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Mitotic cells generate protrusive extracellular forces to divide in three-dimensional microenvironments

Nature Physics volume 14, pages 621–628 (2018)Cite this article

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Abstract

During mitosis, or cell division, mammalian cells undergo extensive morphological changes, including elongation along the mitotic axis, which is perpendicular to the plane that bisects the two divided cells. Although much is known about the intracellular dynamics of mitosis, it is unclear how cells are able to divide in tissues, where the changes required for mitosis are mechanically constrained by surrounding cells and extracellular matrix. Here, by confining cells three dimensionally in hydrogels, we show that dividing cells generate substantial protrusive forces that deform their surroundings along the mitotic axis, clearing space for mitotic elongation. When forces are insufficient to create space for mitotic elongation, mitosis fails. We identify one source of protrusive force as the elongation of the interpolar spindle, an assembly of microtubules aligned with the mitotic axis. Another source of protrusive force is shown to be contraction of the cytokinetic ring, the polymeric structure that cleaves a dividing cell at its equator, which drives expansion along the mitotic axis. These findings reveal key functions for the interpolar spindle and cytokinetic ring in protrusive extracellular force generation, and explain how dividing cells overcome mechanical constraints in confining microenvironments, including some types of tumour.

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Fig. 1: Cells dividing in confining microenvironments generate protrusive extracellular forces that deform the surrounding microenvironments.
Fig. 2: Most cells at metaphase in stiff and elastic hydrogels fail to complete mitosis.
Fig. 3: Morphological analysis reveals conservation of cell volume during cell division and buckling of interpolar spindles, indicating two mechanisms underlying extracellular force generation.
Fig. 4: Interpolar spindle elongation and lateral contraction of the cytokinetic ring both contribute to protrusive force generation.
Fig. 5: Spindle elongation drives extracellular force generation during early stages of mitosis, and mitotic expansion due to cytokinetic ring contraction dominates force generation at later stages.
Fig. 6: Proposed mechanisms for protrusive extracellular force generation during mitosis.

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Acknowledgements

The authors thank the members of the Chaudhuri laboratory, J. Nelson (Stanford University) and D. Fletcher (University of California, Berkeley) for helpful discussions, and M. Levenston (Stanford University) for use of the rheometer. This work was supported by a Samsung Scholarship for S.N., and a grant from the National Science Foundation (CMMI-1536736) to O.C.

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  1. Department of Mechanical Engineering, Stanford University, Stanford, CA, USA

    Sungmin Nam & Ovijit Chaudhuri

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Contributions

S.N. and O.C. designed the experiments and analysed the data. S.N. conducted the experiments and ran the simulations. S.N. and O.C. wrote the manuscript.

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Correspondence to Ovijit Chaudhuri.

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Supplementary information

Supplementary Information

Supplementary Figures 1–14, Supplementary Table 1, Supplementary Notes 1–3, Supplementary References 44–49

Life Sciences Reporting Summary

Videos

Supplementary Video 1: Dividing cells deform the surrounding matrices as they progress mitosis

Single dividing MDA-MB-2.1 cell within a hydrogel exert outward forces and pushes away the surrounding hydrogels along the mitotic axis. Matrix deformation was visualized with microbeads embedded in the hydrogels. White arrows point to specific microbeads, which are notably displaced. Images were shown at the indicated mitotic stages. Scale bar is 10µm.

Supplementary Video 2: Another example of matrix deformation during mitosis

Another movie of cell division within a hydrogel. Scale bar is 10µm.

Supplementary Video 3: Matrix deformation associated with mitotic swelling.

Cells undergoing mitotic swelling expand their size and generate outward forces, consequently deforming the surrounding matrices. Cells are arrested at mitosis by introducing S-trityl-L-cysteine. Matrix deformation was visualized by microbeads embedded in the hydrogels. White arrows point microbeads, which are notably displaced. Scale bar is 10µm.

Supplementary Video 4: Cell at metaphase does not progress through division in stiff and elastic 3D hydrogels.

Single dividing MDA-MB-2.1 cell within stiff and elastic hydrogels does not progress through mitosis, failing to divide. Matrix deformation was visualized with microbeads embedded in the hydrogels. White arrows point to specific microbeads, which are notably displaced inwardly. Scale bar is 10µm.

Supplementary Video 5: Another example of failure of division of cell at metaphase in stiff and elastic 3D hydrogels.

Single dividing MDA-MB-2.1 cell within stiff and elastic hydrogels does not progress through mitosis, failing to divide. Matrix deformation was visualized with microbeads embedded in the hydrogels. White arrows point to specific microbeads, which are notably displaced inwardly. Scale bar is 10µm.

Supplementary Video 6: Cells dividing in stiff and elastic hydrogels often undergo cell death.

Example of cell at metaphase in a stiff and elastic gel, which fails to undergo mitosis and later undergoes apoptosis. Scale bar is 10µm.

Supplementary Video 7: 3D reconstruction of a dividing cell.

Time-lapse images of a dividing cell were three-dimensionally reconstructed. Scale bar is 10µm.

Supplementary Video 8: Demonstration of outward force generation by lateral contraction using water balloon.

A macroscopic analogy to the outward forces generated by lateral contraction of the cytokinetic ring would be squeezing of a spherical water balloon at its equator, which leads to longitudinal expansion at the poles. In the movie, a green balloon contains water inside and is wrapped with a cable. The green balloon and the cable represent a cell and cytokinetic ring. The cable was pulled by a hand. Red arrows indicate ingression of the balloon equator due to pulling of the cable. Black arrows indicate longitudinal expansion of the balloon due to the water flow induced by pulling of the cable.

Supplementary Video 9: Buckled interpolar spindles observed for cells dividing in 3D hydrogels.

Cells dividing in 3D hydrogels were confined by the surrounding hydrogels and often exhibited very curved spindles at the end of cell division, indicative of buckling under compression. Yellow arrows indicate compressive reaction forces from hydrogels, in response to spindle-driven forces. White arrow points high curvature of interpolar spindle. Scale bar is 10µm.

Supplementary Video 10: Cells dividing in 2D culture exhibit straight interpolar spindles.

Cells dividing on 2D cell culture plates were free from confinement and did not show curved spindles, in contrast to cells dividing in 3D hydrogels. Scale bar is 10µm.

Supplementary Video 11: Relaxation of hydrogel deformation after interpolar spindle ablation.

Laser ablation was used to sever the interpolar spindles for cells entering anaphase. Relaxation of hydrogel deformation was visualized by microbeads embedded in the hydrogels. The position of beads retracts immediately in the direction of the cell after the spindles were ablated. White line represents the region of laser ablation, and white arrowheads indicate spindles ablated. White arrows point to specific microbeads, which are notably displaced inwardly. Scale bar is 10µm.

Supplementary Video 12: Another example of laser ablation at anaphase, imaging from metaphase.

Single dividing cell within a hydrogel generate outward forces and pushes away beads embedded in the hydrogel along the mitotic axis, when the cell progresses mitosis from metaphase to anaphase B. However, the position of the beads immediately retracts in the direction of the cell after the spindle was severed by laser ablation. White line represents the region of laser ablation, and white arrowheads indicate spindles ablated. White arrows point to specific microbeads, which are notably displaced. Images were shown at the indicated mitotic stages. Scale bar is 10µm.

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Nam, S., Chaudhuri, O. Mitotic cells generate protrusive extracellular forces to divide in three-dimensional microenvironments. Nature Phys 14, 621–628 (2018). https://doi.org/10.1038/s41567-018-0092-1

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