CELL SCAFFOLDING: This composite super-resolution microscopy image shows actin (on a scale from blue to magenta/red, for earlier to later time points of imaging) in a living pig kidney cell.© TALLEY LAMBERT/SCIENCE SOURCE

It is well known that some human diseases are related to changes in mechanical properties of tissues. In patients suffering from arteriosclerosis, the arteries lose some of their elasticity and become thicker and stiffer. In liver or lung fibrosis, excessive fibrous connective tissue has a similar hardening effect on those organs. And patients with aneurysms have balloon-like bulges in their blood vessels that, if left untreated, can expand under pressure until they burst.

Of course, mechanical properties and forces aren’t just important in disease, but in health as well. Almost all living cells and tissues exert and experience physical forces that influence biological function. The magnitudes of those forces vary among different cell and tissue types, as do cells’ sensitivities to changes in magnitudes, frequencies, and durations of the forces. Touch, hearing, proprioception, and certain other senses are well-known examples of specialized force sensors. But force detection and sensing are not limited to these special cases; rather, they are shared by all living cells in all tissues and organs.  The underlying mechanisms of force generation and detection are not well understood, however, leaving many open questions about force dynamics; the distance over which a force exerts its impact; and how cells convert mechanical signals into biochemical signals and changes in gene expression.

Applied forces concentrate at actin stress fibers and propagate over longer distances in the cytoplasm.

In recent years, biologists have begun to uncover the molecular players that mediate force sensation and propagation at the cellular level, and they’re collecting clues as to how mechanical stimuli influence biological function. Such work could pave the way for a deeper understanding of how physical forces influence biological functions in embryonic development, normal physiology, and complex diseases. Translating this research into the clinic may help create new ways of treating certain diseases using mechanics- and engineering-based tools.

Generating and sensing physical forces

In the early 1980s, Donald Ingber of Harvard University and Mina Bissell of Lawrence Berkeley National Laboratory independently proposed that the extracellular matrix (ECM) that surrounds and supports cells could affect cell/tissue organization and function as well as gene expression. But at the time, experimental evidence was scarce, and the mechanism was not clear.

Over the next several years, researchers began to report that cells experience mechanical stimuli via cell surface receptors. In 1986, MIT’s Richard Hynes and colleagues cloned one such receptor, which they called integrin, that turned out to be the primary transmembrane molecule that mediates cell adhesion to the ECM. Most researchers at the time presumed that integrins (there are now 24 known subtypes) primarily functioned in chemical signaling. But in the early 1990s, while working as a postdoc in Ingber’s lab, I provided the first experimental evidence that integrins, and associated intracellular protein complexes known as focal adhesions, mediate mechanical force transmission to the cytoskeleton.1

Using Arg-Gly-Asp peptide–coated magnetic beads that clustered integrins and induced the formation of focal adhesions beneath the beads on the inner surface of the cell membrane, I applied measured amounts of stress to the surfaces of living cells and found that cell stiffness increased with the magnitude of the forces. By disrupting cytoskeletal filaments such as filamentous actin (F-actin), I could abolish the transmission of force into the cell. This study changed the scientific community’s view of integrins, which are now recognized as key molecular force sensors.

Subsequently, using a laser tweezer, Mike Sheetz’s lab at Columbia University independently confirmed that focal adhesions transmit external forces into the cell.2 In addition, two other groups—those of Yu-Li Wang, now at Carnegie Mellon University, and Benny Geiger at the Weizmann Institute of Science—found that focal adhesions also transmit forces generated inside the cell by powerful molecular motors such as myosin II, which binds to F-actin in the cytoskeleton, out into the ECM. This research showed that focal adhesion–mediated transmission of mechanical signals is bidirectional.3,4

Another class of mechanosensors used by the cell are stretch-activated ion channels on the plasma membrane. Over the past decade, Martin Chalfie of Columbia University and other labs have worked on several candidate channels that, in response to stretch, open to allow ions to flow into the cytoplasm. The result is mechanoelectrical transduction—analogous to a neuronal action potential—that can activate enzymes or proteins in the cytoplasm to affect intracellular activities, or even influence gene expression. The detailed mechanisms of this are still unclear, however.

More recently, many labs have searched for intracellular mechanosensors downstream of integrins at focal adhesions. For example, Sheetz and his colleagues have found evidence of a mechanosensing role for the focal adhesion protein talin,5 while Martin Schwartz’s group at Yale has demonstrated a similar role for the focal adhesion protein vinculin.6 But a fundamental question remains: How does a living cell integrate forces sensed by different mechanosensors and respond in a coherent manner?

Ingber first proposed the model of cellular tensegrity (tensional integrity) in the early 1980s, emphasizing the importance of tension among cytoskeletal structures in the cell’s ability to configure a holistic sense of the forces at play. If there is tension in the cytoskeletal filaments, then a local change in one part of the filament is quickly transmitted to all connected parts.

Experimental evidence now supports this model. In the early 2000s, collaborating with the Ingber lab, my group used chemicals to either contract or relax the cytoskeleton of cultured human smooth muscle cells, which systematically varied the cells’ inherent tension, or “prestress,” without changing their shape. We found that the cell stiffness changed accordingly. In other words, cell stiffness is determined by the cytoskeleton’s tension.7 Moreover, after disrupting the cell’s microtubules with specific drugs, we found that the force transmitted out of the cell increased.8 This suggests that microtubules, which are relatively stiff components of the cytoskeleton, are essentially balancing some of the cell’s endogenous prestress, and when they are disturbed, that tension is transferred to the ECM.

Mechanotransduction at a distance

FOLLOWING THE FORCE
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© THOM GRAVES
For many years, the prevailing view in the field of mechanotransduction was that forces transmit only a short distance in living cells, and thus a local force can only exert significant effects at the periphery of the cell. From a materials science point of view, this limited reach would be reasonable if the material was homogeneous and isotropic—in other words, there is no difference in its stiffness or other mechanical properties when the force direction is changed. In this case, a local stress would rapidly decay as the distance increases. However, the cytoplasm of a living cell is neither homogeneous nor isotropic; it is heterogeneous and anisotropic, meaning that the material’s mechanical properties do depend on the direction of force. Importantly, there are stiff, prestressed actin bundles (also called stress fibers) in the cell. Applied forces concentrate at these actin bundles and propagate over longer distances in the cytoplasm.

Since the early 2000s, my group has demonstrated that forces do propagate across relatively vast cellular distances—on the order of tens of micrometers—in living cells, and that this long-distance signal is dependent on the inherent tension in the cytoskeleton.9,10 Just as a violin string can only ring with the correct resonance and sound the right note if it has proper tension, when the prestressed actin bundles are disrupted, force propagation becomes short-range (acting over only a few μms). The higher the tension, the farther the force will be propagated.

Most recently, we have found that specific signaling molecules—in particular, the tyrosine kinase Src and the GTPase Rac1—can be activated at distances of more than 60 μm away from the site of the local force application via integrins at the cell membrane.11,12 Importantly, this activation is fast, taking less than 300 ms from force application to the activation of Src and Rac1, making mechanotransduction much faster than the 10 to 20 seconds it takes a soluble growth factor–induced signal to travel over the same distance.11

Mechanotransduction in the nucleus

In contrast to the emerging picture of force propagation in the cytoplasm, we know very little about nuclear mechanotransduction. The nuclear envelope is physically tethered to the actin cytoskeleton via the LINC (linker of nucleoskeleton and cytoskeleton) complex. In the late 1990s, Ingber and colleagues published the first evidence that force-carrying connections reach from the plasma membrane to the nucleus, perhaps playing a role in the regulation of gene expression. Using a micropipette coated with fibronectin to attach to the cell surface, the researchers pulled on the cell and found that the nuclear envelope distorted.13 Later, my group revealed a force-induced protein-protein dissociation inside the nucleus.14 This change was dependent on both a properly stressed cytoskeleton and an intact nuclear lamina, a layer of intermediate filament proteins called lamins that line the inside of the bilamellar nuclear envelope. Subsequent research has demonstrated that lamins are mechanosensors critical for extracellular matrix stiffness–directed differentiation,15 and for regulation of transcription factors.16

Mechanomedicine is poised to emerge as an exciting branch of medicine that uses mechanics- and engineering-based prin­ciples and technologies for precision diagnostics and effective therapeutics.

To more directly investigate whether a physiologically relevant force can directly deform chromatin structure in a living cell to regulate specific gene expression, my group recently teamed up with Andy Belmont’s lab at the University of Illinois at Urbana-Champaign. Belmont’s team used bacterial artificial chromosomes to insert multiple green fluorescent proteins and the gene for dihydrofolate reductase (DHFR), an essential enzyme for the synthesis of thymine, into the same chromatin domain in Chinese hamster ovary (CHO) cells. My lab applied a local force to those modified cells via integrins. Sure enough, we measured an uptick in DHFR transcription in response to the applied force. Conversely, disrupting cytoskeletal tension, or the force transmission pathways from the cell surface to lamins and to the nuclear structural proteins that connect to the chromatin, abolished force-induced DHFR expression.17

This work provides the first evidence that externally applied forces can stretch chromatin and promote gene expression. As expected with physical force–mediated processes, the response was rapid; we were able to quantify DHFR transcription upregulation within 15 seconds after force application. Interestingly, force-triggered transcription is sensitive to the angle and direction of force relative to the actin bundles: the higher the stress angle, the greater the transcription. Because endogenous forces are constantly generated inside a living cell, these findings suggest that gene expression might be incessantly regulated by physical forces via this direct structural pathway and the indirect pathways of matrix rigidity–dependent nuclear translocation of certain factors, such as yes-associated protein (YAP) and TWIST1. More research is needed to understand the relative contributions of each of these mechanisms in determining overall gene expression levels in any given cell.

From mechanobiology to mechanomedicine

TUMOR POTENTIAL: When melanoma cells were injected into embryonic zebrafish pericardium (red cells indicated by white arrow), the membrane that surrounds the heart, the cells travel to the tail in just one hour post injection (hpi). Differentiated melanoma cells cultured on stiff substrates (Cont) were less efficient than undifferentiated tumor-repopulating cells (TRCs) cultured on a soft matrix at establishing new tumors.SCI REP, 6:19304, 2016.Mechanobiology is becoming increasingly relevant to stem cell biology. For many years, researchers have cultured cells on top of rigid plastic or glass coverslips. However, it is well known that various types of living cells in soft tissues attach to matrices of varying stiffness. Tuning the substrate stiffness in a controlled manner, Yu-Li Wang and colleagues demonstrated that the size and dynamics of focal adhesion complexes as well as the migration of living cells are dramatically altered by substrates of different rigidity.3 Later, Adam Engler of the University of California, San Diego, and Dennis Discher of the University of Pennsylvania reported that mesenchymal stem cell differentiation can be directed by extracellular matrix stiffness.18 And my lab has demonstrated that applying local force can spur the differentiation of a single embryonic stem cell.19 Physical forces also appear critical in the patterning and organization of germ layers during early mammalian embryonic development.

Researchers are also considering mechanical forces in cancer research. For example, despite decades of study, it is still unclear why only a few cancer cells out of thousands are able to metastasize. The answer may lie in the tumor’s physical environment. Scientists have shown that, in primary tumors, high mechanical tension and matrix stiffening are important in cancer progression, and high fluid/solid pressure in the primary tumor often accompanies tumor growth. However, secondary metastatic sites of tumors appear to be softer—suggesting that they have lower forces—than the surrounding normal tissues.

See “The Forces of Cancer,” The Scientist, April 2016.

Using a 3-D soft matrix made of fibrin gels, my group has managed to isolate and grow cells that are highly tumorigenic and malignant, called tumor-repopulating cells (TRCs), from several murine or human cancer cell lines.20 Interestingly, melanoma TRCs cultured in soft 3-D matrices are less differentiated—and thus more tumorigenic—than melanoma cells grown in stiff matrices or on rigid plastic, suggesting that low matrix stiffness drives TRC growth.21 These soft-cultured melanoma TRCs also move out of the blood vessels in zebrafish to secondary sites more efficiently than more-differentiated melanoma cells cultured on stiff substrates.22 These findings suggest a common thread in metastatic colonization of malignant tumors: a few tumorigenic cells are able to survive, metastasize, and grow at the secondary sites of soft matrices because these cells are undifferentiated.

And the role of physical forces in biology is by no means limited to stem cells and cancer biology. Across the life sciences, researchers are continuing to draw on insights into mechanobiology to better understand and treat a wide variety of conditions. Human organs-on-a-chip for novel drug screening, shear force–activated cleaning of thrombosis, mechanically tuned hydrogels for bone formation, and tumor cell membrane–derived therapeutic microparticles for reversing cancer drug resistance are just a few recent examples of clinical applications of mechanobiology-based technologies. Mechanobiology-based medicine (mechanomedicine) is poised to emerge as an exciting branch of medicine that uses mechanics- and engineering-based principles and technologies for precision diagnostics and effective therapeutics of diseases that are beyond the reach of existing toolboxes. 

Ning Wang is the Leonard C. and Mary Lou Hoeft Professor in the Department of Mechanical Science and Engineering at the University of Illinois at Urbana-Champaign and adjunct professor at Huazhong University of Science and Technology.

References

  1. N. Wang et al., “Mechanotransduction across the cell surface and through the cytoskeleton,” Science, 260:1124-27, 1993.
  2. D. Choquet et al., “Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages,” Cell, 88:39-48, 1997.
  3. R.J. Pelham Jr., Y.L. Wang, “Cell locomotion and focal adhesions are regulated by substrate flexibility,” PNAS, 94:13661-65, 1997.
  4. N.Q. Balaban et al., “Force and focal adhesion assembly: A close relationship studied using elastic micropatterned substrates,” Nat Cell Biol, 3:466-72, 2001.
  5. A. del Rio et al., “Stretching single talin rod molecules activates vinculin binding,” Science, 323:638-41, 2009.
  6. C. Grashoff et al., “Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics,” Nature, 466:263-66, 2010.
  7. N. Wang et al., “Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells,” Am J Physiol Cell Physiol, 282:C606-C616, 2002.
  8. N. Wang et al., “Mechanical behavior in living cells consistent with the tensegrity model,” PNAS, 98:7765-70, 2001.
  9. S. Hu et al., “Intracellular stress tomography reveals stress focusing and structural anisotropy in the cytoskeleton of living cells,” Am J Physiol Cell Physiol, 285:C1082-C1090, 2003.
  10. S. Hu et al., “Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device,” Am J Physiol Cell Physiol, 287:C1184-C1191, 2004.
  11. S. Na et al., “Rapid signal transduction in living cells is a unique feature of mechanotransduction,” PNAS, 105:6626-6631, 2008.
  12. Y.C. Poh et al., “Rapid activation of Rac GTPase in living cells by force is independent of Src,” PLOS ONE, 4:e7886, 2009.
  13. A.J. Maniotis et al., “Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure,” PNAS, 94:849-54, 1997.
  14. Y.C. Poh et al., “Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells,” Nat Commun, 3:866, 2012.
  15. J. Swift et al., “Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation,” Science, 341:1240104, 2013.
  16. C.Y. Ho et al., “Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics,” Nature, 497:507-11, 2013.
  17. A. Tajik et al., “Transcription upregulation via force-induced direct stretching of chromatin,” Nat Mater, 15:1287-96, 2016.
  18. A.J. Engler et al., “Matrix elasticity directs stem cell lineage specification,” Cell, 126:677-89, 2006.
  19. F. Chowdhury et al., “Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells,” Nat Mater, 9:82-88, 2010.
  20. J. Liu et al., “Soft fibrin gels promote selection and growth of tumorigenic cells,” Nat Mater, 11:734-41, 2012.
  21. Y. Tan et al., “Matrix softness regulates plasticity of tumour-repopulating cells via H3K9 demethylation and Sox2 expression,” Nat Commun, 5:4619, 2014.
  22. J. Chen, “Efficient extravasation of tumor-repopulating cells depends on cell deformability,” Sci Rep, 6:19304, 2016