Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.     

Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells.

Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 +/- 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.     

Myosin dynamics on the millisecond time scale

Myosin is a motor protein associating with actin and ATP. It translates along actin filaments against a force by transduction of free energy liberated with ATP hydrolysis. Various myosin crystal structures define time points during ATPase showing the protein undergoes large conformation change during transduction over a cycle with approximately 10 ms periodicity. The protein conformation trajectory between two intermediates in the cycle is surmised by non-equilibrium Monte Carlo simulation utilizing free-energy minimization. The trajectory shows myosin transduction of free energy to mechanical work giving evidence for: (i) a causal relationship between product release and work production in the native isoform that is correctly disrupted in a chemically modified protein, (ii) the molecular basis of ATP-sensitive tryptophan fluorescence enhancement and acrylamide quenching, (iii) an actin-binding site peptide containing the free-energy barrier to ATPase product release defining the rate limiting step and, (iv) a scenario for actin-activation of myosin ATPase.     

Myosin II function in non-muscle cells

Amongst the remarkable variety of motility that cells display, cytokinesis (cell division) is particularly striking. Dramatic changes in cell shape occur before, during and after cytokinesis. Myosin II is implicated in the ‘rounding up’ of cells prior to cytokinesis, and is essential in the formation of the contractile cleavage furrow during cytokinesis. Now it appears that myosin II plays a role in all stages of cytokinesis, as a recent report⁽¹⁾ suggests that myosin II drives post-mitotic cell spreading. A similar type of motile mechanism operating in cell spreading may occur in other cell types in other situations.     

Myosin I and actin dynamics: the frogs weigh in

In this issue of Developmental Cell, Sokac et al. (2006) describe an intriguing new role for an actin-based motor protein in restraining actin polymerization during endocytosis in Xenopus oocytes.     

Myosin II Activity Softens Cells in Suspension

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.     

Myosin II dynamics in Dictyostelium: determinants for filament assembly and translocation to the cell cortex during chemoattractant responses

In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G-protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP-myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP-myosin fusions, and with latrunculin A-treated cells, demonstrated that F-actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A "headless" GFP-myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor-based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant-stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant-regulated myosin II filament assembly and translocation.     

Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation

Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation. acid secretion; cytoskeleton; ion channels and pumps     

Collective dynamics of elastically coupled Myosin v motors

Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain.     

An improved inverse dynamics formulation for estimation of external and internal loads during human sagittal plane movements

Planar musculoskeletal models are common in the inverse dynamics analysis of human movements such as walking, running and jumping. The continued interest in such models is justified by their simplicity and computational efficiency. Related to a human planar model, a unified formulation for both the flying and support phases of the sagittal plane movements is developed. The actuation involves muscle forces in the lower limbs and the resultant muscle torques in the other body joints. The dynamic equations, introduced in absolute coordinates of the segments, are converted into useful compact forms using the projective technique. The solution to a determinate inverse dynamics problem allows for the explicit determination of the external reactions (presumed to vanish during the flying phases) and the resultant muscle torques in all the model joints. The indeterminate inverse dynamics problem is then focused on the assessment of muscle forces and joint reaction forces selectively in the supporting lower limb. Numerical results of the inverse dynamics simulation of sample sagittal plane movements are reported to illustrate the validity and effectiveness of the improved formulation.     

Competition in the presence of a lethal external inhibitor

The study considers two organisms competing for a nutrient in an open system in the presence of an inhibitor (or toxicant). The inhibitor is input at a constant rate and is lethal to one competitor while being taken up by the other without harm. This is in contrast to previous studies, where the inhibitor decreases the reproductive rate of one of the organisms. The mathematical result of the lethal effect, modeled by a mass action term, is that the system cannot be reduced to a monotone dynamical system of one order lower as is common with chemostat-like problems. The model is described by four non-linear, ordinary differential equations and we seek to describe the asymptotic behavior as a function of the parameters of the system. Several global exclusion results are presented with mathematical proofs. However, in the case of coexistence, oscillatory behavior is possible and the study proceeds with numerical examples. The model is relevant to bioremediation problems in nature and to laboratory bio-reactors.     

Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.     

Structural dynamics of the cell wall precursor lipid II in the presence and absence of the lantibiotic nisin

Representing a physiological “Achilles' heel”, the cell wall precursor lipid II (L_II) is a prime target for various classes of antibiotics. Over the years L_II-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying L_II functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of L_II, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded L_II in the absence and presence of the L_II-binding lantibiotic nisin. In a series of 2 × 4 independent, unbiased 100 ns MD simulations we sampled the conformational dynamics of nine L_II as well as nine L_II–nisin complexes embedded in an aqueous 150 mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to L_II induces a reduction of L_II mobility and flexibility, an outward shift of the L_II pentapeptide, an inward movement of the L_II disaccharide section, and an overall deeper insertion of the L_II tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and L_II interaction remain similar to the 1WCO L_II–nisin NMR solution structure.