplemental/

View larger version (20K): [in a new window]   Fig. 2. The data obtained in microinjection experiments (shown in Fig. 1) are summarized. Results obtained by microinjection of GSTMad1F10 and Mad2C were pooled.

View larger version (63K): [in a new window]   Fig. 3. Merotelic orientation of lagging chromosomes after microinjection of Mad1F10. PtK1 cells recovering from a nocodazole-induced mitotic block were microinjected with GST-Mad1F10 to inactivate the mitotic checkpoint and to induce precocious anaphase. Cells were followed by time-lapse microscopy and fixed in mid-anaphase if they possessed one or more lagging chromosomes. Cells were then immunostained for CREST and -tubulin. After immunostaining, cells were re-localized to verify if lagging chromosomes were merotelically oriented. Two cells showing lagging chromosomes after microinjection of GST-Mad1F10 are shown. The frame showing microinjection, the last frame photographed by time-lapse microscopy before fixation, the phase contrast/CREST overlay, and the CREST/-tubulin overlays are shown in the four columns. (A) Microinjected cell showing one merotelically oriented lagging chromosome at anaphase. (B) Microinjected cell showing multiple merotelically oriented lagging chromosomes. Asterisks indicate the tip of the needle at the moment of microinjection. Scale bars: 10 µm.

View larger version (127K): [in a new window]   Fig. 4. PtK1 cells immunostained for Mad2 (red) and -tubulin (green). The overlay of phase contrast images, Mad2, and -tubulin staining is shown. (A) Cell arrested in mitosis by a 3-hour nocodazole treatment. (B-D) Cells fixed after 1, 5 and 15 minutes recovery from the nocodazole-induced mitotic arrest. Scale bars: 10 µm.

View larger version (100K): [in a new window]   Fig. 5. PtK1 cells immunostained for Mad2 and -tubulin. The -tubulin staining is shown in the left column and the overlay of phase contrast images and Mad2 immunostaining is shown in the right column. (A,A'). Cell fixed after a 3 hour nocodazole treatment. (B,B'-D,D'. Cells fixed after 1, 5 and 15 minutes recovery from the nocodazole-induced mitotic arrest. Scale bar: 10 µm.

View larger version (90K): [in a new window]   Fig. 6. PtK1 cells in early mitosis immunostained for CREST and -tubulin (left column). DNA staining by DAPI is shown in the right column. (A,A',B,B' Cells fixed immediately after nuclear envelope breakdown. Two forming microtubule organizing centers can be seen (arrows) in these cells. The CREST staining shows that kinetochores are distributed around the spindle poles and are oriented in many different directions with respect to the poles (B). Furthermore, some chromosomes are oriented with their centromere axis through sister kinetochores nearly parallel to the spindle axis (yellow arrowhead in A), whereas other sister pairs are nearly perpendicular (white arrowheads in A). (C,C') Prometaphase control cell. Only two microtubule organizing centers can be identified (spindle poles) in this cell, as in the vast majority of untreated PtK1 mitotic cells. Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 7. Effect of a prolonged metaphase on the presence of anaphase lagging chromosomes. (A) Both control PtK1 cells (CTRL) and cells recovering from a nocodazole (NOC)-induced mitotic block were either fixed untreated or delayed in metaphase by a 2-hour MG-132 treatment and then fixed after recovery. Anaphase lagging chromosomes were analyzed by CREST staining. N, total number of cells analyzed. (B) Frequencies of single and multiple lagging chromosomes in anaphase PtK1 cells recovering from a nocodazole-induced mitotic block (-MG-132), or in which anaphase onset was delayed by a 2-hour MG-132 treatment (+MG-132) during recovery from nocodazole.

View larger version (86K): [in a new window]   Fig. 8. Examples of merotelic kinetochore orientations in prometaphase/metaphase analyzed by confocal microscopy and 3D image deconvolution. Images on each row show three different angles of the same cell. Arrows indicate merotelically oriented kinetochores. Note that when a merotelically oriented kinetochore is present, it appears still connected to kinetochore microtubules coming from both poles when the image is rotated of 12° (middle column) and 24° (right column). CTRL, control; NOC-R, 30 minutes recovery from a nocodazole-induced mitotic arrest; MG-132, 2 hours treatment with 5 µM MG-132; NOC-R + MG-132, nocodazole (2 µM) mitotic arrest + 25 minutes recovery + 2 hours MG-132 treatment. Scale bar: 10 µm.

View larger version (21K): [in a new window]   Fig. 9. Frequencies of merotelic orientations in late prometaphase/metaphase PtK1 cells and in `delayed' metaphases (see text for details). (A) Frequencies of merotelic orientations in prometaphase/metaphase PtK1 cells (-MG-132), and metaphases `delayed' by a 2 hour MG-132 treatment (+MG-132). Both in control (CTRL) and nocodazole-recovering (NOC) cells, the frequency of merotelic orientations after a 2-hour MG-132 treatment was lower than the frequency before the MG-132 treatment. N, total number of cells analyzed. (B) Frequencies of single and multiple merotelic kinetochore orientations in PtK1 cells recovering from nocodazole, with or without MG-132 treatment.

View larger version (17K): [in a new window]   Fig. 10. (A) Frequencies of merotelically oriented kinetochores based on the assumption that every PtK1 cell analyzed had the normal complement of 12 pairs of sister chromatids and 24 kinetochores. (B) Percentage merotelic kinetochore data (shown in A) normalized by values measured in prometaphase.

View larger version (16K): [in a new window]   Fig. 11. (A) Diagram showing how merotelic kinetochore orientation might be favored in chromosomes oriented with their centromere axis perpendicular to the interpolar spindle axis. (B) The aurora B/INCENP complex might play a role in merotelic orientation correction because of its proximity to the part of the merotelic kinetochore attached to the incorrect pole. (C) Correct chromosome segregation in the presence of merotelic orientation can occur either because the pulling force in one direction prevails on the force in the opposite direction (middle) or because the pulling forces on the kinetochore induce its breakage (bottom).