Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore

Abstract

The attachment of spindle fibres to the kinetochore is an important process that ensures successful completion of the cell division. The Ca²⁺ concentration increases during the mitotic phase and contributes microtubule stability. However, its role in the spindle organisation in mitotic cells remains controversial. Here, we investigated the role of Ca²⁺ on kinetochore fibres in living cells. We found that depletion of Ca²⁺ during mitosis reduced kinetochore fibre stability. Reduction of kinetochore fibre stability was not due to direct inhibition of microtubule polymerisation by Ca²⁺-depletion but due to elimination of one dynamic component of kinetochore, CENP-F from the kinetochore. This compromised the attachment of kinetochore fibres to the kinetochore which possibly causes mitotic defects induced by the depletion of Ca²⁺.

Figure 1

Ca²⁺-depletion reduces the stability of spindle fibres. (a) HeLa^WT cells arrested at metaphase using MG132 were treated with 25 µM BAPTA-AM or 10 mM BAPTA and 5 µM ionomycin to reduce intracellular Ca²⁺ then stained with anti-Ac-tubulin antibody. (b) A graph presenting the relative Ac-tubulin intensity at kinetochore of each treatment. (c) Fluorescence images of metaphase cells in control, BAPTA-AM and BAPTA (with ionomycin) treatments after exposure to low temperature. (d) A graph presenting the relative cold-stable microtubule intensity at kinetochore of each treatment. (e) A bar graph showing the intensity ratio of Fura-2 excited at 340 and 380 nm measured in mitotic cells, which represent intracellular calcium levels. Error bars indicate standard deviations derived from three independent experiments. Bar, 5 µm.

Figure 2

BAPTA-AM treatment inhibits microtubule re-polymerisation. (a–c) Time-lapse images of U2OS cells expressing CENP-A-GFP and mCherry-α-tubulin during microtubule de-/re-polymerisation assay. Cells were released from nocodazole into fresh medium containing drugs as indicated. (d) A graph presenting the relative mCherry-α-tubulin at kinetochore of each treatment. Error bars indicate standard errors (n = 15 cells for control, n = 12 cells for BAPTA-AM, and n = 8 cells for BAPTA). Bar, 5 µm.

Figure 3

Ca²⁺-depletion does not alter H3K9me3 and HP1α localisation. Fluorescence images of chromosome spreads prepared from control and BAPTA-AM treated cells for the localisation of H3K9me3 (a) and HP1α (b). Bar graphs below indicate H3K9me3 centromere-to-arm signal intensity (c) and HP1α/CREST signal intensity (d). Error bars indicate standard deviations derived from three independent experiments. Bar, 5 µm.

Figure 4

Ca²⁺-depletion causes defects in the localisation of CENP-F at the outer kinetochore. Fluorescence images of metaphase cells after control, BAPTA-AM and BAPTA (with ionomycin) treatments for the localisation of Aurora B (a), Mis12 (b), Hec1 (c) and CENP-F (d). Bar, 5 µm.