The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses.

Smooth muscle exhibits biophysical characteristics and physiological behaviors that are not readily explained by present paradigms of cytoskeletal and cross-bridge mechanics. There is increasing evidence that contractile activation of the smooth muscle cell involves an array of cytoskeletal processes that extend beyond cross-bridge cycling and the sliding of thick and thin filaments. We review here the evidence suggesting that the biophysical and mechanical properties of the smooth muscle cell reflect the integrated interactions of an array of highly dynamic cytoskeletal processes that both react to and transform the dynamics of cross-bridge interactions over the course of the contraction cycle. The activation of the smooth muscle cell is proposed to trigger dynamic remodeling of the actin filament lattice within cellular microdomains in response to local mechanical and pharmacological events, enabling the cell to adapt to its external environment. As the contraction progresses, the cytoskeletal lattice stabilizes, solidifies, and forms a rigid structure well suited for transmission of tension generated by the interaction of myosin and actin. The integrated molecular transitions that occur within the contractile cycle are interpreted in the context of microscale agitation mechanisms and resulting remodeling events within the intracellular microenvironment. Such an interpretation suggests that the cytoskeleton may behave as a glassy substance whose mechanical function is governed by an effective temperature.     

Normal and cystic fibrosis airway surface liquid homeostasis. The effects of phasic shear stress and viral infections

Mammalian airways normally regulate the volume of a thin liquid layer, the periciliary liquid (PCL), to facilitate the mucus clearance component of lung defense. Studies under standard (static) culture conditions revealed that normal airway epithelia possess an adenosine-regulated pathway that blends Na+ absorption and Cl- secretion to optimize PCL volume. In cystic fibrosis (CF), the absence of CF transmembrane conductance regulator results in a failure of adenosine regulation of PCL volume, which is predicted to initiate mucus stasis and infection. However, under conditions that mimic the phasic motion of the lung in vivo, ATP release into PCL was increased, CF ion transport was rebalanced, and PCL volume was restored to levels adequate for lung defense. This ATP signaling system was vulnerable, however, to insults that trigger CF bacterial infections, such as viral (respiratory syncytial virus) infections, which up-regulated extracellular ATPase activity and abolished motion-dependent ATP regulation of CF PCL height. These studies demonstrate (i) how the normal coordination of opposing ion transport pathways to maintain PCL volume is disrupted in CF, (ii) the hitherto unknown role of phasic motion in regulating key aspects of normal and CF innate airways defense, and (iii) that maneuvers directed at increasing motion-induced nucleotide release may be therapeutic in CF patients.     

Filamin-a and rheological properties of cultured melanoma cells

Here we report the rheological properties of cultured hsFLNa (filamin-A)-expressing (FIL+) and hsFLNa-deficient (FIL-) melanoma cells. Using magnetic twisting cytometry over a wide range of probing frequencies, and targeting either cortical or deeper cytoskeletal structures, we found that differences in stiffness of FIL+ versus FIL- cells were remarkably small. When probed through deep cytoskeletal structures, FIL+ cells were, at most, 30% stiffer than FIL- cells, whereas when probed through more peripheral cytoskeletal structures FIL- cells were not different except at very high frequencies. The loss tangent, expressed as an effective cytoskeletal temperature, was systematically greater in FIL- than FIL+ cells, but these differences were small and showed that the FIL+ cells were only slightly closer to a solidlike state. To quantify cytoskeletal remodeling, we measured spontaneous motions of beads bound to cortical cytoskeletal structures and found no difference in FIL+ versus FIL- cells. Although mechanical differences between FIL+ and FIL- cells were evident both in cortical and deeper structures, these differences were far smaller than expected based on measurements of the rheology of purified actin-filamin solutions. These findings do not rule out an important contribution of filamin to the mechanical properties of the cortical cytoskeleton, but suggest that effects of filamin in the cortex are not exerted on the length scale of the probe used here. These findings would appear to rule out any important contribution of filamin to the bulk mechanical properties of the cytoplasm, however. Although filamin is present in the cytoplasm, it may be inactive, its mechanical effects may be small compared with other crosslinkers, or mechanical properties of the matrix may be dominated by an overriding role of cytoskeletal prestress.     

A finite element model of cell deformation during magnetic bead twisting.

Magnetic twisting cytometry probes mechanical properties of an adherent cell by applying a torque to a magnetic bead that is tightly bound to the cell surface. Here we have used a three-dimensional finite element model of cell deformation to compute the relationships between the applied torque and resulting bead rotation and lateral bead translation. From the analysis, we computed two coefficients that allow the cell elastic modulus to be estimated from measurements of either bead rotation or lateral bead translation, respectively, if the degree of bead embedding and the cell height are known. Although computed strains in proximity of the bead can be large, the relationships between applied torque and bead rotation or translation remain virtually linear up to bead rotations of 15 degrees, above which geometrical nonlinearities become significant. This appreciable linear range stands in contrast to the intrinsically nonlinear force-displacement relationship that is observed when cells are indented during atomic force microscopy. Finally, these computations support the idea that adhesive forces are sufficient to keep the bead firmly attached to the cell surface throughout the range of working torques.     

Dynamic equilibration of airway smooth muscle contraction during physiological loading.

Airway smooth muscle contraction is the central event in acute airway narrowing in asthma. Most studies of isolated muscle have focused on statically equilibrated contractile states that arise from isometric or isotonic contractions. It has recently been established, however, that muscle length is determined by a dynamically equilibrated state of the muscle in which small tidal stretches associated with the ongoing action of breathing act to perturb the binding of myosin to actin. To further investigate this phenomenon, we describe in this report an experimental method for subjecting isolated muscle to a dynamic microenvironment designed to closely approximate that experienced in vivo. Unlike previous methods that used either time-varying length control, force control, or time-invariant auxotonic loads, this method uses transpulmonary pressure as the controlled variable, with both muscle force and muscle length free to adjust as they would in vivo. The method was implemented by using a servo-controlled lever arm to load activated airway smooth muscle strips with transpulmonary pressure fluctuations of increasing amplitude, simulating the action of breathing. The results are not consistent with classical ideas of airway narrowing, which rest on the assumption of a statically equilibrated contractile state; they are consistent, however, with the theory of perturbed equilibria of myosin binding. This experimental method will allow for quantitative experimental evaluation of factors that were previously outside of experimental control, including sensitivity of muscle length to changes of tidal volume, changes of lung volume, shape of the load characteristic, loss of parenchymal support and inflammatory thickening of airway wall compartments.     

Cytoskeleton dynamics: fluctuations within the network

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.     

Directional memory and caged dynamics in cytoskeletal remodelling

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.     

Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.     

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.     

Cytoskeletal mechanics in adherent human airway smooth muscle cells: probe specificity and scaling of protein-protein dynamics

We probed elastic and loss moduli in the adherent human airway smooth muscle cell through a variety of receptor systems, each serving as a different molecular window on cytoskeletal dynamics. Coated magnetic microbeads were attached to the cell surface via coating-receptor binding. A panel of bead coatings was investigated: a peptide containing the sequence RGD, vitronectin, urokinase, activating antibody against ₁-integrin, nonactivating antibody against ₁-integrin, blocking antibody against ₁-integrin, antibody against ₁-integrin, and acetylated low-density lipoprotein. An oscillatory mechanical torque was applied to the bead, and resulting lateral displacements were measured at baseline, after actin disruption by cytochalasin D, or after contractile activation by histamine. As expected, mechanical moduli depended strongly on bead type and bead coating, differing at the extremes by as much as two orders of magnitude. In every case, however, elastic and loss moduli increased with frequency f as a weak power law, f ^x–1. Moreover, with few exceptions, data could be scaled such that elastic and frictional responses depended solely on the power law exponent x. Taken together, these data suggest that power law behavior represents a generic feature of underlying protein-protein dynamics. actin; cytoskeleton; magnetic twisting cytometry; scale free; viscoelasticity