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Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin

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Abstract

The shape of adherent cells is known to be a key determinant of cellular processes such as proliferation, apoptosis, and differentiation. Manipulation of cell shape affects stem cell differentiation, gene expression, and the response of cells to mechanical stimulation. Shape sensing is at least partially due to mechanically-sensitive signaling within focal adhesions (FAs). Therefore, we evaluate the dependence of cellular force generation on cellular geometry by measuring loads across the FA protein vinculin using an engineered Förster Resonance Energy Transfer-based biosensor. To control cellular geometry, vinculin deficient mouse embryonic fibroblasts stably expressing the vinculin sensor were confined to specific shapes of the same area using photopatterning techniques. It was observed that the tension across vinculin increases at the edges of the cells. However, vinculin supporting compressive loads was found in the center of cells, with an increase in compression observed with increasing aspect ratio. This phenomena is consistent with observations of compressive forces directly under the nucleus and supported by our observation of increased nuclear deformation and enhanced apical actin organization, though this is the first time compressive loads across vinculin have been shown to maintain FA assembly. This suggests a new paradigm for mechanosensitivity in adhesion mechanobiology, where the compressive and tensile loads across particular proteins must be considered.

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Acknowledgments

The authors thank Drs. Ben Fabry, Wolfgang Goldman, and Wolfgang Ziegler for providing MEFs; Dr. George Dubay for his assistance with fluorometric data acquisition; The Franz lab for the use of their UV–Vis spectrophotometer; Dr. Nicolas Christoforou for his work creating the initial VinTS lentiviral construct; and Vidya Venkataramanan and Aarti Urs for production of stable cell lines and other technical support. This work was supported by a Searle Scholar Award, a Basil O’Connor Starter Scholar Research Award (March of Dimes Foundation) Award, and National Science Foundation CAREER Award to Dr. Brenton Hoffman; a National Science Foundation Graduate Research Fellowship awarded to Katheryn Rothenberg; and a Grand Challenge Scholar Grant awarded to Shane Neibart.

Conflict of interest

Katheryn E. Rothenberg, Shane S. Neibart, Andrew S. LaCroix, and Brenton D. Hoffman declare they have no conflicts of interest.

Human and Animal Rights and Informed Consent

No human or animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Katheryn E. Rothenberg, Shane S. Neibart, Andrew S. LaCroix & Brenton D. Hoffman

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  1. Katheryn E. Rothenberg
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  2. Shane S. Neibart
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Correspondence to Brenton D. Hoffman.

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Associate Editor Cynthia A. Reinhart-King oversaw the review of this article.

This article is part of the 2015 Young Innovators Issue.

Dr. Brenton Hoffman is an Assistant Professor in the Department of Biomedical Engineering at Duke University where he is the principal investigator of the Cell and Molecular Mechanobiology Laboratory. He received a B.S. Degree in Chemical Engineering from Lehigh University and a PhD in Chemical and Biomolecular Engineering from the University of Pennsylvania. His thesis work focused on adapting soft matter physics techniques to probe the active mechanics of the cytoskeleton. Then he completed Post-doctoral training in the Cardiovascular Research Center at the University of Virginia, receiving a Post-doctoral Fellowship from the American Heart Association. This work centered on the creation and use of optically-based biosensors that report forces across specific proteins in living cells. Dr. Hoffman has won several prestigious awards, including a Basil O’Connor Starter Scholar Award from the March of Dimes, a Searle Scholar Award, and National Science Foundation CAREER Award. He has published peer-reviewed articles in Nature, Current Biology, PNAS, Angewandte Chemie, Journal of Cell Science, Physical Review Letters, and Biophysical Journal. His current research focuses on the development and use of force-sensitive biosensors to understand the mechanisms cell use to sense, detect, and respond to the cellular microenvironment.

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Rothenberg, K.E., Neibart, S.S., LaCroix, A.S. et al. Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin. Cel. Mol. Bioeng. 8, 364–382 (2015). https://doi.org/10.1007/s12195-015-0404-9

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