Figure 4.

Side-attached kinetochores are more compact specifically when pulled toward minus ends. (A) Time-lapse images showing flow-induced reorientation of a microtubule (magenta) attached by its side to a surface-assembled kinetochore. Both the Ndc80-GFP kinetochore marker (cyan) and the Atto565 label on the centromeric DNA (yellow) oscillated with the direction of buffer flow. Horizontal dashed lines indicate approximate positions of the DNA tether point on the coverslip. (B) Schematic of a surface-assembled kinetochore attached to the side of a microtubule. Oscillating the direction of flow caused the kinetochore and its captured microtubule to flip back and forth, reorienting by 180° with each reversal of the flow. (C) Example records of position versus time for an Ndc80-GFP kinetochore attached to the side of a microtubule. Displacements of both the Ndc80-GFP spot and the Atto565 label on the wild type centromeric DNA (CEN^WT) relative to the tether point were estimated by averaging during the intervals when the microtubule orientation was steady. Black symbols represent mean ± SD from N = 60 tracked positions during each interval. Positions recorded during the reorientation of the microtubule were omitted from the averaging and are indicated here by gray shading. (D) Distributions of displacement for the indicated fluorescent components within tip-attached kinetochores (from N = 74–116 intervals), fit with single Gaussian functions. The mean ± SEM for each Gaussian is indicated. These data and fits are replotted from Fig. 3 E with an expanded vertical scale. (E) Distributions of displacement for the indicated fluorescent components within side-attached kinetochores (from N = 32–87 intervals), fit with either a single Gaussian (CEN^WT DNA) or a double Gaussian function (Ndc80-GFP). The mean ± SEM for each Gaussian is indicated. The distribution of Ndc80-GFP displacements for side-attached kinetochores is wider in comparison to the tip-attached kinetochores in panel D, and apparently bimodal, including a sub-population very close to the tether with a mean displacement of only 18 ± 2 nm. (F) Distributions of displacement for Ndc80-GFP within kinetochores attached to the sides of polarity-marked microtubules. Boxes extend from first to third quartiles with medians indicated by central horizontal solid lines. Medians for kinetochores pulled toward plus and minus ends were 36 and 12 nm, respectively (from N = 45 and 30 intervals). Whiskers extend ± one SD from the mean.