Abstract
A molecular force sensing cassette (stFRET) was incorporated into actinin, filamin, and spectrin in vascular endothelial cells (BAECs) and into collagen-19 in Caenorhabditis elegans. To estimate the stress sensitivity of stFRET in solution, we used DNA springs. A 60-mer loop of single stranded DNA was covalently linked to the external cysteines of the donor and acceptor. When the complementary DNA was added it formed double stranded DNA with higher persistence length, stretching the linker and substantially reducing FRET efficiency. The probe stFRET detected constitutive stress in all cytoskeletal proteins tested, and in migrating cells the stress was greater at the leading edge than the trailing edge. The stress in actinin, filamin and spectrin could be reduced by releasing focal attachments from the substrate with trypsin. Inhibitors of actin polymerization produced a modest increase in stress on the three proteins suggesting they are mechanically in parallel. Local shear stress applied to the cell with a perfusion pipette showed gradients of stress leading from the site of perfusion. Transgenic C. elegans labeled in collagen-19 produced a behaviorally and anatomically normal animal with constitutive stress in the cuticle. Stretching the worm visibly stretched the probe in collagen showing that we can trace the distribution of mean tissue stress in specific molecules. stFRET is a general purpose dynamic sensor of mechanical stress that can be expressed intracellularly and extracellularly in isolated proteins, cells, tissues, organs and animals.
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An, X., M. C. Lecomte, J. A. Chasis, N. Mohandas, and W. Gratzer. Shear-response of the spectrin dimer-tetramer equilibrium in the red blood cell membrane. J. Biol. Chem. 277:31796–31800, 2002.
Avvisato, C. L., et al. Mechanical force modulates global gene expression and beta-catenin signaling in colon cancer cells. J. Cell Sci. 120:2672–2682, 2007.
Benz, P. M., et al. Cytoskeleton assembly at endothelial cell–cell contacts is regulated by alphaII-spectrin-VASP complexes. J. Cell Biol. 180:205–219, 2008.
Besch, S. R., T. Suchyna, and F. Sachs. High-speed pressure clamp. Pflugers Arch.-Eur. J. Physiol. 445:161–166, 2002.
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77:71–94, 1974.
Brown, A. E. X., R. I. Litvinov, D. E. Discher, and J. W. Weisel. Forced unfolding of coiled-coils in fibrinogen by single-molecule AFM. Biophys. J. 92:L39–L41, 2007.
Carter, N. J., and R. A. Cross. Kinesin’s moonwalk. Curr. Opin. Cell Biol. 18:61–67, 2006.
Croft, D. R., et al. Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. J. Cell Biol. 168:245–255, 2005.
Cunningham, C. C., et al. Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255:325–327, 1992.
Esue, O., Y. Tseng, and D. Wirtz. Alpha-actinin and filamin cooperatively enhance the stiffness of actin filament networks. PLoS One 4:e4411, 2009.
Ferrer, J. M., et al. Measuring molecular rupture forces between single actin filaments and actin-binding proteins. Proc. Natl Acad. Sci. USA 105:9221–9226, 2008.
Forde, N. R., D. Izhaky, G. R. Woodcock, G. J. Wuite, and C. Bustamante. Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase. Proc. Natl Acad. Sci. USA 99:11682–11687, 2002.
Furuike, S., T. Ito, and M. Yamazaki. Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy. FEBS Lett. 498:72–75, 2001.
Gardel, M. L., et al. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells. Proc. Natl Acad. Sci. USA 103:1762–1767, 2006.
Gosse, C., and V. Croquette. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82:3314–3329, 2002.
Huang, S., and D. E. Ingber. The structural and mechanical complexity of cell-growth control. Nat. Cell Biol. 1:E131–E138, 1999.
Johnson, C. P., H. Y. Tang, C. Carag, D. W. Speicher, and D. E. Discher. Forced unfolding of proteins within cells. Science 317:663–666, 2007.
Kainulainen, T., et al. Cell death and mechanoprotection by filamin a in connective tissues after challenge by applied tensile forces. J. Biol. Chem. 277:21998–22009, 2002.
Kramer, J. M. Structures and functions of collagens in Caenorhabditis elegans. FASEB J. 8:329–336, 1994.
Kramer, J. M., J. J. Johnson, R. S. Edgar, C. Basch, and S. Roberts. The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis. Cell 55:555–565, 1988.
Lamaze, C., L. M. Fujimoto, H. L. Yin, and S. L. Schmid. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J. Biol. Chem. 272:20332–20335, 1997.
Leikina, E., M. V. Mertts, N. Kuznetsova, and S. Leikin. Type I collagen is thermally unstable at body temperature. Proc. Natl Acad. Sci.USA 99:1314–1318, 2002.
Liu, L., M. P. Jedrychowski, S. P. Gygi, and P. F. Pilch. Role of insulin-dependent cortical fodrin/spectrin remodeling in glucose transporter 4 translocation in rat adipocytes. Mol. Biol. Cell 17:4249–4256, 2006.
MacDonald, R. I., and E. V. Pozharski. Free energies of urea and of thermal unfolding show that two tandem repeats of spectrin are thermodynamically more stable than a single repeat. Biochemistry 40:3974–3984, 2001.
Martin, P., and S. M. Parkhurst. Development: may the force be with you. Science 300:63–65, 2003.
Matthews, B. D., D. R. Overby, R. Mannix, and D. E. Ingber. Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels. J. Cell Sci. 119:508–518, 2006.
Mello, C. C., J. M. Kramer, D. Stinchcomb, and V. Ambros. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10:3959–3970, 1991.
Meng, F., T. M. Suchyna, and F. Sachs. A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ. FEBS J. 275:3072–3087, 2008.
Min, W., et al. Fluctuating enzymes: lessons from single-molecule studies. Acc. Chem. Res. 38:923–931, 2005.
Myllyharju, J., and K. I. Kivirikko. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 20:33–43, 2004.
Na, S., et al. Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc. Natl Acad. Sci. USA 105:6626–6631, 2008.
Nagai, T., et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20:87–90, 2002.
Ohta, Y., N. Suzuki, S. Nakamura, J. H. Hartwig, and T. P. Stossel. The small GTPase RalA targets filamin to induce filopodia. Proc. Natl Acad. Sci. USA 96:2122–2128, 1999.
Okreglak, V., and D. G. Drubin. Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway. J. Cell Biol. 188:769–777, 2010.
Primalov, P. Physical basis of the stability of folded conformations of proteins. New York: Freeman, 1992.
Qu, H., C. Y. Tseng, Y. Wang, A. J. Levine, and G. Zocchi. The elastic energy of sharply bent nicked DNA. EPL 90:1–5, 2010.
Rief, M., J. Pascual, M. Saraste, and H. E. Gaub. Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles. J. Mol. Biol. 286:553–561, 1999.
Rizzo, M. A., G. H. Springer, B. Granada, and D. W. Piston. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22:445–449, 2004.
Sarkar, A., S. Caamano, and J. M. Fernandez. The elasticity of individual titin PEVK exons measured by single molecule atomic force microscopy. J. Biol. Chem. 280:6261–6264, 2005.
Schmoller, K. M., O. Lieleg, and A. R. Bausch. Internal stress in kinetically trapped actin bundle networks. Soft Matter 4:2365–2367, 2008.
Schwaiger, I., C. Sattler, D. R. Hostetter, and M. Rief. The myosin coiled-coil is a truly elastic protein structure. Nat. Mater. 1:232–235, 2002.
Schwaiger, I., M. Schleicher, A. A. Noegel, and M. Rief. The folding pathway of a fast-folding immunoglobulin domain revealed by single-molecule mechanical experiments. EMBO Rep. 6:46–51, 2005.
Soncini, M., et al. Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study. Biomech. Model. Mechanobiol. 6:399–407, 2007.
Suchyna, T. M., and F. Sachs. Mechanosensitive channel properties and membrane mechanics in mouse dystrophic myotubes. J. Physiol. 581:369–387, 2007.
Thein, M. C., et al. Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology. Dev. Dyn. 226:523–539, 2003.
Trepat, X., et al. Universal physical responses to stretch in the living cell. Nature 447:592–595, 2007.
Wakatsuki, T., B. Schwab, N. C. Thompson, and E. L. Elson. Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114:1025–1036, 2001.
Wang, Y., A. Wang, H. Qu, and G. Zocchi. Protein–DNA chimeras: synthesis of two-arm chimeras and non-mechanical effects of the DNA spring. J. Phys. Condens. Matter 21:1–11, 2009.
Wang, A., and G. Zocchi. Elastic energy driven polymerization. Biophys. J. 96:2344–2352, 2009.
Wiggins, P. A., et al. High flexibility of DNA on short length scales probed by atomic force microscopy. Nat. Nanotechnol. 1:137–141, 2006.
Zhang, Z., S. A. Weed, P. G. Gallagher, and J. S. Morrow. Dynamic molecular modeling of pathogenic mutations in the spectrin self-association domain. Blood 98:1645–1653, 2001.
Acknowledgments
We acknowledge the assistance of the Confocal Microscope and Flow Cytometry Facility in the School of Medicine and Biomedical Sciences, University at Buffalo and support from the NIH.
Author contributions
Fanjie Meng—Designed and constructed stFRET probes, DNA stretching of stFRET, Imaging and analysis of constitutive strain, Wrote paper.
Thomas Suchyna—Designed performed mechanical stimulation experiments, FRET systems calibration/characterization, Imaging and analysis of constitutive strain, Wrote paper.
Elena Lazakovitch—Injected C. elegans oocytes and cultured worms, detergent solubilization of worms.
Richard M. Gronostajski—Collagen construct design, worm injection, edited paper.
Frederick Sachs—Project design, data analysis, Wrote paper.
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Associate Editor Yingxiao Peter Wang and Peter Butler oversaw the review of this article.
F. Meng and T. M. Suchyna are co-first authors.
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Meng, F., Suchyna, T.M., Lazakovitch, E. et al. Real Time FRET Based Detection of Mechanical Stress in Cytoskeletal and Extracellular Matrix Proteins. Cel. Mol. Bioeng. 4, 148–159 (2011). https://doi.org/10.1007/s12195-010-0140-0
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DOI: https://doi.org/10.1007/s12195-010-0140-0