Figure 1—figure supplement 4. | Human kinetochores are swivel joints that mediate microtubule attachments

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Human kinetochores are swivel joints that mediate microtubule attachments

Figure 1—figure supplement 4.

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University of Warwick, United Kingdom
Figure 1—figure supplement 4.
Download figureOpen in new tabFigure 1—figure supplement 4. Chromatic shift correction precision and accuracy in the dynamic live assay to analyse intra-kinetochore measurements.

(A) Histograms representing the compilation of all measurements of x- (ζx; light blue), y- (ζy; purple), and z-directional (ζz; pink) chromatic shift, defined as the distance between the diffraction-limited spot centres of the two fluorophores, in cells expressing eGFP-CENP-A and mCherry-CENP-A, for 18 imaging sessions. Each distribution of measurements of chromatic shift for a given imaging session was shifted by its median (ζ¯x, i.e. the measured chromatic shift for that imaging session) to centre it at zero. Values given are means of the standard deviations for each of these distributions (n = 18). (B) Schematic demonstrating the expected orientation of a kinetochore’s inner (green circles) and outer domains (red circles) relative to its spindle pole (black crossed circles) in both the xy- (top) and xz-plane (bottom). Blue lines represent the k-fibres which bind the kinetochores and spindle poles. Thin dashed red circles represent examples of biological variance in outer domain localisation, thick red circles represent the expected outer domain localisation. Dark blue arrows represent measurements of x- (Δx), y- (Δy) and z-directional (Δz) intra-kinetochore distance. (C) Histograms of measurements of x- (Δx; light blue), y- (Δy; purple) and z-directional (Δz; pink) intra-kinetochore distance from dynamic live movies of cells expressing eGFP-CENP-A and Ndc80-tagRFP. Values given are medians ± standard error, which are close to zero as expected from the schematic in (B).

DOI: http://dx.doi.org/10.7554/eLife.16159.007