Supplementary Figure 4: The Ndc80 FRET sensor is located between the CH domain and the Loop domain.
From: How the kinetochore couples microtubule force and centromere stretch to move chromosomes
(a) Method of establishment of the Ndc80FRET sensor cells. (b) PCR using primers in (a) amplified in control or Ndc80 FRET biosensor genome. (c) Representative images of mECFP and mYPet in Ndc80 FRET sensor cell, Ndc80-mECFP cell, and Ndc80-mYPet cell. (d) Confirmation of FRET probe position within the Ndc80 protein by the nm-scale heat-map method (see Methods). For this analysis, we established the Ndc80-GFP (M, at the aa 410) cell line, where GFP was inserted into the same position as the FRET probe. In short, the peak intensity (brightest pixel) of each fluorescent spot was determined and the coordinate of each spot was plotted relative to the spindle pole body (see Methods). The coordinates of over 100 images were used to generate a positional density map representing the distribution of Ndc80 as a fraction of the distance (nm) from the center of the spindle pole. (e) Representative images of GFP in Ndc80-GFP (C-terminal) cell, and Ndc80-GFP (M) cell (left). The ratio of GFP intensities in Ndc80-GFP cell and Ndc80-GFP (M) cell (right). n = 100 kinetochores. Error bars are SD from the means. Scale bars are 5 μm. The mean values were calculated using the data pooled from 2 independent experiments.
