Figure 3: Acceptor photobleaching of CENP-C-TSMod reporters and force estimate.
From: Chromosome biorientation produces hundreds of piconewtons at a metazoan kinetochore
(a) Representative examples of acceptor photobleaching of the internal TSMod. Comparable channels are displayed with identical contrast and brightness scaling. (b) FRET efficiency measurements from acceptor photobleaching experiments. C-terminal data are from three independent experiments; n=238 interphase centromeres, n=229 metaphase centromeres/kinetochores. Internal data are from four independent experiments; n=318 interphase centromeres, n=346 metaphase centromeres/kinetochores for untreated cells and two independent experiments for kinesin-5 RNAi-treated cells; n=198 metaphase centromeres/kinetochores. (c) Based on the published calibration of the TSMod reporter39, a 20.7% FRET efficiency of the internal TSMod reporter corresponds to the application of ∼1.2–1.4 pN. The red and blue crosshairs denote the mean FRET efficiency values of the internal reporter in interphase and metaphase respectively. (d) FRET efficiency measurements from acceptor photobleaching experiments. Dhc RNAi data are from five independent experiments; n=454 bioriented kinetochores in DMSO-treated cells, n=433 bioriented kinetochores in taxol-treated cells. Scale bar, 1 μm. Error bars are s.e.m. Two-tailed P value of Student’s t-test is reported for the C-terminal data in b otherwise P values from Mann–Whitney Wilcoxon t-tests are reported: not significant (NS) P value>0.05, *P value<0.05, ***P value<0.0005.
