The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.

Calcific aortic valve disease (CAVD) results in impaired function through the inability of valves to fully open and close, but the causes of this pathology are unknown. Stiffening of the aorta is associated with CAVD and results in exposing the aortic valves to greater mechanical strain. Transforming growth factor β1 (TGF-β1) is enriched in diseased valves and has been shown to combine with strain to synergistically alter aortic valve interstitial cell (AVIC) phenotypes. Therefore, we investigated the role of strain and TGF-β1 on the calcification of AVICs. Following TGF-β1 pretreatment, strain induced intact monolayers to aggregate and calcify. Using a wound assay, we confirmed that TGF-β1 increases tension in the monolayer in parallel with α-smooth muscle actin (αSMA) expression. Continual exposure to strain accelerates aggregates to calcify into mature nodules that contain a necrotic core surrounded by an apoptotic ring. This phenotype appears to be mediated by strain inhibition of AVIC migration after the initial formation of aggregates. To better interpret the extent to which externally applied strain physically impacts this process, we modified the classical Lamé solution, derived using principles from linear elasticity, to reveal strain magnification as a novel feature occurring in a mechanical environment that supports nodule formation. These results indicate that strain can impact multiple points of nodule formation: by modifying tension in the monolayer, remodeling cell contacts, migration, apoptosis, and mineralization. Therefore, strain induced nodule formation provides new directions for developing strategies to address CAVD.

Objective Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MEK1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of TGF-β1-induced cadherin-11 expression in calcific nodule formation. Methods and Results As shown previously, porcine AVICs treated with TGF-β1 prior to cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, TGF-β1 treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in TGF-β1 treated AVICs possessing cadherin-11. Furthermore, when siRNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell-cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets. Conclusions These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2 dependent pathway.

A subset of PSGL-1 is constitutively associated with L-selectin and signals through Src family kinases to activate LFA-1, which regulates neutrophil slow rolling and recruitment.

Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament's approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near nonadherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend >35 m, comparable with the average, mass-weighted filament length. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments by using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. However, explaining depletion profiles and concentration dependence (power-law of ؊1) requires modifying the rigid rod model. Within cells, surface depletion should slow membrane-associated F-actin reactions another Ϸ10-fold beyond hydrodynamically slowed diffusion of filaments (Ϸ10-fold). In addition, surface depletion should cause membranes to bend spontaneously toward filaments. Such depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.confocal microscopy ͉ cytoskeleton ͉ fluorescence ͉ polymer physics ͉ polymer depletion

No abstract

No abstract

Calcific Aortic Valve Disease (CAVD) is the third most common cause of cardiovascular disease, affecting nearly 5 million people in the United States alone. It is now the most common form of acquired valvular disease in industrialized countries and will likely affect more individuals in the coming years as the prevalence increases with life expectancy. It is known that the progression of CAVD is closely related to the behavior of aortic valve interstitial cells (AVICs); however the cellular mechanobiological mechanisms leading to dysfunction remain unclear. Generally, CAVD is characterized by the formation of calcified AVIC aggregates with an apoptotic core. These aggregates increase the leaflet stiffness and impede normal valve function. Multiple studies have investigated the effects of various biochemical cues on this process, such as transformation growth factor β1 (TGF-β1), on the regulation of nodule formation [1]. Additionally, Yip et al revealed that matrix stiffness controls nodule formation in vitro, with stiffer substrates promoting apoptotic nodule formation, while compliant substrates generated nodules containing cells with osteoblast markers [2]. This suggests that matrix stiffness is involved in the regulatory mechanisms of nodule formation and may initiate different types of nodule formation (i.e. osteogenic vs. dystrophic). In the current study, we examined the synergistic role of strain and TGF-β1 in the generation of calcified nodules AVICs.