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MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis

Abstract

Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein–dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasis.

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Figure 1: MAP4 is required for correct spindle architecture.
Figure 2: Loss of MAP4 leads to spindle position and orientation defects.
Figure 3: MAP4 depletion increases cortical astral-microtubule and lateral spindle-pole movements.
Figure 4: MAP4 suppresses dynein-dependent force generation.
Figure 5: Depletion of CLASP1 leads to spindle-positioning defects in the x y plane, but does not affect spindle orientation in z.
Figure 6: CLASP1, but not CLASP2, rescues the spindle mispositioning in CLASP1-depleted cells.
Figure 7: MAP4- and CLASP1-mediated control of cortical force generators ensures an accurate cell-division axis.

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Acknowledgements

We thank A. Garrod for selecting the double stable EB3–tdTomato eGFP–CENP-A cell line, K. Kaseda for selecting the mCherry– α-tubulin cell line and D. Roth for assistance with cloning. We are grateful to I. Titley at the Institute of Cancer Research in Sutton, UK, for help with cell sorting, N. Galjart for the gift of CLASP1 antiserum and A. Akhmanova for providing CLASP1 and CLASP2 rescue constructs. This work was supported by programme grants to A.D.M. and A.S. from Marie Curie Cancer Care and a Fundação para a Ciência e Tecnologia fellowship (C.P.S.). L.C. is funded by the NSF-sponsored Institute for Cellular Engineering IGERT programme grant DGE-0654128 and J.L.R. is supported by a Cottrell Scholars Award from the Research Corporation.

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Contributions

This project was co-directed by A.S. and A.D.M. Project conception, planning and data interpretation was carried out by C.P.S., B.M., A.S. and A.D.M. Live- and fixed-cell imaging of spindle geometry, positioning and mitotic progression, as well as co-immunoprecipitation experiments, were carried out and analysed by C.P.S. Live-cell imaging of astral-microtubule dynamics, as well as cloning and purification of MAP4, was carried out and analysed by B.M. Dynein purification and gliding assays were carried out by L.C. and J.L.R. and patterned-substrate experiments by A.S. and A.D.M. The manuscript was prepared by A.S. and A.D.M. with contributions by C.P.S., B.M. and J.L.R.

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Correspondence to Anne Straube or Andrew D. McAinsh.

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Samora, C., Mogessie, B., Conway, L. et al. MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis. Nat Cell Biol 13, 1040–1050 (2011). https://doi.org/10.1038/ncb2297

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