Co-reporter:Michael P. Murrell;Margaret L. Gardel
PNAS 2012 Volume 109 (Issue 51 ) pp:20820-20825
Publication Date(Web):2012-12-18
DOI:10.1073/pnas.1214753109
Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin)
network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive
and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence
length to <1 μm. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent
of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between
tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin
with a radius of curvature ∼300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility
with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses
can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range
transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling
between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin
to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general
mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle
and nonmuscle cells.
Co-reporter:Melanie Norstrom and Margaret L. Gardel
Soft Matter 2011 vol. 7(Issue 7) pp:3228-3233
Publication Date(Web):24 Jan 2011
DOI:10.1039/C0SM01157F
The material properties of cytoskeletal F-actin networks facilitate a broad range of cellular behaviors, whereby in some situations cell shape is preserved in the presence of force and, at other times, force results in irreversible shape change. These behaviors strongly suggest that F-actin networks can variably deform elastically or viscously. While a significant amount is known about the regulation of the elastic stiffness of F-actin networks, our understanding of the regulation of viscous behaviors of F-actin networks is largely lacking. Here, we study the rheological behavior of F-actin networks formed with heavy meromyosin non-muscle IIB (NMMIIB). We show that NMMIIB quenched with ADP crosslinks F-actin into networks that, for sufficient densities, display stress stiffening behavior. By performing a series of creep tests, we show that densely crosslinked actin/NMMIIB–ADP networks undergo viscous deformation over a wide range of stresses, ranging from 0.001 to 10 Pa. At high stresses, networks that stress stiffen are also observed to shear thicken, whereby the effective viscosity increases as a function of stress. Shear thickening results in a reduction in the extent of irreversible, viscous deformation in actin/NMMIIB–ADP networks at high stresses compared to that expected for a linear viscoelastic material. Thus, viscous deformation contributes less to the overall mechanical response at high levels of applied force. Our results indicate mechanisms by which the fluid-like nature of the actomyosin cytoskeleton can be reduced under high load.
Co-reporter:Venkat Maruthamuthu;Margaret L. Gardel;Ulrich S. Schwarz;Benedikt Sabass
PNAS 2011 Volume 108 (Issue 12 ) pp:4708-4713
Publication Date(Web):2011-03-22
DOI:10.1073/pnas.1011123108
Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency
of forces sustained at cell-ECM and cell–cell adhesions are unknown. In this paper, we demonstrate that the endogenous force
sustained at the cell–cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the
cell–cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations
in cell–cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM
and the endogenous cell–cell force exists, indicating that the cell–cell tension is a constant fraction of the cell-ECM traction.
Thus, modulation of ECM properties that impact cell-ECM traction alters cell–cell tension. Finally, we show in a minimal model
of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent
of cell-ECM and cell–cell adhesion. This interdependence of cell–cell and cell-ECM forces has significant implications for
the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.
Co-reporter:Patrick W Oakes, Margaret L Gardel
Current Opinion in Cell Biology (October 2014) Volume 30() pp:68-73
Publication Date(Web):1 October 2014
DOI:10.1016/j.ceb.2014.06.003
Focal adhesion assembly and maturation often occurs concomitantly with changes in force generated within the cytoskeleton or extracellular matrix. To coordinate focal adhesion dynamics with force, it has been suggested that focal adhesion dynamics are mechanosensitive. This review discusses current understanding of the regulation of focal adhesion assembly and force transmission, and the limits to which we can consider focal adhesion plaques as mechanosensitive entities.
Co-reporter:Venkat Maruthamuthu, Yvonne Aratyn-Schaus, Margaret L Gardel
Current Opinion in Cell Biology (October 2010) Volume 22(Issue 5) pp:583-588
Publication Date(Web):1 October 2010
DOI:10.1016/j.ceb.2010.07.010
Adhesions are a central mechanism by which cells mechanically interact with the surrounding extracellular matrix (ECM) and neighboring cells. In both cell–ECM and cell–cell adhesions, forces generated within the actin cytoskeleton are transmitted to the surrounding environment and are essential for numerous morphogenic processes. Despite differences in many molecular components that regulate cell–cell and cell–ECM adhesions, the roles of F-actin dynamics and mechanical forces in adhesion regulation are surprisingly similar. Moreover, force transmission at adhesions occurs concomitantly with dynamic F-actin; proteins comprising the adhesion of F-actin to the plasma membrane must accommodate this movement while still facilitating force transmission. Thus, despite different molecular architectures, integrin and cadherin-mediated adhesions operate with common biophysical characteristics to transmit and respond to mechanical forces in multicellular tissue.
Co-reporter:Todd Thoresen, Martin Lenz, Margaret L. Gardel
Biophysical Journal (8 June 2011) Volume 100(Issue 11) pp:
Publication Date(Web):8 June 2011
DOI:10.1016/j.bpj.2011.04.031
Contractile actomyosin bundles are critical for numerous aspects of muscle and nonmuscle cell physiology. Due to the varying composition and structure of actomyosin bundles in vivo, the minimal requirements for their contraction remain unclear. Here, we demonstrate that actin filaments and filaments of smooth muscle myosin motors can self-assemble into bundles with contractile elements that efficiently transmit actomyosin forces to cellular length scales. The contractile and force-generating potential of these minimal actomyosin bundles is sharply sensitive to the myosin density. Above a critical myosin density, these bundles are contractile and generate large tensile forces. Below this threshold, insufficient cross-linking of F-actin by myosin thick filaments prevents efficient force transmission and can result in rapid bundle disintegration. For contractile bundles, the rate of contraction decreases as forces build and stalls under loads of ∼0.5 nN. The dependence of contraction speed and stall force on bundle length is consistent with bundle contraction occurring by several contractile elements connected in series. Thus, contraction in reconstituted actomyosin bundles captures essential biophysical characteristics of myofibrils while lacking numerous molecular constituents and structural signatures of sarcomeres. These results provide insight into nonsarcomeric mechanisms of actomyosin contraction found in smooth muscle and nonmuscle cells.
Co-reporter:Jonathan Stricker, Tobias Falzone, Margaret L. Gardel
Journal of Biomechanics (5 January 2010) Volume 43(Issue 1) pp:9-14
Publication Date(Web):5 January 2010
DOI:10.1016/j.jbiomech.2009.09.003
Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is critical to numerous physical cellular processes, including cell adhesion, migration and division. Each of these processes require precise regulation of cell shape and mechanical force generation which, to a large degree, is regulated by the dynamic mechanical behaviors of a diverse assortment of F-actin networks and bundles. In this review, we review the current understanding of the mechanics of F-actin networks and identify areas of further research needed to establish physical models. We first review our understanding of the mechanical behaviors of F-actin networks reconstituted in vitro, with a focus on the nonlinear mechanical response and behavior of “active” F-actin networks. We then explore the types of mechanical response measured of cytoskeletal F-actin networks and bundles formed in living cells and identify how these measurements correspond to those performed on reconstituted F-actin networks formed in vitro. Together, these approaches identify the challenges and opportunities in the study of living cytoskeletal matter.
Co-reporter:Todd Thoresen, Martin Lenz, Margaret L. Gardel
Biophysical Journal (5 February 2013) Volume 104(Issue 3) pp:
Publication Date(Web):5 February 2013
DOI:10.1016/j.bpj.2012.12.042
Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction.
Co-reporter:Venkat Maruthamuthu, Margaret L. Gardel
Biophysical Journal (5 August 2014) Volume 107(Issue 3) pp:
Publication Date(Web):5 August 2014
DOI:10.1016/j.bpj.2014.06.028
Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.
Co-reporter:Samantha Stam, Margaret L. Gardel
Developmental Cell (25 August 2014) Volume 30(Issue 4) pp:365-366
Publication Date(Web):25 August 2014
DOI:10.1016/j.devcel.2014.08.013
Intracellular transport of organelles and proteins is driven by multiple ATP-dependent processes. Recently in Cell, Guo et al. (2014) developed a technique, force-spectrum microscopy, to measure intracellular forces and demonstrate that large motion of cellular components can be produced by random ATP-dependent fluctuations within the cytoplasm.
Co-reporter:Jonathan Stricker, Yvonne Aratyn-Schaus, Patrick W. Oakes, Margaret L. Gardel
Biophysical Journal (22 June 2011) Volume 100(Issue 12) pp:
Publication Date(Web):22 June 2011
DOI:10.1016/j.bpj.2011.05.023
Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.
Co-reporter:Samantha Stam, Jon Alberts, Margaret L. Gardel, Edwin Munro
Biophysical Journal (21 April 2015) Volume 108(Issue 8) pp:
Publication Date(Web):21 April 2015
DOI:10.1016/j.bpj.2015.03.030
Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) arrays to generate contractile forces in muscle and nonmuscle cells. How myosin II force production is shaped by isoform-specific motor properties and environmental stiffness remains poorly understood. Here, we used computer simulations to analyze force production by an ensemble of myosin motors against an elastically tethered actin filament. We found that force output depends on two timescales: the duration of F-actin attachment, which varies sharply with the ensemble size, motor duty ratio, and external load; and the time to build force, which scales with the ensemble stall force, gliding speed, and environmental stiffness. Although force-dependent kinetics were not required to sense changes in stiffness, the myosin catch bond produced positive feedback between the attachment time and force to trigger switch-like transitions from transient attachments, generating small forces, to high-force-generating runs. Using parameters representative of skeletal muscle myosin, nonmuscle myosin IIB, and nonmuscle myosin IIA revealed three distinct regimes of behavior, respectively: 1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness; 2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force buildup times that exceed physiological timescales; and 3) small assemblies of low-duty ratio motors operating at intermediate speeds are poised to respond sharply to changes in mechanical context—at low force or stiffness, they serve as low-affinity cross-links, but they can transition to force production via the positive-feedback mechanism described above. Together, these results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment.
Co-reporter:Patrick W. Oakes, Shiladitya Banerjee, M. Cristina Marchetti, Margaret L. Gardel
Biophysical Journal (19 August 2014) Volume 107(Issue 4) pp:
Publication Date(Web):19 August 2014
DOI:10.1016/j.bpj.2014.06.045
Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries.
Co-reporter:Tobias T. Falzone, Patrick W. Oakes, Jennifer Sees, David R. Kovar, Margaret L. Gardel
Biophysical Journal (16 April 2013) Volume 104(Issue 8) pp:
Publication Date(Web):16 April 2013
DOI:10.1016/j.bpj.2013.01.017
Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties.