Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts
Edited by Dominique C. Bergmann, Stanford University, Stanford, CA, and approved November 6, 2020 (received for review May 5, 2020)
Significance
In plants, microtubules largely determine the direction of cell expansion and the orientation of cell division planes. However, what processes orient the microtubules has remained debated. Here, we used microfabricated wells to confine and deform wallless plant cells in a controlled way to analyze the response of microtubules to cell geometry and surface tension. We demonstrate that microtubules align with cell geometry by default, whereas when surface tension increases (e.g. when turgor pressure increases), they align with the direction of maximal tension. Not only does this explain many observations in plant tissues, but it also provides a simple mechanism at the core of plant morphogenesis, in which microtubules can spontaneously align with tension, in a typical self-organized system.
Abstract
In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.
Data Availability
All study data are included in the article and supporting information.
Acknowledgments
We thank the MBI, Singapore, for hosting A.C. and O.H. for 6 months, where the original experiments were performed. We also thank our colleagues for constructive discussions about this project and for critical reading of the manuscript. We thank Platim for help with imaging and Gianluca Grenci for the design of the microwell casts at MBI. This work was supported by European Research Council Grant 615739 “MechanoDevo” (to O.H.), Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas “Plant-Structure Optimization Strategy” GrantJP18H05484) (to S.T.) and MBI seed grant support (to V.V. and T.E.S.).
Supporting Information
Appendix (PDF)
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Movie S1.
Stack: Confined protoplast expressing pUbq10::LTI6b-TdTomato in a 15 × 20 μm microwell for 2 hours in 600 mOsm/L mannitol
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Movie S2.
Stack: Confined protoplast expressing pUbq10::LTI6b-TdTomato in a 15 × 20 μm microwell for 2 hours in 280 mOsm/L mannitol
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Movie S3.
Kinetics of CMT orientation in a protoplast confined in a 12×40 μm microwell after transfer from 800 to 400 mOsm/L mannitol.
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Movie S4.
Kinetics of CMT orientation in a protoplast confined in a 12×40 μm microwell after successive transfers from 600 to 280 mOsm/L mannitol. Example 1.
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Movie S5.
Kinetics of CMT orientation in a protoplast confined in a 12×40 μm microwell after successive transfers from 600 to 280 mOsm/L mannitol. Example 2.
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- 1.39 MB
Movie S6.
Kinetics of CMT orientation in a protoplast confined in a 12×40 μm microwell after successive transfers from 600 to 280 mOsm/L mannitol. Example 3.
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- 2.17 MB
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Copyright © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data Availability
All study data are included in the article and supporting information.
Submission history
Published online: December 7, 2020
Published in issue: December 22, 2020
Keywords
Acknowledgments
We thank the MBI, Singapore, for hosting A.C. and O.H. for 6 months, where the original experiments were performed. We also thank our colleagues for constructive discussions about this project and for critical reading of the manuscript. We thank Platim for help with imaging and Gianluca Grenci for the design of the microwell casts at MBI. This work was supported by European Research Council Grant 615739 “MechanoDevo” (to O.H.), Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas “Plant-Structure Optimization Strategy” GrantJP18H05484) (to S.T.) and MBI seed grant support (to V.V. and T.E.S.).
Notes
This article is a PNAS Direct Submission.
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Competing Interests
The authors declare no competing interest.
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Cite this article
Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts, Proc. Natl. Acad. Sci. U.S.A.
117 (51) 32731-32738,
https://doi.org/10.1073/pnas.2008895117
(2020).
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