Abstract
Accurate chromosome segregation during meiosis depends on the assembly of a microtubule-based spindle of proper shape and size. Current models for spindle-size control focus on reaction diffusion–based chemical regulation and balance in activities of motor proteins. Although several molecular perturbations have been used to test these models, controlled mechanical perturbations have not been possible. Here we report a piezoresistive dual cantilever–based system to test models for spindle-size control and examine the mechanical features, such as deformability and stiffness, of the vertebrate meiotic spindle. We found that meiotic spindles prepared in Xenopus laevis egg extracts were viscoelastic and recovered their original shape in response to small compression. Larger compression resulted in plastic deformation, but the spindle adapted to this change, establishing a stable mechanical architecture at different sizes. The technique we describe here may also be useful for examining the micromechanics of other cellular organelles.
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Acknowledgements
This work was supported in part by Grant-in-Aid for Young Scientists (B) and Grant-in-Aid for Scientific Research on Priority Areas (T.I.), and Grant-in-Aid for Scientific Research (A), The 21st Century Center Of Excellence program and “Establishment of Consolidated Research Institute for Advanced Science and Medical Care” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.I.), and a Research Grant from the Human Frontier Science Program (S.I. and T.M.K.). T.M.K. also acknowledges support from National Institutes of Health–National Institute of General Medical Sciences (GM65933).
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T.I. performed the experiments and data analysis. J.T. provided considerable experimental assistance. T.I., T.M.K. and S.I. wrote the manuscript. H.O., K.K. and I.S. contributed to design and provide the piezo-resistive cantilevers. J.G. and Y.S. contributed to the initial project planning and experiment design. All authors discussed the results and commented on the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–4 (PDF 613 kb)
Supplementary Video 1
Time-lapse observation of fluorescent meiotic spindle during small deformation. The meiotic spindle, which was sandwiched between the manipulating (left) and the force-sensing (right) cantilevers, was compressed perpendicular to the pole-to-pole axis by moving the manipulating cantilever horizontally with an amplitude of 2 μm on this image plane. Images were acquired every 1 s. Force-dependent deformability and the force response of the spindle are shown in Fig. 2 and Supplementary Fig. 1. Scale bar is 10 μm. (MOV 706 kb)
Supplementary Video 2
Time-lapse observation of fluorescent meiotic spindle during pole-to-pole compression. The meiotic spindle, which was sandwiched between the manipulating (left) and the force-sensing (right) cantilevers, was compressed to the pole-to-pole axis by moving the manipulating cantilever horizontally with an amplitude of 2 to 10-μm range in periodic cycles. Images were acquired every 1 s. Force-dependent deformability and the force response of the spindle at an amplitude of 2 μm are shown in Fig. 3. Scale bar is 10 μm. (MOV 4517 kb)
Supplementary Video 3
Time-lapse observation of fluorescent meiotic spindle during large deformation. The meiotic spindle, which was sandwiched between the manipulating (left) and the force-sensing (right) cantilevers, was compressed perpendicular to the pole-to-pole axis by moving the manipulating cantilever horizontally with an amplitude of 8 to 12-μm range in successive cycles. Images were acquired every 1 s. Force-dependent deformability and the force response of the spindle are shown in Fig. 4 and Supplementary Fig. 3. Scale bar is 10 μm. (MOV 4850 kb)
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Itabashi, T., Takagi, J., Shimamoto, Y. et al. Probing the mechanical architecture of the vertebrate meiotic spindle. Nat Methods 6, 167–172 (2009). https://doi.org/10.1038/nmeth.1297
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DOI: https://doi.org/10.1038/nmeth.1297
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