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.     

Dynamic response of the isolated passive rat diaphragm strip.

To further our understanding of the mechanisms underlying chest wall mechanics, we investigated the dynamic response of the isolated passive rat diaphragm strip. Stress adaptation of the tissue was measured from 0.05 to 60 s after subjecting the strips to strain steps of normalized strain amplitudes from 0.005 to 0.04. The tissue resistance (R), elastance (E), and hysteresivity (eta) were measured in the same range of amplitudes by sinusoidally straining the strip at frequencies from 0.03125 to 10 Hz. The stress (T) depended exponentially on the strain (epsilon) and relaxed and recovered linearly with the logarithm of time. E increased linearly with the logarithm of frequency and decreased with increasing amplitude. R fell hyperbolically with frequency and showed an amplitude dependence similar to that of E. To interpret the strong nonlinear behavior, we extended the viscoelastic model of Hildebrandt (J. Appl. Physiol. 28: 365-372, 1970) to include an exponential stress-strain relationship. Accordingly, the step response was described by T - Tr = Tr(e alpha delta epsilon - 1)(1 - gamma log t), where delta epsilon is the strain amplitude, Tr is the initial operating stress, alpha is a measure of the stress-strain nonlinearity, and gamma is the rate of stress adaptation. The oscillatory response of the model was computed by applying Fung's quasi-linear viscoelastic theory. This quasi-linear viscoelastic model fitted the step and oscillatory data fairly well but only if alpha depended negatively on delta epsilon, as might be expected in a plastic material.     

Mechanical impedance of the lung periphery

Hantos, Z., F. Peták, Á. Adamicza, T. Asztalos, J. Tolnai, and J. J. Fredberg. Mechanical impedance ofthe lung periphery. J. Appl. Physiol.83(5): 1595-1601, 1997.The mechanics of the regional airways andtissues was studied in isolated dog lobes by means of a modifiedwave-tube technique. Small-amplitude pseudorandom forced oscillationsbetween 0.1 and 48 Hz were applied through catheters wedged in2-mm-diameter bronchi in three regions of each lobe at translobarpressures (PL) of 10, 7, 5, 3, 2, and 1 cmH₂O. The measuredregional input impedances were fitted by a model containing theresistance (R₁) and inertance(I) of the regular (segmental) airways, the resistance of thecollateral channels (R₂), andthe damping (G) and elastance (H) of the local tissues. This model gavefar better fits to the data on impedance of the lung periphery thanwhen G and H were replaced by a single tissue compliance, whichexplains why interruption of segmental flow did not lead tomonoexponential pressure decay in previous studies. The interlobar andintralobar variances of the parameters were equally significant, andpoor correlations were found between the airway parametersR₁ andR₂ and between any airway andtissue parameter (e.g., R₁ and H).R₂ was on average ~10 timeshigher than R₁, although theR₂-to-R₁ratios and their dependencies on PL were regionally highlyvariable. However, for the total of 33 regions studied, thePL dependence was the same forR₁ and R₂, which may reflect similarmorphological structures for the regular and collateral airways. Thedependencies of G and H on PLshowed high interregional variations; generally, however, they assumedtheir minima at medium PL values(~5 cmH₂O).

     

Preferential axial flow during high-frequency oscillations: effects of gas density

Allen et al. (J. Clin. Invest. 76: 620-629, 1985) reported that regional phasic lung distension during high-frequency oscillations (HFO) is substantially and systemically heterogeneous when both frequency (f) and tidal volume (VT) are large. They hypothesized that this phenomenon was attributable to central airway geometry and preferential axial flow induced therein by the momentum flux of the inspiratory gas stream. According to that hypothesis, the observed distribution of phasic lung distension would depend on the ratio VT/VD* (where VD* is an index of anatomic dead space), independent of gas density (rho), when f is scaled in proportion to lung resonant frequency, fo. To test this hypothesis, we used the methods of Allen et al. (ibid.) to study six excised dog lungs during HFO (f = 2-32 Hz; VT = 5-80 ml) using gases of different densities. Alveolar pressure excursions (PA) were measured as rho spanned a 12-fold range using He, air, and SF6. The apex-to-base and right-to-left ratios of PA were used as indexes of regional heterogeneity of phasic lung distension. For each gas at low f, distension of the lung base was favored slightly independent of VT, but at higher f distension of the lung apex was favored when VT was small, whereas distension of the lung base was favored when VT was large. In addition, we observed substantial right-to-left differences in apical lobes during oscillation at high f not seen before.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interdependent regional lung emptying during forced expiration: a transistor model

We recognized similarities between isovolume pressure-flow curves of the lung and emitter-collector voltage-current characteristics of bipolar transistors, and used this analogy to model expiratory flow limitation in a two-generation branching network with parallel nonhomogeneity. In this model, each of two bronchi empty parenchymal compliances through a common trachea, and each branch includes resistances upstream and downstream of a flow-limiting site. Properties of each airway are specified independently, allowing simulation of differences between the tracheal and bronchial generations and between the parallel bronchial paths. Simulations of four types of parallel asymmetry were performed: unilateral peripheral bronchoconstriction; unilateral central bronchoconstriction; asymmetric redistribution of parenchymal compliance; and unilateral alteration of the bronchial area-transmural pressure characteristic. Our results indicate that multiple axial choke points can exist simultaneously in a symmetric lung when large airway opening-pleural pressure gradients exist; despite severe nonhomogeneity of regional lung emptying, flow interdependence among parallel branches tends to maintain a near normal configuration of the overall maximal expiratory flow-volume (MEFV) curve throughout a large fraction of the vital capacity; and sudden changes of slope of the MEFV curve ("knees" or "bumps") may reflect choking in one branch in a nonuniform lung, but need not be obvious even when severe heterogeneity of lung emptying exists.     

Periodic flow at airway bifurcations. I. Development of steady pressure differences

In studies of large-amplitude periodic flows at an airway bifurcation, we found an appreciable steady-state pressure difference between the terminal units. To elucidate the fluid dynamic origins of such steady-state pressure differences, we studied single asymmetric bifurcation models with various area ratios and branching angles. The daughter ducts were identical in size and were terminated into identical elastic loads. Sinusoidal flow oscillations were applied at the parent duct so that the upstream Reynolds number ranged from 30 to 77,000 and the Womersley parameter from 2 to 30. The steady-state component (time averaged) of the pressure measured at the terminal with the smaller branching angle was found to be consistently higher than that at the other terminal. This steady-state pressure difference scaled approximately as a fixed fraction of the parent duct dynamic head. Guided by the results of flow-visualization studies, we modeled such behavior based on the temporal and spatial differences of head loss between the two branches of the bifurcation. Our results suggest that interlobar heterogeneity of mean alveolar-pressure observed in excised canine lungs during high frequency oscillation (Allen et al., J. Appl. Physiol. 62: 223-228, 1987) arises solely from fluid dynamic origins: differential head loss due to asymmetry of central airway branching structure.