Ramaswamy Krishnan

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.

Reinforcement versus Fluidization in Cytoskeletal Mechanoresponsiveness

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

Propulsion and navigation within the advancing monolayer sheet

As a wound heals, or a body plan forms, or a tumor invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge or, paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space.

Cell Elasticity Determines Macrophage Function

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood. Principal Findings In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize. Conclusions Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.

Dilatation of the Constricted Human Airway by Tidal Expansion of Lung Parenchyma

RATIONALE In the normal lung, breathing and deep inspirations potently antagonize bronchoconstriction, but in the asthmatic lung this salutary effect is substantially attenuated or even reversed. To explain these findings, the prevailing hypothesis focuses on contracting airway smooth muscle and posits a nonlinear dynamic interaction between actomyosin binding and the tethering forces imposed by tidally expanding lung parenchyma. OBJECTIVE This hypothesis has never been tested directly in bronchial smooth muscle embedded within intraparenchymal airways. Our objective here is to fill that gap. METHODS We designed a novel system to image contracting intraparenchymal human airways situated within near-normal lung architecture and subjected to dynamic parenchymal expansion that simulates breathing. MEASUREMENTS AND MAIN RESULTS Reversal of bronchoconstriction depended on the degree to which breathing actually stretched the airway, which in turn depended negatively on severity of constriction and positively on the depth of breathing. Such behavior implies positive feedbacks that engender airway instability. OVERALL CONCLUSIONS These findings help to explain heterogeneity of airflow obstruction as well as why, in people with asthma, deep inspirations are less effective in reversing bronchoconstriction.

Mechanical responsiveness of the endothelial cell of Schlemm's canal: scope, variability and its potential role in controlling aqueous humour outflow

Primary open-angle glaucoma is associated with elevated intraocular pressure, which in turn is believed to result from impaired outflow of aqueous humour. Aqueous humour outflow passes mainly through the trabecular meshwork (TM) and then through pores formed in the endothelium of Schlemms canal (SC), which experiences a basal-to-apical pressure gradient. This gradient dramatically deforms the SC endothelial cell and potentially contributes to the formation of those pores. However, mechanical properties of the SC cell are poorly defined. Using optical magnetic twisting cytometry and traction force microscopy, here we characterize the mechanical properties of primary cultures of the human SC cell, and for the first time, the scope of their changes in response to pharmacological agents that are known to modulate outflow resistance. Lysophosphatidic acid, sphingosine-1-phosphate (S1P) and thrombin caused an increase in cell stiffness by up to 200 per cent, whereas in most cell strains, exposure to latrunculin A, isoproterenol, dibutryl cyclic-AMP or Y-27632 caused a decrease in cell stiffness by up to 80 per cent, highlighting that SC cells possess a remarkably wide contractile scope. Drug responses were variable across donors. S1P, for example, caused 200 per cent stiffening in one donor strain but only 20 per cent stiffening in another. Isoproterenol caused dose-dependent softening in three donor strains but little or no response in two others, a finding mirrored by changes in traction forces and consistent with the level of expression of β2-adrenergic receptors. Despite donor variability, those drugs that typically increase outflow resistance systematically caused cell stiffness to increase, while in most cases, those drugs that typically decrease outflow resistance caused cell stiffness to decrease. These findings establish the endothelial cell of SC as a reactive but variable mechanical component of the aqueous humour outflow pathway. Although the mechanism and locus of increased outflow resistance remain unclear, these data suggest the SC endothelial cell to be a modulator of outflow resistance.

Collective migration and cell jamming

Our traditional physical picture holds with the intuitive notion that each individual cell comprising the cellular collective senses signals or gradients and then mobilizes physical forces in response. Those forces, in turn, drive local cellular motions from which collective cellular migrations emerge. Although it does not account for spontaneous noisy fluctuations that can be quite large, the tacit assumption has been one of linear causality in which systematic local motions, on average, are the shadow of local forces, and these local forces are the shadow of the local signals. New lines of evidence now suggest a rather different physical picture in which dominant mechanical events may not be local, the cascade of mechanical causality may be not so linear, and, surprisingly, the fluctuations may not be noise as much as they are an essential feature of mechanism. Here we argue for a novel synthesis in which fluctuations and non-local cooperative events that typify the cellular collective might be illuminated by the unifying concept of cell jamming. Jamming has the potential to pull together diverse factors that are already known to contribute but previously had been considered for the most part as acting separately and independently. These include cellular crowding, intercellular force transmission, cadherin-dependent cell-cell adhesion, integrin-dependent cell-substrate adhesion, myosin-dependent motile force and contractility, actin-dependent deformability, proliferation, compression and stretch.

Airway smooth muscle and bronchospasm: fluctuating, fluidizing, freezing.

We review here four recent findings that have altered in a fundamental way our understanding of airways smooth muscle (ASM), its dynamic responses to physiological loading, and their dominant mechanical role in bronchospasm. These findings highlight ASM remodeling processes that are innately out-of-equilibrium and dynamic, and bring to the forefront a striking intersection between topics in condensed matter physics and ASM cytoskeletal biology. By doing so, they place in a new light the role of enhanced ASM mass in airway hyper-responsiveness as well as in the failure of a deep inspiration to relax the asthmatic airway. These findings have established that (i) ASM length is equilibrated dynamically, not statically; (ii) ASM dynamics closely resemble physical features exhibited by so-called soft glassy materials; (iii) static force-length relationships fail to describe dynamically contracted ASM states; (iv) stretch fluidizes the ASM cytoskeleton. Taken together, these observations suggest that at the origin of the bronchodilatory effect of a deep inspiration, and its failure in asthma, may lie glassy dynamics of the ASM cell.