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Abstract

Introduction

Protocol

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Acknowledgements

Materials

References

Bioengineering

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Published: November 2nd, 2018

DOI:

10.3791/58619

1Department of Biomedical Engineering, Duke University

Here, we present a protocol for the simultaneous use of Förster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the Förster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

The extracellular environment is a rich source of both biochemical and physical cues that dictate cell behavior. In particular, the physical nature of the microenvironment can mediate key cellular functions, including cell growth, migration, and differentiation1,2,3,4. Dysregulation of the mechanics of the microenvironment is a critical component to many diseases that do not yet have adequate treatments, such as cancer5, atherosclerosis6, and fibrosis7. A complete unders....

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1. Generate Samples for Imaging

  1. Stably express tension sensor construct in desired cell type.
    1. Clone tension sensor construct into pRRL vector or other viral expression plasmid.
      NOTE: Several different molecular cloning tools are available to achieve this step including the use of restriction enzymes, overlap extension, and Gibson Assembly35. The pRRL vector is used in lenti viral transduction and enables a substantial degree of protein production through the use of .......

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FRET-FRAP is made up of the combination of two fluorescent techniques, FRET and FRAP. As we focused on the effects of protein load, we used FRET-based tension sensors34,46. These sensors are often based on a tension sensing module consisting of two fluorescent proteins, such as mTFP1 and VenusA206K, connected by a flagelliform linker (Figure 1A). When the module is placed between the head and tail dom.......

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The FRET-FRAP method allows for direct measurement of force-sensitive protein dynamics, a property that has been difficult to directly probe inside living cells. The sensitivity of protein dynamics to molecular load is critical to the protein's function as a force transmitter or transducer. Loading is required for the transmission of both internally-generated and externally-applied forces, called mechanotransmission, and for the conversion of those forces into biochemically-detectable signals, called mechanotransduct.......

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This work was supported by a National Science Foundation CAREER Award (NSF-CMMI-14-54257) as well as grants from the American Heart Association (16GRNT30930019) and National Institutes of Health (R01GM121739-01) awarded to Dr. Brenton Hoffman and a National Science Foundation Graduate Research Fellowship awarded to Katheryn Rothenberg. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF or NIH.

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Name Company Catalog Number Comments
0.05% Trypsin-EDTA Thermo Fisher 25300062
16% Paraformaldehyde Electron Microscopy Sciences 30525-89-4
60x Objective NA1.35 Olympus UPLSAPO 60XO
Antibiotic-Antimycotic Solution (100x) Gibco 15240-062
Automated Stage Prior Scientific H117EIX3
Custom Dichroic Mirror Chroma Technology Corp T450/514rpc
Custom mTFP1 Emission Filter Chroma Technology Corp ET485/20m
Custom mTFP1 Excitation Filter Chroma Technology Corp ET450/30x
Custom Venus Excitation Filter Chroma Technology Corp ET514/10x
DMEM-gfp Live Cell Visualization Medium Sapphire MC102
Dulbecco's Modified Eagle's Medium  Sigma Aldrich D5796 with L-glutamine and sodium bicarbonate
Fetal Bovine Serum HyClone SH30396.03
Fibronectin, Human Corning 47743-654
FRAPPA Calibration Slide Andor provided along with FRAPPA unit
FRAPPA System with 515 nm Laser Andor
Glass-bottomed Fluoro Dishes World Precision Instruments FD35
HEK293-T Cells ATCC CRL-3216
Hexadimethrine Bromide, Polybrene Sigma Aldrich H9268-5G
High-glucose Dulbecco's Modified Eagle's Medium Sigma Aldrich D6429
Inverted Fluorescent Microscope Olympus IX83
JMP Pro Software SAS
Lambda 10-3 Motorized Filter Wheels Sutter Instruments LB10-NW
LambdaLS Arc Lamp with 300W Ozone-Free Xenon Bulb Sutter Instruments LS/OF30
Lipofectamine 2000 Invitrogen 11668-027
MATLAB Software Mathworks
MEM Non-Essential Amino Acids Thermo Fisher 11140050
MetaMorph for Olympus Olympus
Micro-Humidification System Bioptechs 130708
MoFlo Astrios EQ Cell Sorter Beckman Coulter B25982
Objective Heater Medium Bioptechs 150819-13
OptiMEM Thermo Fisher 31985070
Phosphate Buffered Saline Sigma Aldrich D8537
pMD2.G Envelope Plasmid Addgene 12259
pRRL Vector gift from Dr. Kam Leong (Columbia University)
psPax2 Packaging Plasmid Addgene 12260
sCMOS ORCA-Flash4.0 V2 Camera Hamamatsu Photonics C11440-22CU
Sorvall Legend XT/XF Centrifuge Thermo Fisher 75004505
Stable Z Stage Warmer Bioptechs 403-1926
Venus Emission Filter Semrock FF01-571/72

  1. DuFort, C. C., Paszek, M. J., Weaver, V. M. Balancing forces: architectural control of mechanotransduction. Nature Reviews Molecular Cell Biology. 12 (5), 308-319 (2011).
  2. Mammoto, T., Ingber, D. E. Mechanical control of tissue and organ development. Development. 137 (9), 1407-1420 (2010).
  3. Sun, Y., Chen, C. S., Fu, J. Forcing stem cells to behave: a biophysical perspective of the cellular microenvironment. Annual Review of Biophysics. 41, 519-542 (2012).
  4. Wozniak, M. A., Chen, C. S. Mechanotransduction in development: a growing role for contractility. Nature Reviews Molecular Cell Biology. 10 (1), 34-43 (2009).
  5. Broders-Bondon, F., Nguyen Ho-Bouldoires, T. H., Fernandez-Sanchez, M. E., Farge, E. Mechanotransduction in tumor progression: The dark side of the force. The Journal of cell biology. 217 (5), 1571-1587 (2018).
  6. Simmons, R. D., Kumar, S., Jo, H. The role of endothelial mechanosensitive genes in atherosclerosis and omics approaches. Archives of Biochemistry and Biophysics. 591, 111-131 (2016).
  7. Wells, R. G. Tissue mechanics and fibrosis. Biochimica et Biophysica Acta. 1832 (7), 884-890 (2013).
  8. Lele, T. P., et al. Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells. Journal of Cellular Physiology. 207 (1), 187-194 (2006).
  9. Hoffman, B. D., Grashoff, C., Schwartz, M. A. Dynamic molecular processes mediate cellular mechanotransduction. Nature. 475 (7356), 316-323 (2011).
  10. Dembo, M., Torney, D. C., Saxman, K., Hammer, D. The reaction-limited kinetics of membrane-to-surface adhesion and detachment. Proceedings of the Royal Society of London B: Biological Sciences. 234 (1274), 55-83 (1988).
  11. LaCroix, A. S., Rothenberg, K. E., Hoffman, B. D. Molecular-Scale Tools for Studying Mechanotransduction. Annual Review of Biomedical Engineering. 17, 287-316 (2015).
  12. Neuman, K. C., Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods. 5 (6), 491-505 (2008).
  13. Kong, F., Garcia, A. J., Mould, A. P., Humphries, M. J., Zhu, C. Demonstration of catch bonds between an integrin and its ligand. The Journal of Cell Biology. 185 (7), 1275-1284 (2009).
  14. Rakshit, S., Zhang, Y., Manibog, K., Shafraz, O., Sivasankar, S. Ideal catch, and slip bonds in cadherin adhesion. Proceedings of the National Academy of Sciences. 109 (46), 18815-18820 (2012).
  15. del Rio, A., et al. Stretching Single Talin Rod Molecules Activates Vinculin Binding. Science. 323 (5914), 638-641 (2009).
  16. Seddiki, R., et al. Force-dependent binding of vinculin to alpha-catenin regulates cell-cell contact stability and collective cell behavior. Molecular Biology of the Cell. 29 (4), 380-388 (2018).
  17. Huang, D. L., Bax, N. A., Buckley, C. D., Weis, W. I., Dunn, A. R. Vinculin forms a directionally asymmetric catch bond with F-actin. Science. 357 (6352), 703-706 (2017).
  18. Chrzanowska-Wodnicka, M., Burridge, K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. The Journal of Cell Biology. 133 (6), 1403-1415 (1996).
  19. Choquet, D., Felsenfeld, D. P., Sheetz, M. P. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell. 88 (1), 39-48 (1997).
  20. Liu, Z., et al. Mechanical tugging force regulates the size of cell-cell junctions. Proceedings of the National Academy of Science. 107 (22), 9944-9949 (2010).
  21. Riveline, D., et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. The Journal of Cell Biology. 153 (6), 1175-1186 (2001).
  22. Brevier, J., Vallade, M., Riveline, D. Force-extension relationship of cell-cell contacts. Physical Review Letters. 98 (26), 268101 (2007).
  23. Bershadsky, A., Kozlov, M., Geiger, B. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Current opinion in cell biology. 18 (5), 472-481 (2006).
  24. Chan, C. E., Odde, D. J. Traction dynamics of filopodia on compliant substrates. Science. 322 (5908), 1687-1691 (2008).
  25. Wu, Z., Plotnikov, S. V., Moalim, A. Y., Waterman, C. M., Liu, J. Two distinct actin networks mediate traction oscillations to confer focal adhesion mechanosensing. Biophysical Journal. 112 (4), 780-794 (2017).
  26. Fritzsche, M., Charras, G. Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching. Nature Protocols. 10 (5), 660-680 (2015).
  27. McNally, J. G. Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods of Cell Biology. 85, 329-351 (2008).
  28. Wehrle-Haller, B. Analysis of integrin dynamics by fluorescence recovery after photobleaching. Methods in Molecular Biology. 370, 173-202 (2007).
  29. Carisey, A., Stroud, M., Tsang, R., Ballestrem, C. Fluorescence recovery after photobleaching. Methods of Molecular Biology. 769, 387-402 (2011).
  30. Carisey, A., et al. Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Current Biology. 23 (4), 271-281 (2013).
  31. Wolfenson, H., Bershadsky, A., Henis, Y. I., Geiger, B. Actomyosin-generated tension controls the molecular kinetics of focal adhesions. Journal of Cell Science. 124, 1425-1432 (2011).
  32. Dumbauld, D. W., et al. How vinculin regulates force transmission. Proceedings of the National Academy of Sciences. 110 (24), 9788-9793 (2013).
  33. Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., Geiger, B. Functional atlas of the integrin adhesome. Nature Cell Biology. 9 (8), 858-867 (2007).
  34. Grashoff, C., et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 466 (7303), 263-266 (2010).
  35. LaCroix, A. S., Rothenberg, K. E., Berginski, M. E., Urs, A. N., Hoffman, B. D. Construction, imaging, and analysis of FRET-based tension sensors in living cells. Biophysical Methods in Cell Biology. 125, 161-186 (2015).
  36. Meng, F., Suchyna, T. M., Sachs, F. A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ. The FEBS Journal. 275 (12), 3072-3087 (2008).
  37. Rothenberg, K. E., Scott, D. W., Christoforou, N., Hoffman, B. D. Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration. Biophysical journal. 114 (7), 1680-1694 (2018).
  38. Austen, K., et al. Extracellular rigidity sensing by talin isoform-specific mechanical linkages. Nature Cell Biology. 17 (12), 1597-1606 (2015).
  39. Kumar, A., et al. Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity. The Journal of Cell Biology. 213 (3), 371-383 (2016).
  40. Acharya, B. R., et al. Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility. Cell Reports. 18 (12), 2854-2867 (2017).
  41. Borghi, N., et al. E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch. Proceedings of the National Academy of Sciences. 109 (31), 12568-12573 (2012).
  42. Conway, D. E., Williams, M. R., Eskin, S. G., McIntire, L. V. Endothelial cell responses to atheroprone flow are driven by two separate flow components: low time-average shear stress and fluid flow reversal. American Journal of Physiology-Heart and Circulatory Physiology. 298 (2), H367-H374 (2010).
  43. Arsenovic, P. T., et al. Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension. Biophysical Journal. 110 (1), 34-43 (2016).
  44. Ye, N., et al. Direct observation of alpha-actinin tension and recruitment at focal adhesions during contact growth. Experimental Cell Research. 327 (1), 57-67 (2014).
  45. Paszek, M. J., et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature. 511 (7509), 319-325 (2014).
  46. Gayrard, C., Borghi, N. FRET-based Molecular Tension Microscopy. Methods. 94, 33-42 (2016).
  47. de Beco, S., Gueudry, C., Amblard, F., Coscoy, S. Endocytosis is required for E-cadherin redistribution at mature adherens junctions. Proceedings of the National Academy of Sciences. 106 (17), 7010-7015 (2009).
  48. Östlund, C., et al. Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins. Journal of Cell Science. 122 (22), 4099-4108 (2009).
  49. Coffin, J. M., Hughes, S. H., Varmus, H. E. . The interactions of retroviruses and their hosts. , (1997).
  50. Komatsubara, A. T., Matsuda, M., Aoki, K. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer). Scientific Reports. 5, 13283 (2015).
  51. Nasri, M., Karimi, A., Farsani, M. A. Production purification and titration of a lentivirus-based vector for gene delivery purposes. Cytotechnology. 66 (6), 1031-1038 (2014).
  52. . Generating Stable Cell Lines with Lentivirus Available from: https://www.addgene.org/protocols/generating-stable-cell-lines (2016)
  53. Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry: The Journal of the International Society for Analytical Cytology. 45 (3), 194-205 (2001).
  54. Malkani, N., Schmid, J. A. Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation. PLoS One. 6 (4), e18586 (2011).
  55. Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S., Ikeda, S. R. Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophysical journal. 91 (5), L39-L41 (2006).
  56. Rothenberg, K. E., Neibart, S. S., LaCroix, A. S., Hoffman, B. D. Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin. Cellular and Molecular Bioengineering. 8 (3), 364-382 (2015).
  57. Carnell, M., Macmillan, A., Whan, R. Fluorescence recovery after photobleaching (FRAP): acquisition, analysis, and applications. Methods of Molecular Biology. 1232, 255-271 (2015).
  58. Trembecka, D. O., Kuzak, M., Dobrucki, J. W. Conditions for using FRAP as a quantitative technique--influence of the bleaching protocol. Cytometry A. 77 (4), 366-370 (2010).
  59. Lavelin, I., et al. Differential effect of actomyosin relaxation on the dynamic properties of focal adhesion proteins. PLoS One. 8 (9), e73549 (2013).
  60. Foote, H. P., Sumigray, K. D., Lechler, T. FRAP analysis reveals stabilization of adhesion structures in the epidermis compared to cultured keratinocytes. PLoS One. 8 (8), e71491 (2013).
  61. Zal, T., Gascoigne, N. R. Photobleaching-corrected FRET efficiency imaging of live cells. Biophysical Journal. 86 (6), 3923-3939 (2004).
  62. Hodgson, L., Shen, F., Hahn, K. Biosensors for characterizing the dynamics of rho family GTPases in living cells. Current Protocols in Cell Biology. 46 (1), (2010).
  63. Day, R. N. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods. 66 (2), 200-207 (2014).
  64. Rapsomaniki, M. A., et al. easyFRAP: an interactive, easy-to-use tool for qualitative and quantitative analysis of FRAP data. Bioinformatics. 28 (13), 1800-1801 (2012).
  65. Zamir, E., et al. Molecular diversity of cell-matrix adhesions. Journal of cell science. 112 (11), 1655-1669 (1999).
  66. Bakolitsa, C., et al. Structural basis for vinculin activation at sites of cell adhesion. Nature. 430 (6999), 583-586 (2004).
  67. De Los Santos, C., Chang, C. W., Mycek, M. A., Cardullo, R. FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Molecular Reproduction and Development. 82 (7-8), 587-604 (2015).
  68. Periasamy, A., Wallrabe, H., Chen, Y., Barroso, M. Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy. Methods in Cell Biology. 89, 569-598 (2008).
  69. Stehbens, S., Pemble, H., Murrow, L., Wittmann, T. Imaging intracellular protein dynamics by spinning disk confocal microscopy. Methods in Enzymology. , 293-313 (2012).
  70. Zeug, A., Woehler, A., Neher, E., Ponimaskin, E. G. Quantitative intensity-based FRET approaches--a comparative snapshot. Biophysical Journal. 103 (9), 1821-1827 (2012).
  71. Ehrlicher, A. J., Nakamura, F., Hartwig, J. H., Weitz, D. A., Stossel, T. P. Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A. Nature. 478 (7368), 260-263 (2011).
  72. Guo, B., Guilford, W. H. Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction. Proceedings of the National Academy of Sciences. 103 (26), 9844-9849 (2006).
  73. Lee, C. Y., et al. Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds. Proceedings of the National Academy of Sciences. 110 (13), 5022-5027 (2013).
  74. Cost, A. L., Ringer, P., Chrostek-Grashoff, A., Grashoff, C. How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors. Cellular and Molecular Bioengineering. 8 (1), 96-105 (2015).
  75. Bertocchi, C., et al. Nanoscale architecture of cadherin-based cell adhesions. Nature Cell Biology. 19 (1), 28-37 (2017).
  76. Case, L. B., et al. Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nature Cell Biology. 17 (7), 880-892 (2015).
  77. Chen, H., Cohen, D. M., Choudhury, D. M., Kioka, N., Craig, S. W. Spatial distribution and functional significance of activated vinculin in living cells. The Journal of Cell Biology. 169 (3), 459-470 (2005).
  78. Kim, T. J., et al. Dynamic visualization of alpha-catenin reveals rapid, reversible conformation switching between tension states. Current Biology. 25 (2), 218-224 (2015).
  79. Yao, M., et al. Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation. Scientific reports. 4, 4610 (2014).

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