Biophysical Measurements Reveal Fusion of Sister Kinetochores during Meiosis I

Abstract

Sister chromatids move toward opposite poles of the spindle during mitosis. Strikingly however, they must instead co-migrate toward the same pole during phase I of meiosis, the specialized cell division that produces sperm and eggs. Sister co-migration in budding yeast requires a meiosis I-specific protein complex called monopolin that possibly acts by mechanically fusing sister kinetochores (on the corresponding sister chromatids). Since sister kinetochore fusion might bring together twice as many microtubule-binding elements, we hypothesized that meiosis I kinetochores could form stronger attachments to microtubules than mitotic or meiosis II kinetochores. To test this idea we purified kinetochore particles from meiotic or mitotic budding yeast, attached them to individual dynamic microtubules in vitro, and then measured their rupture strength at the single particle level using a computer-controlled laser trap operating in force-ramp mode. Consistent with the fusion model, meiosis I kinetochores ruptured at 13.09 ± 0.47 pN (mean ± s.e.m) on average, significantly higher than the strengths of mitotic and meiosis II kinetochores, 9.61 ± 0.56 pN and 9.30 ± 0.73 pN, respectively. Blocking DNA replication during pre-meiotic S-phase reduced the high strength of meiosis I kinetochores, making them rupture at mitotic-like levels, 9.31 ± 1.22 pN, confirming that sister chromatid's presence is essential. High strength of meiosis I kinetochores was also lost when monopolin was disrupted and conversely, strength of mitotic kinetochores upon ectopic expression of monopolin increased to 11.28 ± 0.90 pN, an intermediate value suggesting a mix of fused sisters and individual, un-fused kinetochores. Addition of recombinant monopolin to mitotic kinetochores in vitro was also sufficient to increase their strength to meiosis I-like levels, 13.00 ± 0.86 pN. Taken together, these data provide strong direct evidence for a monopolin-dependent mechanical fusion of sister kinetochores during meiosis I.