Steady-state kinetics of smooth muscle myosin.

The kinetic properties of purified smooth muscle myosin, free of actin, have been examined. Analysis of the steady-state kinetic data revealed an intermediary plateau region on the substrate saturation curves. In addition, these data, when analyzed by Hill and Lineweaver and Burk plots, indicate both positive and negative cooperativity, suggesting at least four substrate binding sites. The plateau region was abolished when the kinetic measurements were made at pH 5.5 and 9.0. Both positive and negative cooperative effects were absent at pH 9.0 and hyperbolic kinetics was observed. In contrast, at pH 5.5, although the plateau region was abolished, the enzyme exhibited positive cooperativity of substrate binding. When either heated or urea treated enzyme was used for kinetic measurements: (i) the plateau region shifted toward higher substrate concentration range; (ii) the cooperativity of binding sites was lost at low substrate concentrations but was instead seen at higher concentrations; and (iii) the Vmax was doubled. These data have been interpreted as due to ligand-induced conformational changes in the enzyme according to J. Teipel and D. E. Koshland, Jr. (1969).     

Cryo-atomic force microscopy of smooth muscle myosin.

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.     

Tethering forces of secretory granules measured with optical tweezers

Fusion of a vesicle with its target membrane is preceded by tethering or docking. However, the physical mechanism of vesicle-tethering is unknown. To study this mechanism, we used eosinophil secretory granules, which undergo stimulated homotypic fusion events inside the cell during degranulation. Using a dual optical trap system, we observed tether formation between isolated eosinophil secretory granules. The results show that secretory granules interact stochastically with a target membrane forming physical tethers linking the vesicle and target membrane, rather than via interactions with the cytoskeleton. The necessary components are membrane-associated, and the addition of cytosolic components is not required. Tether-lifetime measurements as a function of applied mechanical force revealed at least three kinetically distinct tethered states. The tethered-state lifetimes of isolated eosinophil granules match the residence times of chromaffin granules at the plasma membrane in intact cells, suggesting that the tethering mechanisms reported here may represent the physiological mechanisms of vesicle-tethering in the cell.     

Compliance of bacterial polyhooks measured with optical tweezers.

In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.     

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.     

Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.     

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm-2), medium (1000 cells cm-2), and high (10,000 cells cm-2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60% of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.     

Comparison of steady-state kinetics of thiophosphorylated versus unphosphorylated smooth muscle myosin

(i) The steady-state kinetic data obtained with purified gizzard and uterus smooth muscle myosins indicated the presence of a plateau region on the substrate-saturation curves. Hill plots of these data provided evidence for mixed positive and negative cooperative interactions. In contrast, when gizzard myosin was prepared according to the method of A. Sobieszek and R.D. Bremel (1975, Eur. J. Biochem.55, 49–60), the saturation curve in the presence of CaATP was hyperbolic and no cooperativity of the binding site(s) was discerned. However, in the presence of MgATP although the curve appeared hyperbolic the Hill plot of the data was biphasic with negative cooperativity at low MgATP concentration, (ii) When thiophosphorylated gizzard myosin was used for kinetic analysis, the plateau region in the presence of MnATP was eliminated from the saturation curve and this curve became hyperbolic. However, in the presence of MgATP, although the plateau was almost eliminated, the saturation curve was still biphasic with either no or greatly reduced negative cooperativity of binding sites at low MgATP concentrations but positive cooperativity of binding at high MgATP concentrations. In addition, the thiophosphorylation of myosin also increased the K_m and V of MgATP and MnATP, thus indicating weaker affinity for these substrates with thiophosphorylated myosin. (iii) Gizzard myosin also hydrolyzed other nucleotides (the order of rates being CTP = ITP > ATP = UTP > GTP), therefore saturation kinetics using different nucleotides as substrates was also carried out. The saturation curves with each nucleotide were different i.e., hyperbolic with CTP, sigmoid with GTP, hyperbolic with biphasic Hill plot with ITP, and possessing plateau with UTP. In addition, it was observed that the kinetic pattern with each nucleotide was very sensitive to temperature and pH.     

Physical properties of Escherichia coli P pili measured by optical tweezers

The mechanical behavior of individual P pili of uropathogenic Escherichia coli has been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of approximately 10(3) PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. They are particularly important in the pathogenesis of E. coli colonizing the upper urinary tract and kidneys. A biological model system has been established for in situ measurements of the forces that occur during mechanical stretching of pili. A mathematical model of the force-versus-elongation behavior of an individual pilus has been developed. Three elongation regions of pili were identified. In region I, P pili stretch elastically, up to a relative elongation of 16 +/- 3%. The product of elasticity modulus and area of a P pilus, EA, was assessed to 154 +/- 20 pN (n=6). In region II, the quaternary structure of the PapA rod unfolds under a constant force of 27 +/- 2 pN (n approximately 100) by a sequential breaking of the interactions between adjacent layers of PapA subunits. This unfolding can elongate the pilus up to 7 +/- 2 times. In region III, pili elongate in a nonlinear manner as a result of stretching until the bond ruptures.     

Mechanical force characterization in manipulating live cells with optical tweezers

Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach.     

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.     

Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle

Urinary bladder (detrusor) smooth muscle is active in the absence of an external stimulus. Tone occurs even "at rest" during the filling phase, and it is elevated in patients with overactive bladder. This study examined the role of muscle length on tone and the level of basal myosin light chain phosphorylation (MLC(20P)). MLC(20P) was 23.9 +/- 1% (n = 58) at short lengths (zero preload; L(z)). An increase in length from L(z) to the optimal length for contraction (L(o)) caused a reduction in MLC(20P) to 15.8 +/- 1% (n = 49). Whereas 10 microM staurosporine reduced MLC(20P) at L(z), 1 microM staurosporine, a Ca(2+)-free solution, and inhibitors of MLC kinase, protein kinase C (PKC) and RhoA kinase (ROK) did not. However, 1 microM staurosporine and inhibitors of ROK inhibited MLC(20P) and tone at L(o). These data support the hypothesis that a Ca(2+)-independent kinase, possibly ZIP-like kinase, regulates MLC(20P) at L(z), whereas in detrusor stretched to L(o), additional kinases, such as ROK, participate.     

Stretching short sequences of DNA with constant force axial optical tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.     

Filamentous smooth muscle myosin is regulated by phosphorylation

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody- stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.     

Interaction forces between F-actin and titin PEVK domain measured with optical tweezers

Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.     

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

We tested the hypothesis that increases in force at a given cytosolic Ca(2+) concentration (i.e., Ca(2+) sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in alpha-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 microM Ca(2+) in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 microM) plus GTP (1 microM) significantly shifted the Ca(2+)-force relationship curve to the left (EC(50): 0.39 +/- 0.06 to 0.078 +/- 0.006 microM Ca(2+)) and significantly increased the maximum force (104.4 +/- 4.8 to 120.2 +/- 2.8%; n = 6 observations). The Ca(2+)-rMLC phosphorylation relationship curve was also shifted to the left (EC(50): 1.26 +/- 0.57 to 0.13 +/- 0.04 microM Ca(2+)) and upward (maximum rMLC phosphorylation: 70.9 +/- 7.9 to 88.5 +/- 5. 1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca(2+) concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca(2+) sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.     

Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results

Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1-10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010-3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.     

Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.