Fig. 1: Disruption of the CENP-T-Mis12C interaction in cells lacking the CENP-C-Mis12C interaction leads to severe growth defects.
From: Recruitment of two Ndc80 complexes via the CENP-T pathway is sufficient for kinetochore functions
a Recruiting pathway of KMN network via CENP-C or CENP-T. b Schematic representation of chicken CENP-T. Ndc80C- and Mis12C-binding domains were deleted in CENP-T∆90 and CENP-T∆121-240, respectively. c Schematic representation of chicken CENP-C. The Mis12C-binding domain was deleted in CENP-C∆73. d Immunoblot analyses of various CENP-C or CENP-T in cKO-CENP-T (Tet-Off) cells. Expression of the CENP-T transgene (Tet-CENP-T) was terminated by 24 h after Tet addition. mScarlet-CENP-C∆73 was expressed in CENP-C knockout cells. The 3X FLAG-fused CENP-T mutants were stably expressed. CENP-C migration was slightly slow, due to CENP-C phosphorylation in mitosis. Alpha-tubulin (tubulin) was used as the loading control. The asterisks indicate nonspecific bands. None depicts parental cKO-CENP-T cells. e The growth of cKO-CENP-T cells expressing 3X FLAG-CENP-T∆121-240 in the absence or presence of Tet (–Tet or +Tet) with CENP-C WT (left) or mScarlet-CENP-C∆73 (right). f Schematic representation of each CENP-T mutant. g GFP-Dsn1 levels at kinetochores in cKO-CENP-T cells expressing mScarlet-CENP-C∆73 and various CENP-T mutants in the presence of Tet for 30 h. None indicates parental cKO-CENP-T/CENP-C∆73/GFP-Dsn1 cells. Subtract values are presented using signal intensities of None. DNA was stained with DAPI. Error bars show the mean and standard deviation. p values were calculated by one-way ANOVA followed by Tukey’s test. h The growth of cKO-CENP-T cells expressing CENP-CΔ73 and either CENP-TΔ121-160, CENP-TΔ161-200, CENP-TΔ201-216, CENP-TΔ217-230, or CENP-TΔ231-240 in the absence or presence of Tet. Some time points (N.D.) were missing because of no viable cells. i Cell cycle distribution of cKO-CENP-T/CENP-C∆73 (None), cKO-CENP-T/CENP-C∆73 expressing CENP-TWT or CENP-T∆121-240 in the presence of Tet for 30 h. j Chromosome alignment values in cKO-CENP-T/CENP-C∆73 (None), cKO-CENP-T/CENP-C∆73 expressing CENP-TWT, or CENP-T∆121-240. The cells were cultured in the presence of Tet for 30 h and MG132 for last 3 h. Error bars indicate the mean and standard deviation. p values were calculated as in g. k Numbers of spindle pole stained by anti-γ-tubulin in cKO-CENP-T/CENP-C∆73 (None), cKO-CENP-T/CENP-C∆73 expressing CENP-TWT, or CENP-T∆121-240 in the presence of Tet for 30 h.
