Figure 4: Measurements of the CENP-C-TR reporters.
From: Chromosome biorientation produces hundreds of piconewtons at a metazoan kinetochore
(a) Schematic of the proposed effects of force on the structural organization of the TR domain and the potential exposure of VH-binding sites in the TR domain based on a theoretical model of the chicken talin rod domain used here (aa 482–889)56. The red regions represent VH-binding domains in TR and the TR-binding domain in VH. (b) Experimental design of the internal CENP-C-TR reporter in cells co-expressing soluble VH-EGFP. The dashed lines represent potential VH–TR interactions. (c) Quantification of the percentage of MG132-treated cells with metaphase plates and >1 misaligned chromosomes in control and endogenous CENP-C depleted internal CENP-C-TR expressing cells (representative western blot—upper panel). Mean values from three independent experiments; Control RNAi; n=162 cells, CENP-C 3′-UTR RNAi; n=162 cells. (d) VH-EGFP localizes to kinetochores in cells expressing TagRFP-T-CENP-C-TR but not wild-type TagRFP-T-CENP-C. (e) Counting the number of VH-EGFP molecules associated per TR domain in the internal and C-terminal TR reporters in colchicine-treated and metaphase cells. The C-terminal TR data are from five independent experiments; n=146 colchicine-treated cells, n=125 metaphase cells. The internal TR data are from six independent experiments; n=182 colchicine-treated cells, n=179 metaphase cells. (f) Force estimation of the internal CENP-C-TR reporter based on published VH counting from single TR molecule magnetic trap experiments41. Scale bar, 10 μm. Error bars are s.e.m. P values from Mann–Whitney Wilcoxon t-tests are reported: not significant (NS) P value>0.05, ***P value<0.0005.
