Progression through M I in the absence of KT function.

2018-05-07T17:38:07Z (GMT) by Soumya Chaurasia Christian F. Lehner

(A-D) The kinetochore protein Spc105 was depleted in spermatocytes expressing Cid-EGFP and His2Av-mRFP. After time lapse imaging, all eight KTs in a representative spermatocyte were tracked during M I. (A) The KT tracks during consecutive division phases (time in minutes:seconds) are displayed as an overlay on still frames from the end of the corresponding phases. The two KTs associated with the same bivalent are represented by colored spheres with red, yellow, blue and green indicating chr XY, 2, 3 and 4, respectively. Transport of bivalents into the central region followed by aberrant segregation of unseparated KT pairs to opposite poles along horizontal axis is apparent. Scale bar = 3 μm. (B) KT velocities (VKT) plotted over time. (C) Angles (AKT) between the spindle axis and the axis connecting the two KTs of a bivalent plotted over time. (D) Distances (DKT) between the two KTs of a bivalent plotted over time. (E) Spc105 is required for recruitment of Mis12 to the KT. Spc105 was depleted in spermatocytes expressing Mis12-EGFP and His2Av-mRFP. Still frames after live imaging document the time point in prometaphase I where Mis12-EGFP signals at KTs are maximal in controls. (F) Spc105 is required for recruitment of Bub3 to the KT and for SAC function. Spc105 was depleted in spermatocytes expressing EGFP-Bub3 and His2Av-mRFP. Still frames after live imaging document the time point in prometaphase I where EGFP-Bub3 signals at KTs are maximal in controls. Dot plots indicate the duration of M I and M II as well as averages (+/- s.d., n ≥ 10 cells from at least six different cysts) in spermatocytes without (control) and with Spc105 depletion (Spc105 RNAi). (G) Cid-EGFP dot counts per nucleus in daughter cells generated by M I and M II, respectively. Four dots per nucleus are observed in normal cells (control) and variable numbers after Spc105 depletion (Spc105 RNAi). (n) number of analyzed cells. (H) Chromosome transport into the central region is evident after Spc105 depletion (Spc105 RNAi) but not in the presence of colcemid. Representative still frames at NEBD I (00:00) and 15 minutes later are shown with dotted yellow circles indicating the region occupied by chromosomes. Scale bars = 5 μm.