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Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers

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Abstract

Introduction

In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an ‘ultrasound tweezers’-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed “actinin-sstFRET,” to monitor in situ single-cell allostasis in response to transient stimulation in real time.

Methods

The ultrasound tweezers utilize 1 Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis.

Results

By subjecting a 1 Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation.

Conclusions

By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

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References

  1. Alhussein, G., A. Shanti, I. A. Farhat, S. B. Timraz, N. S. Alwahab, Y. E. Pearson, M. N. Martin, N. Christoforou, and J. C. Teo. A spatiotemporal characterization method for the dynamic cytoskeleton. Cytoskeleton 73:221–232, 2016.

    Article  Google Scholar 

  2. Allingham, J. S., R. Smith, and I. Rayment. The structural basis of blebbistatin inhibition and specificity for myosin II. Nat. Struct. Mol. Biol. 12:378, 2005.

    Article  Google Scholar 

  3. Balasubramanian, L., C.-M. Lo, J. S. Sham, and K.-P. Yip. Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction. Am. J. Physiol. Cell Physiol. 304:C382–C391, 2013.

    Article  Google Scholar 

  4. Beningo, K. A., K. Hamao, M. Dembo, Y.-L. Wang, and H. Hosoya. Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase. Arch. Biochem. Biophys. 456:224–231, 2006.

    Article  Google Scholar 

  5. Binnewies, M., E. W. Roberts, K. Kersten, V. Chan, D. F. Fearon, M. Merad, L. M. Coussens, D. I. Gabrilovich, S. Ostrand-Rosenberg, and C. C. Hedrick. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat. Med. 24:541–550, 2018.

    Article  Google Scholar 

  6. Blanchoin, L., R. Boujemaa-Paterski, C. Sykes, and J. Plastino. Actin dynamics, architecture, and mechanics in cell motility. Physiol. Rev. 94:235–263, 2014.

    Article  Google Scholar 

  7. Bonakdar, N., R. Gerum, M. Kuhn, M. Spörrer, A. Lippert, W. Schneider, K. E. Aifantis, and B. Fabry. Mechanical plasticity of cells. Nat. Mater. 15:1090, 2016.

    Article  Google Scholar 

  8. Broussard, J. A., B. Rappaz, D. J. Webb, and C. M. Brown. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nat. Protoc. 8:265, 2013.

    Article  Google Scholar 

  9. Brown, R., R. Prajapati, D. McGrouther, I. Yannas, and M. Eastwood. Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates. J. Cell. Physiol. 175:323–332, 1998.

    Article  Google Scholar 

  10. Chen, W., S. G. Allen, W. Qian, Z. Peng, S. Han, X. Li, Y. Sun, C. Fournier, L. Bao, and R. H. Lam. Biophysical phenotyping and modulation of ALDH + inflammatory breast cancer stem-like cells. Small 15:1802891, 2019.

    Article  Google Scholar 

  11. Chen, D., Y. Sun, C. X. Deng, and J. Fu. Improving survival of disassociated human embryonic stem cells by mechanical stimulation using acoustic tweezing cytometry. Biophys. J. 108:1315–1317, 2015.

    Article  Google Scholar 

  12. Chen, D., Y. Sun, M. S. Gudur, Y.-S. Hsiao, Z. Wu, J. Fu, and C. X. Deng. Two-bubble acoustic tweezing cytometry for biomechanical probing and stimulation of cells. Biophys. J. 108:32–42, 2015.

    Article  Google Scholar 

  13. Chowdhury, F., S. Na, D. Li, Y.-C. Poh, T. S. Tanaka, F. Wang, and N. Wang. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. Nat. Mater. 9:82, 2010.

    Article  Google Scholar 

  14. Chrzanowska-Wodnicka, M., and K. Burridge. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133:1403–1415, 1996.

    Article  Google Scholar 

  15. Collinsworth, A. M., C. E. Torgan, S. N. Nagda, R. J. Rajalingam, W. E. Kraus, and G. A. Truskey. Orientation and length of mammalian skeletal myocytes in response to a unidirectional stretch. Cell Tissue Res. 302:243–251, 2000.

    Article  Google Scholar 

  16. Coste, B., B. Xiao, J. S. Santos, R. Syeda, J. Grandl, K. S. Spencer, S. E. Kim, M. Schmidt, J. Mathur, and A. E. Dubin. Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483:176, 2012.

    Article  Google Scholar 

  17. Dietzel, I., and U. Heinemann. Dynamic variations of the brain cell microenvironment in relation to neuronal hyperactivity. Ann. N. Y. Acad. Sci. 481:72–84, 1986.

    Article  Google Scholar 

  18. Du Roure, O., A. Saez, A. Buguin, R. H. Austin, P. Chavrier, P. Siberzan, and B. Ladoux. Force mapping in epithelial cell migration. Proc. Natl. Acad. Sci. USA 102:2390–2395, 2005.

    Article  Google Scholar 

  19. Fan, Z., Y. Sun, D. Chen, D. Tay, W. Chen, C. X. Deng, and J. Fu. Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility. Sci. Rep. 3:2176, 2013.

    Article  Google Scholar 

  20. Fan, Z., X. Xue, R. Perera, S. Nasr Esfahani, A. A. Exner, J. Fu, and C. X. Deng. Acoustic actuation of integrin-bound microbubbles for mechanical phenotyping during differentiation and morphogenesis of human embryonic stem cells. Small 14:1803137, 2018.

    Article  Google Scholar 

  21. Faust, U., N. Hampe, W. Rubner, N. Kirchgessner, S. Safran, B. Hoffmann, and R. Merkel. Cyclic stress at mHz frequencies aligns fibroblasts in direction of zero strain. PLoS ONE 6:e28963, 2011.

    Article  Google Scholar 

  22. Fu, J., Y.-K. Wang, M. T. Yang, R. A. Desai, X. Yu, Z. Liu, and C. S. Chen. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7:733, 2010.

    Article  Google Scholar 

  23. Gattazzo, F., A. Urciuolo, and P. Bonaldo. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim. Biophys. Acta 2506–2519:2014, 1840.

    Google Scholar 

  24. Ghigo, A., M. Laffargue, M. Li, and E. Hirsch. PI3 K and calcium signaling in cardiovascular disease. Circ. Res. 121:282–292, 2017.

    Article  Google Scholar 

  25. Goldstein, D. S., and B. McEwen. Allostasis, homeostats, and the nature of stress. Stress 5:55–58, 2002.

    Article  Google Scholar 

  26. Gunst, S. J., and W. Zhang. Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction. Am. J. Physiol. Cell Physiol. 295:C576–C587, 2008.

    Article  Google Scholar 

  27. Hayakawa, K., H. Tatsumi, and M. Sokabe. Actin stress fibers transmit and focus force to activate mechanosensitive channels. J. Cell Sci. 121:496–503, 2008.

    Article  Google Scholar 

  28. Heureaux, J., D. Chen, V. L. Murray, C. X. Deng, and A. P. Liu. Activation of a bacterial mechanosensitive channel in mammalian cells by cytoskeletal stress. Cell. Mol. Bioeng. 7:307–319, 2014.

    Article  Google Scholar 

  29. Hoffman, B. D., C. Grashoff, and M. A. Schwartz. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475:316, 2011.

    Article  Google Scholar 

  30. Hsu, H.-J., C.-F. Lee, and R. Kaunas. A dynamic stochastic model of frequency-dependent stress fiber alignment induced by cyclic stretch. PLoS ONE 4:e4853, 2009.

    Article  Google Scholar 

  31. Humphrey, J. D., E. R. Dufresne, and M. A. Schwartz. Mechanotransduction and extracellular matrix homeostasis. Nat. Rev. Mol. Cell Biol. 15:802, 2014.

    Article  Google Scholar 

  32. Kaksonen, M., C. P. Toret, and D. G. Drubin. A modular design for the clathrin-and actin-mediated endocytosis machinery. Cell 123:305–320, 2005.

    Article  Google Scholar 

  33. Kato, S., T. Osa, and T. Ogasawara. Kinetic model for isometric contraction in smooth muscle on the basis of myosin phosphorylation hypothesis. Biophys. J. 46:35, 1984.

    Article  Google Scholar 

  34. Kaunas, R., P. Nguyen, S. Usami, and S. Chien. Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc. Natl. Acad. Sci. USA 102:15895–15900, 2005.

    Article  Google Scholar 

  35. Kim, T.-J., C. Joo, J. Seong, R. Vafabakhsh, E. L. Botvinick, M. W. Berns, A. E. Palmer, N. Wang, T. Ha, and E. Jakobsson. Distinct mechanisms regulating mechanical force-induced Ca2+ signals at the plasma membrane and the ER in human MSCs. Elife 4:e04876, 2015.

    Article  Google Scholar 

  36. Kranenburg, O., M. Poland, M. Gebbink, L. Oomen, and W. H. Moolenaar. Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA. J. Cell Sci. 110:2417–2427, 1997.

    Google Scholar 

  37. Labernadie, A., T. Kato, A. Brugués, X. Serra-Picamal, S. Derzsi, E. Arwert, A. Weston, V. González-Tarragó, A. Elosegui-Artola, and L. Albertazzi. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat. Cell Biol. 19:224, 2017.

    Article  Google Scholar 

  38. Lam, R. H., Y. Sun, W. Chen, and J. Fu. Elastomeric microposts integrated into microfluidics for flow-mediated endothelial mechanotransduction analysis. Lab Chip 12:1865–1873, 2012.

    Article  Google Scholar 

  39. Livne, A., E. Bouchbinder, and B. Geiger. Cell reorientation under cyclic stretching. Nat. Commun. 5:3938, 2014.

    Article  Google Scholar 

  40. Mann, J. M., R. H. Lam, S. Weng, Y. Sun, and J. Fu. A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response. Lab Chip 12:731–740, 2012.

    Article  Google Scholar 

  41. McEwen, B. S. Stress, adaptation, and disease: allostasis and allostatic load. Ann. N. Y. Acad. Sci. 840:33–44, 1998.

    Article  Google Scholar 

  42. McEwen, B. S., and J. C. Wingfield. The concept of allostasis in biology and biomedicine. Horm. Behav. 43:2–15, 2003.

    Article  Google Scholar 

  43. Meng, F., and F. Sachs. Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor. J. Cell Sci. 124:261–269, 2011.

    Article  Google Scholar 

  44. Milewicz, D. M., K. M. Trybus, D.-C. Guo, H. L. Sweeney, E. Regalado, K. Kamm, and J. T. Stull. Altered smooth muscle cell force generation as a driver of thoracic aortic aneurysms and dissections. Arterioscler. Thromb. Vasc. Biol. 37:26–34, 2017.

    Article  Google Scholar 

  45. Murrell, M., P. W. Oakes, M. Lenz, and M. L. Gardel. Forcing cells into shape: the mechanics of actomyosin contractility. Nat. Rev. Mol. Cell Biol. 16:486, 2015.

    Article  Google Scholar 

  46. Palmer, A. E., and R. Y. Tsien. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1:1057, 2006.

    Article  Google Scholar 

  47. Pasterkamp, G., D. P. de Kleijn, and C. Borst. Arterial remodeling in atherosclerosis, restenosis and after alteration of blood flow: potential mechanisms and clinical implications. Cardiovasc. Res. 45:843–852, 2000.

    Article  Google Scholar 

  48. Porter, K. E., and K. Riches. The vascular smooth muscle cell: a therapeutic target in type 2 diabetes? Clin. Sci. 125:167–182, 2013.

    Article  Google Scholar 

  49. Pyle, A. L., and P. P. Young. Atheromas feel the pressure: biomechanical stress and atherosclerosis. Am. J. Pathol. 177:4–9, 2010.

    Article  Google Scholar 

  50. Qian, W., L. Gong, X. Cui, Z. Zhang, A. Bajpai, C. Liu, A. B. Castillo, J. C. Teo, and W. Chen. Nanotopographic regulation of human mesenchymal stem cell osteogenesis. ACS Appl. Mater. Interfaces. 9:41794–41806, 2017.

    Article  Google Scholar 

  51. Raftopoulou, M., and A. Hall. Cell migration: Rho GTPases lead the way. Dev. Biol. 265:23–32, 2004.

    Article  Google Scholar 

  52. Rahimzadeh, J., F. Meng, F. Sachs, J. Wang, D. Verma, and S. Z. Hua. Real-time observation of flow-induced cytoskeletal stress in living cells. Am. J. Physiol. Cell Physiol. 301:C646–C652, 2011.

    Article  Google Scholar 

  53. Retailleau, K., F. Duprat, M. Arhatte, S. S. Ranade, R. Peyronnet, J. R. Martins, M. Jodar, C. Moro, S. Offermanns, and Y. Feng. Piezo1 in smooth muscle cells is involved in hypertension-dependent arterial remodeling. Cell Rep. 13:1161–1171, 2015.

    Article  Google Scholar 

  54. Riches, K., P. Warburton, D. J. O’Regan, N. A. Turner, and K. E. Porter. Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: possible role of differential RhoA activity. Cardiovasc. Revasc. Med. 15:141–148, 2014.

    Article  Google Scholar 

  55. Ritsma, L., S. I. Ellenbroek, A. Zomer, H. J. Snippert, F. J. de Sauvage, B. D. Simons, H. Clevers, and J. van Rheenen. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507:362, 2014.

    Article  Google Scholar 

  56. Schwartz, M. W., R. J. Seeley, M. H. Tschöp, S. C. Woods, G. J. Morton, M. G. Myers, and D. D’alessio. Cooperation between brain and islet in glucose homeostasis and diabetes. Nature 503:59, 2013.

    Article  Google Scholar 

  57. Shao, Y., J. M. Mann, W. Chen, and J. Fu. Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction. Integr. Biol. 6:300–311, 2014.

    Article  Google Scholar 

  58. Shyu, K.-G. Cellular and molecular effects of mechanical stretch on vascular cells and cardiac myocytes. Clin. Sci. 116:377–389, 2009.

    Article  Google Scholar 

  59. Sukharev, S., M. Betanzos, C.-S. Chiang, and H. R. Guy. The gating mechanism of the large mechanosensitive channel MscL. Nature 409:720, 2001.

    Article  Google Scholar 

  60. Sun, Z., S. S. Guo, and R. Fässler. Integrin-mediated mechanotransduction. J. Cell Biol. 215:445–456, 2016.

    Article  Google Scholar 

  61. Topal, T., X. Hong, X. Xue, Z. Fan, N. Kanetkar, J. T. Nguyen, J. Fu, C. X. Deng, and P. H. Krebsbach. Acoustic tweezing cytometry induces rapid initiation of human embryonic stem cell differentiation. Sci. Rep. 8:12977, 2018.

    Article  Google Scholar 

  62. Waddingham, M. T., A. J. Edgley, H. Tsuchimochi, D. J. Kelly, M. Shirai, and J. T. Pearson. Contractile apparatus dysfunction early in the pathophysiology of diabetic cardiomyopathy. World J. Diabetes 6:943, 2015.

    Article  Google Scholar 

  63. Wang, Y., E. L. Botvinick, Y. Zhao, M. W. Berns, S. Usami, R. Y. Tsien, and S. Chien. Visualizing the mechanical activation of Src. Nature 434:1040, 2005.

    Article  Google Scholar 

  64. Wang, Y., J. Y.-J. Shyy, and S. Chien. Fluorescence proteins, live-cell imaging, and mechanobiology: seeing is believing. Annu. Rev. Biomed. Eng. 10:1–38, 2008.

    Article  Google Scholar 

  65. Wang, Y., and N. Wang. FRET and mechanobiology. Integr. Biol. 1:565–573, 2009.

    Article  Google Scholar 

  66. Webster, K. D., W. P. Ng, and D. A. Fletcher. Tensional homeostasis in single fibroblasts. Biophys. J. 107:146–155, 2014.

    Article  Google Scholar 

  67. Weng, S., Y. Shao, W. Chen, and J. Fu. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis. Nat. Mater. 15:961, 2016.

    Article  Google Scholar 

  68. Xue, X., X. Hong, Z. Li, C. X. Deng, and J. Fu. Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation. Biomaterials 134:22–30, 2017.

    Article  Google Scholar 

  69. Xue, X., Y. Sun, A. M. Resto-Irizarry, Y. Yuan, K. M. A. Yong, Y. Zheng, S. Weng, Y. Shao, Y. Chai, and L. Studer. Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nat. Mater. 17:633–641, 2018.

    Article  Google Scholar 

  70. Yang, M. T., J. Fu, Y.-K. Wang, R. A. Desai, and C. S. Chen. Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity. Nat. Protoc. 6:187, 2011.

    Article  Google Scholar 

  71. Yang, M. T., D. H. Reich, and C. S. Chen. Measurement and analysis of traction force dynamics in response to vasoactive agonists. Integr. Biol. 3:663–674, 2011.

    Article  Google Scholar 

  72. Zhang, Y., A. Gordon, W. Qian, and W. Chen. Engineering nanoscale stem cell niche: Direct stem cell behavior at cell–matrix interface. Adv. Healthc. Mater. 4:1900–1914, 2015.

    Article  Google Scholar 

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Acknowledgments

We acknowledge financial support from the Department of Mechanical and Aerospace Engineering at New York University, the American Heart Association Scientist Development Grant (16SDG31020038), the National Science Foundation (CBET 1701322), and the National Institute of Health (R21EB025406).

Conflict of interest

Weiyi Qian and Weiqiang Chen declare that they have no conflicts of interest.

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This study does not involve any human studies and animal studies by any author in this article.

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  1. Department of Mechanical and Aerospace Engineering, New York University, Brooklyn, NY, 11201, USA

    Weiyi Qian & Weiqiang Chen

  2. Department of Biomedical Engineering, New York University, Brooklyn, NY, 11201, USA

    Weiqiang Chen

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Weiqiang Chen is an Assistant Professor in the Departments of Mechanical and Aerospace Engineering and Biomedical Engineering at New York University. He received his B.S. in Physics from Nanjing University and M.S. degrees from Shanghai Jiao Tong University and Purdue University, both in Electrical Engineering. He earned his Ph.D. in Mechanical Engineering from the University of Michigan in 2014. Research in the Chen’s lab focuses on developing new biomaterials, microfluidics, and organ-on-a-chip systems to address emerging biomedical problems in cell mechanobiology, cancer biology, immune engineering, and stem cell-based regenerative medicine. He is the recipient of the American Heart Association Scientist Development Award, the Lab on a Chip Emerging Investigator Award, the New York University Whitehead Fellowship in Biomedical and Biological Sciences, the Goddard Junior Faculty Fellowship, the Baxter Young Investigator Award, the University of Michigan Richard F. & Eleanor A. Towner Prize for Outstanding PhD Research, and the ProQuest Distinguished Dissertation Award.

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Qian, W., Chen, W. Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers. Cel. Mol. Bioeng. 12, 415–427 (2019). https://doi.org/10.1007/s12195-019-00578-z

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