Figure 7.
CENP-F directly controls Nde1 loading to kinetochores. (a) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and Ndel1. Scale bar, 5 µm. (b) Quantification of kinetochore Ndel1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (c) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and Nde1. Scale bar, 5 µm. (d) Quantification of kinetochore Nde1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (e) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells expressing eGFP-Lis1, arrested in 330 nM nocodazole for 16 h, and stained with DAPI and an antibody against CENP-C. Scale bar, 5 µm. (f) Quantification of kinetochore eGFP-Lis1 levels after background subtraction in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (g) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and DIC. Scale bar, 5 µm. (h) Quantification of kinetochore DIC levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (i) Cartoon schematic of Nde1-binding mutants. (j) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells transfected with eGFP, CENP-F^mEmerald, or the Nde1-binding mutants, arrested in 330 nM nocodazole for 16 h, and stained with antibodies against CENP-C and Nde1. Scale bar, 5 µm. (k) Quantification of kinetochore Nde1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells transfected with eGFP, CENP-F^mEmerald, or the Nde1-binding mutants and arrested in nocodazole. In b, d, f, h, and k, boxes depict the median and first and third quartiles, and whiskers represent Q1 and Q3 ± 1.5× interquartile range. ****, P < 0.0001.

CENP-F directly controls Nde1 loading to kinetochores. (a) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and Ndel1. Scale bar, 5 µm. (b) Quantification of kinetochore Ndel1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (c) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and Nde1. Scale bar, 5 µm. (d) Quantification of kinetochore Nde1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (e) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells expressing eGFP-Lis1, arrested in 330 nM nocodazole for 16 h, and stained with DAPI and an antibody against CENP-C. Scale bar, 5 µm. (f) Quantification of kinetochore eGFP-Lis1 levels after background subtraction in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (g) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and DIC. Scale bar, 5 µm. (h) Quantification of kinetochore DIC levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (i) Cartoon schematic of Nde1-binding mutants. (j) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells transfected with eGFP, CENP-F^mEmerald, or the Nde1-binding mutants, arrested in 330 nM nocodazole for 16 h, and stained with antibodies against CENP-C and Nde1. Scale bar, 5 µm. (k) Quantification of kinetochore Nde1 levels relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells transfected with eGFP, CENP-F^mEmerald, or the Nde1-binding mutants and arrested in nocodazole. In b, d, f, h, and k, 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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