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Krishnan, Ramaswamy

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.

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.

Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT), and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility, and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons, and that the DIAPH3 network potentially reports taxane sensitivity in human tumors.

As a wound heals, or a body plan forms, or a tumour 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.

Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms–and their failure in asthma–remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

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.

There is growing interest in quantifying vascular cell and tissue stiffness. Most measurement approaches, however, are incapable of assessing stiffness in the presence of physiological flows. We developed a microfluidic approach which allows measurement of shear modulus (G) during flow. The design included a chamber with glass windows allowing imaging with upright or inverted microscopes. Flow was controlled gravitationally to push culture media through the chamber. Fluorescent beads were conjugated to the sample surface and imaged before and during flow. Bead displacements were calculated from images and G was computed as the ratio of imposed shear stress to measured shear strain. Fluid-structure simulations showed that shear stress on the surface did not depend on sample stiffness. Our approach was verified by measuring the moduli of polyacrylamide gels of known stiffness. In human pulmonary microvascular endothelial cells, G was 20.4 ± 12 Pa and decreased by 20% and 22% with increasing shear stress and inhibition of non-muscle myosin II motors, respectively. The G showed a larger intra- than inter-cellular variability and it was mostly determined by the cytosol. Our shear modulus microscopy can thus map the spatial distribution of G of soft materials including gels, cells and tissues while allowing the visualization of microscopic structures such as the cytoskeleleton.

Genetic testing in the clinic and research lab is becoming more routinely used to identify rare genetic variants. However, attributing these rare variants as the cause of disease in an individual patient remains challenging. Here, we report a patient who presented with nephrotic syndrome and focal segmental glomerulosclerosis (FSGS) with collapsing features at age 14. Despite treatment, her kidney disease progressed to end-stage within a year of diagnosis. Through genetic testing, an Y265H variant with unknown clinical significance in alpha-actinin-4 gene (ACTN4) was identified. This variant has not been seen previously in FSGS patients nor is it present in genetic databases. Her clinical presentation is different from previous descriptions of ACTN4 mediated FSGS, which is characterized by sub-nephrotic proteinuria and slow progression to end stage kidney disease. We performed in vitro and cellular assays to characterize this novel ACTN4 variant before attributing causation. We found that ACTN4 with either Y265H or K255E (a known disease-causing mutation) increased the actin bundling activity of ACTN4 in vitro, was associated with the formation of intracellular aggregates, and increased podocyte contractile force. Despite the absence of a familial pattern of inheritance, these similar biological changes caused by the Y265H and K255E amino acid substitutions suggest that this new variant is potentially the cause of FSGS in this patient. Our studies highlight that functional validation in complement with genetic testing may be required to confirm the etiology of rare disease, especially in the setting of unusual clinical presentations.