Cell-generated Mechanical Forces Research Articles

Harnessing atomic force microscopy-based single-cell analysis to advance physical oncology.

Single-cell analysis is an emerging and promising frontier in the field of life sciences, which is expected to facilitate the exploration of fundamental laws of physiological and pathological processes. Single-cell analysis allows experimental access to cell-to-cell heterogeneity to reveal the distinctive behaviors of individual cells, offering novel opportunities to dissect the complexity of severe human diseases such as cancers. Among the single-cell analysis tools, atomic force microscopy (AFM) is a powerful and versatile one which is able to nondestructively image the fine topographies and quantitatively measure multiple mechanical properties of single living cancer cells in their native states under aqueous conditions with unprecedented spatiotemporal resolution. Over the past few decades, AFM has been widely utilized to detect the structural and mechanical behaviors of individual cancer cells during the process of tumor formation, invasion, and metastasis, yielding numerous unique insights into tumor pathogenesis from the biomechanical perspective and contributing much to the field of cancer mechanobiology. Here, the achievements of AFM-based analysis of single cancer cells to advance physical oncology are comprehensively summarized, and challenges and future perspectives are also discussed. RESEARCH HIGHLIGHTS: Achievements of AFM in characterizing the structural and mechanical behaviors of single cancer cells are summarized, and future directions are discussed. AFM is not only capable of visualizing cellular fine structures, but can also measure multiple cellular mechanical properties as well as cell-generated mechanical forces. There is still plenty of room for harnessing AFM-based single-cell analysis to advance physical oncology.

Molecular Tension Probes to Quantify Cell-Generated Mechanical Forces.

Living cells generate, sense, and respond to mechanical forces through their interaction with neighboring cells or extracellular matrix, thereby regulating diverse cellular processes such as growth, motility, differentiation, and immune responses. Dysregulation of mechanosensitive signaling pathways is found associated with the development and progression of various diseases such as cancer. Yet, little is known about the mechanisms behind mechano-regulation, largely due to the limited availability of tools to study it at the molecular level. The recent development of molecular tension probes allows measurement of cellular forces exerted by single ligand-receptor interaction, which has helped in revealing the hitherto unknown mechanistic details of various mechanosensitive processes in living cells. Here, we provide an introductory overview of two methods based on molecular tension probes, tension gauge tether (TGT), and molecular tension fluorescence microscopy (MTFM). TGT utilizes the irreversible rupture of double-stranded DNA tether upon application of force in the piconewton (pN) range, whereas MTFM utilizes the reversible extension of molecular springs such as polymer or single-stranded DNA hairpin under applied pN forces. Specifically, the underlying principle of how molecular tension probes measure cell-generated mechanical forces and their applications to mechanosensitive biological processes are described.

The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

Collagen linearization is a hallmark of aggressive tumors and a key pathogenic event that promotes cancer cell invasion and metastasis. Cell-generated mechanical tension has been proposed to contribute to collagen linearization in tumors, but it is unknown whether other mechanisms play prominent roles in this process. Here, we show that the secretome of cancer cells is by itself able to induce collagen linearization independently of cell-generated mechanical forces. Among the tumor cell-secreted factors, we find a key role in this process for the matricellular protein WISP1 (CCN4). Specifically, WISP1 directly binds to type I collagen to promote its linearization invitro (in the absence of cells) and invivo in tumors. Consequently, WISP1-induced type I collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis, without significantly affecting gene expression. Furthermore, higher WISP1 expression in tumors from cancer patients correlates with faster progression to metastatic disease and poor prognosis. Altogether, these findings reveal a conceptually novel mechanism whereby pro-metastatic collagen linearization critically depends on a cancer cell-secreted factor.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

From static to animated: Measuring mechanical forces in tissues.

Cells are physical objects that exert mechanical forces on their surroundings as they migrate and take their places within tissues. New techniques are now poised to enable the measurement of cell-generated mechanical forces in intact tissues in vivo, which will illuminate the secret dynamic lives of cells and change our current perception of cell biology.

On Buckling Morphogenesis.

Cell-generated mechanical forces drive many of the tissue movements and rearrangements that are required to transform simple populations of cells into the complex three-dimensional geometries of mature organs. However, mechanical forces do not need to arise from active cellular movements. Recent studies have illuminated the roles of passive forces that result from mechanical instabilities between epithelial tissues and their surroundings. These mechanical instabilities cause essentially one-dimensional epithelial tubes and two-dimensional epithelial sheets to buckle or wrinkle into complex topologies containing loops, folds, and undulations in organs as diverse as the brain, the intestine, and the lung. Here, I highlight examples of buckling and wrinkling morphogenesis, and suggest that this morphogenetic mechanism may be broadly responsible for sculpting organ form.

Erratum: Corrigendum: Quantifying cell-generated mechanical forces within living embryonic tissues

Nat. Methods 11, 183–189 (2014); published online 8 December 2013; corrected after print 5 February 2014 In the version of this article initially published, the current affiliation of author Ralph Sperling was not included. His current affiliation is the Fraunhofer ICT-IMM, Mainz, Germany. The errorhas been corrected in the HTML and PDF versions of the article.

Quantifying cell-generated mechanical forces within living embryonic tissues

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. However, little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. Here we present a method to quantify cell-generated mechanical stresses that are exerted locally within living embryonic tissues using fluorescent, cell-sized, oil microdroplets with defined mechanical properties and coated with surface integrin or cadherin receptor ligands. After introducing a droplet between cells in a tissue, local stresses are determined from the droplet shape deformations, which are obtained via fluorescence microscopy and computerized image analysis. Using this method, we quantify the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates and confirm that these stresses (3.4 nN/µm2) are dependent on myosin II activity and more than two-fold larger than the stresses generated by cells of embryonic tooth mesenchyme when analyzed within similar cultured aggregates or in developing whole mouse mandibles.

Traction in smooth muscle cells varies with cell spreading

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell–substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.