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. 2003 Dec 1;17(23):2902–2921. doi: 10.1101/gad.1144403

Figure 7.

Figure 7.

Functional analysis of the COMA and MIND complexes in chromosome segregation. (A) Strains carrying a centromere-proximal chrIV tag and Spc42p-GFP (He et al. 2000) were synchronized with α-factor and released into prewarmed media at 37°C. Cells were fixed after 75 min, and the extent of transient sister separation was determined in metaphase cells (as determined by nuclear morphology and spindle length). Data for ndc80-1 are provided for comparison (He et al. 2001). (B) okp1-5 and mtw1-1 cells containing Ndc80p-GFP (green) and ndc80-1 cells containing Mtw1p-GFP (green) were shifted to 37°C for 3 h, fixed, and imaged as described in the Figure 2E legend. All strains contained Spc42p-CFP (red) to mark the position of the SPBs, which are indicated by the red arrows. (C) Schematic of the chrIV tag and Spc42p-GFP SPB tag showing the spindle-centered cylindrical reference system and three key parameters used to characterize chromosome motion: d1, the distance from the reference spindle pole to one chromatid tag; d2, the distance from the reference spindle pole to the sister chromatid; and d3, the distance between the SPBs. (D-O, left column) Scatter plots of the positions of chromosome tags over 300 sec in a time-lapse movie in wild-type, ndc80-1, okp1-5, and mtw1-1 cells at 37°C. The length of the spindle is shown in red; in general, spindles are longer in cells with kinetochore mutants than in wild-type cells, as a consequence of reduced inward directed spindle forces (Goshima and Yanagida 2000). Separate green and black lines are seen only when chromatids are transiently separated. The positions of chromosomes are viewed along the spindle axis. (D-O, middle column). Scatter plots with the spindle axis rotated -45° and elevated 20°. (D-O, right column) Plots of spindle length, d3 in part C (red), the distance between a reference SPB and the first chromosome tag, d1 (black) and between the reference SPB and the second chromosome tag, d2 (green). Distinct black and green lines are seen only when sister chromatids are undergoing transient separation.