Figure 1.
CENP-F MTBDs are required for K-K tension and stable microtubule attachment. (a) Top: Coiled-coil prediction of the CENP-F^mEmerald protein. Bottom: Schematic showing the position of the microtubule- and kinetochore-binding domains in CENP-F^mEmerald and mutant versions. (b) Quantification of kinetochore CENP-E intensity relative to CENP-C in the rescue experiment shown in c and Fig. S1 a. (c) Immunofluorescence microscopy images of HeLa-K cells treated with either control or CENP-F siRNA, transfected with CENP-F^mEmerald or a microtubule-binding mutant and stained with DAPI and antibodies against CENP-C and CENP-E. Scale bar, 5 µm. Only the CENP-E channel is shown. (d) Quantification of kinetochore eGFP intensity in HeLa-K cells transfected with either CENP-F^mEmerald or a microtubule-binding mutant. (e) Quantification of the CENP-C–based intersister distance in the CENP-F rescue experiment depicted in Fig. S1 b. Dotted gray line indicates the intersister distance in cells treated with CENP-F siRNA and transfected with an empty vector. (f) Quantification of kinetochore oscillatory movements in control and CENP-F siRNA–treated cells using KiT software (Olziersky et al., 2018). Left: Example images of eGFP-CENP-A–expressing HeLa cells and corresponding charts showing tracks from example sister pairs (circled). Right: Autocorrelation plot based on the oscillations of eGFP-CENP-A kinetochores. The orange and purple arrowheads indicate the half and full periods in control cells, respectively (control siRNA: 23 cells, 783 sisters; CENP-F siRNA, 33 cells, 1,137 sisters). Scale bar, 5 µm. (g) Quantification of the kinetochore proximal α-tubulin intensity relative to CENP-C in the cold-stable CENP-F rescue experiment depicted in panel h and Fig. S1 c. Dotted gray line indicates the α-tubulin intensity in cells treated with CENP-F siRNA and rescued with an empty vector. (h) Immunofluorescence microscopy images of the cold-stable CENP-F rescue experiment. HeLa-K cells were treated with either control or CENP-F siRNA and transfected with CENP-F^mEmerald or a microtubule-binding mutant before incubation on ice and fixation. Cells were stained with DAPI and antibodies against CENP-C and α-tubulin. Only the α-tubulin channel is shown. Scale bar, 5 µm. In b, d, e, and g, boxes depict the median and first and third quartiles, and whiskers represent Q1 and Q3 ± 1.5× interquartile range. ****, P < 0.0001.

CENP-F MTBDs are required for K-K tension and stable microtubule attachment. (a) Top: Coiled-coil prediction of the CENP-F^mEmerald protein. Bottom: Schematic showing the position of the microtubule- and kinetochore-binding domains in CENP-F^mEmerald and mutant versions. (b) Quantification of kinetochore CENP-E intensity relative to CENP-C in the rescue experiment shown in c and Fig. S1 a. (c) Immunofluorescence microscopy images of HeLa-K cells treated with either control or CENP-F siRNA, transfected with CENP-F^mEmerald or a microtubule-binding mutant and stained with DAPI and antibodies against CENP-C and CENP-E. Scale bar, 5 µm. Only the CENP-E channel is shown. (d) Quantification of kinetochore eGFP intensity in HeLa-K cells transfected with either CENP-F^mEmerald or a microtubule-binding mutant. (e) Quantification of the CENP-C–based intersister distance in the CENP-F rescue experiment depicted in Fig. S1 b. Dotted gray line indicates the intersister distance in cells treated with CENP-F siRNA and transfected with an empty vector. (f) Quantification of kinetochore oscillatory movements in control and CENP-F siRNA–treated cells using KiT software (Olziersky et al., 2018). Left: Example images of eGFP-CENP-A–expressing HeLa cells and corresponding charts showing tracks from example sister pairs (circled). Right: Autocorrelation plot based on the oscillations of eGFP-CENP-A kinetochores. The orange and purple arrowheads indicate the half and full periods in control cells, respectively (control siRNA: 23 cells, 783 sisters; CENP-F siRNA, 33 cells, 1,137 sisters). Scale bar, 5 µm. (g) Quantification of the kinetochore proximal α-tubulin intensity relative to CENP-C in the cold-stable CENP-F rescue experiment depicted in panel h and Fig. S1 c. Dotted gray line indicates the α-tubulin intensity in cells treated with CENP-F siRNA and rescued with an empty vector. (h) Immunofluorescence microscopy images of the cold-stable CENP-F rescue experiment. HeLa-K cells were treated with either control or CENP-F siRNA and transfected with CENP-F^mEmerald or a microtubule-binding mutant before incubation on ice and fixation. Cells were stained with DAPI and antibodies against CENP-C and α-tubulin. Only the α-tubulin channel is shown. Scale bar, 5 µm. In b, d, e, and g, boxes depict the median and first and third quartiles, and whiskers represent Q1 and Q3 ± 1.5× interquartile range. ****, P < 0.0001.

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