Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome mis-segregation in cancer cells

Abstract

Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed gamma-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells.

Figure 1. CIN cells frequently assemble multipolar spindles in early mitosis.

A. Examples of MIN (HCT116) and CIN (HT-29, SW620) prometaphase cells immunostained for α-tubulin (red, left column) and kinetochores (green, middle column). All the images are maximum intensity projections of stacks of optical sections acquired at 0.6 µm intervals through the cell Z-axis. Merged images are shown in the right column. For each cell line, an example of bipolar prometaphase and one of multipolar prometaphase are shown. Scale bar, 5 µm. B. Frequencies of prometaphase and anaphase cells with multipolar spindles. The data shown here represent means and standard errors (bars) of four independent experiments. Multipolar CIN prometaphases were significantly more frequent than multipolar MIN prometaphases (χ² test, P<0.001 for both HT-29 and SW620 when compared to HCT116). However, multipolar CIN anaphases occurred at low frequencies that did not differ from those of multipolar MIN anaphases.

Figure 2. Most CIN cells divide in a bipolar fashion, but exhibit lagging chromosomes in anaphase.

A. Frequencies of cells exhibiting multipolar chromosome segregation in phase-contrast time-lapse experiments. B. Frequencies of bipolar anaphase MIN (HCT116) and CIN (HT-29, SW620) cells with lagging chromosomes. The data shown here represent means and standard errors (bars) of four independent experiments. Frequencies of anaphase lagging chromosomes were significantly higher in CIN than MIN cells (χ² test, P<0.001 for both HT-29 and SW620 when compared to HCT116). C. Examples of MIN (HCT116) and CIN (HT-29, SW620) anaphase cells immunostained for α-tubulin (red, first column) and kinetochores (green, second column). The images are maximum intensity projections of stacks of optical sections acquired at 0.6 µm intervals through the cell Z-axis. Merged images are shown in the third column. For each cell line, an example of normal anaphase and an anaphase with a lagging chromosome are shown. The column at the far right shows the cells with lagging chromosomes at a single focal plane, in which the contrast has been enhanced to highlight the merotelic connections of the lagging chromosome kinetochore. Scale bar, 5 µm.

Figure 3. Both multipolar prometaphase and bipolar metaphase CIN cells possess multiple centrosomes.

The figure shows examples of multipolar prometaphase (A) and bipolar metaphase (B) CIN cells immunostained for α-tubulin, kinetochores, and γ-tubulin. Images shown were obtained by merging maximum intensity projections of either α-tubulin and CREST (kinetochores) images (left column) or α-tubulin and γ-tubulin images (right column). A. Most multipolar prometaphase cells (exact percentages shown at the bottom right corner of the right panels) exhibited γ-tubulin staining at all spindle poles. B. Examples of bipolar metaphase CIN cells with multiple centrosomes at a single spindle pole (arrows point at γ-tubulin-stained dots). Scale bar, 5 µm. C. Frequencies of bipolar metaphase/early anaphase CIN cells possessing multiple centrosomes (as visualized by γ-tubulin staining) at a single spindle pole.

Figure 4. Multipolar prometaphase CIN cells possess larger numbers of merotelic kinetochores than bipolar prometaphase CIN cells.

A. Single focal plane of an unprocessed HT-29 multipolar prometaphase. Scale bar, 2.5 µm. B. Same cell and same focal plane as in (A) obtained after processing of the images in the two channels by special filtering, merging, and smoothing (see Materials and Methods for details). A merotelic kinetochore is visible in the boxed area. KTs: kinetochores. MTs: microtubules. C. Ratio view of the focal plane shown in (A) and (B). Regions of juxtaposition between the kinetochore and its microtubule bundle(s) appear in green. D. Enlargement (400%) of the boxed area in (B). E. Average number of merotelic kinetochores in bipolar vs. multipolar prometaphase CIN cells. Multipolar prometaphases possess significantly more merotelic kinetochores than bipolar prometaphases (t-test, P<0.001 for both cell lines).

Figure 5. Schematic representation of the mechanism by which multipolarity can lead to merotelic kinetochore attachment and mitotic chromosome mis-segregation.

A. Within a multipolar spindle, a single kinetochore is more likely to face two spindle poles than it would be in a bipolar spindle. B. Because of the multipolar spindle geometry, a single kinetochore can easily bind microtubules emanating from two spindle poles rather than from just one pole. After establishment of merotelic kinetochore attachment, the mitotic spindle bi-polarizes by a process of spindle pole coalescence (or centrosome clustering). C. Merotelic kinetochore attachment can persist through metaphase and into anaphase. D. During anaphase, the merotelic kinetochore attachment can give rise to a lagging chromosome.