Low spring constant regulates P-selectin-PSGL-1 bond rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r_f (≥10² pN/s), defined as the product of spring constant k and retract velocity v, how the low r_f (<10² pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r_f ≤ 20 pN/s with low k (∼10⁻³-10⁻² pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r_f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r_f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 × 10⁻³ pN/nm, indicating that the bond dissociation at low r_f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r_f or k.     

The elastic properties of the structurally characterized myosin II S2 subdomain: a molecular dynamics and normal mode analysis

The elastic properties (stretching and bending moduli) of myosin are expected to play an important role in its function. Of particular interest is the extended α-helical coiled-coil portion of the molecule. Since there is no high resolution structure for the entire coiled-coil, a study is made of the scallop myosin II S2 subdomain for which an x-ray structure is available (Protein Data Bank 1nkn). We estimate the stretching and bending moduli of the S2 subdomain with an atomic level model by use of molecular simulations. Results were obtained from nonequilibrium molecular dynamics simulations in the presence of an external force, from the fluctuations in equilibrium molecular dynamics simulations and from normal modes. In addition, a poly-Ala (78 amino acid residues) α-helix model was examined to test the methodology and because of its interest as part of the lever arm. As expected, both the α-helix and coiled-coil S2 subdomain are very stiff for stretching along the main axis, with the stretching stiffness constant in the range 60-80 pN/nm (scaled to the 60 nm long S2). Both molecules are much more flexible for bending with a lateral stiffness of ∼0.010pN/nm for the S2 and 0.0055pN/nm for the α-helix (scaled to 60 nm). These results are expected to be useful in estimating cross-bridge elasticity, which is required for understanding the strain-dependent transitions in the actomyosin cycle and for the development of three-dimensional models of muscle contraction.     

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (P_i) reduce isometric force, providing support for the notion that the release of P_i from myosin is closely associated with the generation of muscular force. P_i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P_i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P_i on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of P_i, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P_i, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated P_i also caused a >65% reduction in binding-event lifetime, suggesting that P_i induces premature detachment from a strongly bound state. Definitive evidence of a P_i-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which P_i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P_i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P_i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.     

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)₃ that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of ∼21 pN and ∼63 nm, respectively. Stretching of (A1A2A3)₃ yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.     

Mechanical studies of single ribosome/mRNA complexes

Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA(Phe), an analog of the initiator fMet-tRNA(Met), enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 +/- 0.05 nm and the backbone elasticity was 900 +/- 140 pN, similar to values for single-stranded DNA.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

Direct observation of active protein folding using lock-in force spectroscopy

Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study, we present a lock-in force-spectroscopy technique that improves resolution of atomic-force microscopy force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from Dictyostelium discoideum filamin (ddFLN4) refolds against forces of ∼4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide before folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain-length and zero force refolding rate.     

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na⁺-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na⁺). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at ∼ 2200 pN nm) from stall up to a “knee” speed of ∼ 420 Hz, and then falls linearly with speed, extrapolating to zero torque at ∼ 910 Hz. Motors containing one to five stators generated ∼ 200 pN nm per stator at speeds up to ∼ 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to ∼ 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to ∼ 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.     

Stabilization provided by neighboring strands is critical for the mechanical stability of proteins

Single-molecule force spectroscopy studies and steered molecular dynamics simulations have revealed that protein topology and pulling geometry play important roles in determining the mechanical stability of proteins. Most studies have focused on local interactions that are associated with the force-bearing β-strands. Interactions mediated by neighboring strands are often overlooked. Here we use Top7 and barstar as model systems to illustrate the critical importance of the stabilization effect provided by neighboring β-strands on the mechanical stability. Using single-molecule atomic force microscopy, we showed that Top7 and barstar, which have similar topology in their force-bearing region, exhibit vastly different mechanical-stability characteristics. Top7 is mechanically stable and unfolds at ∼150 pN, whereas barstar is mechanically labile and unfolds largely below 50 pN. Steered molecular dynamics simulations revealed that stretching force peels one force-bearing strand away from barstar to trigger unfolding, whereas Top7 unfolds via a substructure-sliding mechanism. This previously overlooked stabilization effect from neighboring β-strands is likely to be a general mechanism in protein mechanics and can serve as a guideline for the de novo design of proteins with significant mechanical stability and novel protein topology.     

Stretching DNA with optical tweezers.

Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.     

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (P_i) reduce isometric force, providing support for the notion that the release of P_i from myosin is closely associated with the generation of muscular force. P_i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P_i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P_i on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of P_i, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P_i, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated P_i also caused a >65% reduction in binding-event lifetime, suggesting that P_i induces premature detachment from a strongly bound state. Definitive evidence of a P_i-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which P_i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P_i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P_i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.     

Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM^hVLC-1) or E56G-mutated hVLC-1 (hVLC-1^E56G; TgM^E56G). hVLC-1 or hVLC-1^E56G expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM^hVLC-1 (1.67 pN/nm and 2.3 μm/s, respectively) were significantly higher than myosin with hVLC-1^E56G prepared from TgM^E56G (1.25 pN/nm and 1.7 μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM^hVLC-1 (80.0 mmHg) were significantly higher than hearts from TgM^E56G (66.2 mmHg) or C57/BL6 (59.3 ± 3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1^E56G ≈ mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.     

Mechanical Distortion of Single Actin Filaments Induced by External Force: Detection by Fluorescence Imaging

Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a “bio-nano strain gauge” that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys³⁷⁴ and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by ∼6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys³⁷⁴ or rhodamine-phalloidin showed only an ∼2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys³⁷⁴ approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αLβ2 Under Force

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin in each experiment. Of fundamental importance, the assay for off-rates does not depend on how the force is applied over time, and remains valid when the rates of dissociation change with different levels of force. In this first article, we present results from tests of a monovalent ICAM-1 probe against immobilized α_Lβ₂ in environments of divalent cations (Ca²⁺, Mg²⁺, and Mn²⁺) and demonstrate in detail the method for assay of off-rates. When extrapolated to zero force, the force-free values for the off-rates are found to be consistent with published solution-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., ∼10⁻²/s in Mg²⁺ or Mn²⁺ and ∼1/s in Ca²⁺. At the same time, as expected for adhesive function, we find that the β₂ integrin bonds activated in Mn²⁺ or Mg²⁺ possess significant and persistent mechanical strength (e.g., >20 pN for >1 s) even when subjected to slow force ramps (<10 pN/s). As discussed in our companion article, using the same assay, we find that although the rates of dissociation for diICAM-1fc bonds to LFA-1 on neutrophils in Mn²⁺ are similar to those for mICAM-1 bonds to recombinant α_Lβ₂ on microspheres, they appear to represent a dimeric attachment to a pair of tightly clustered integrin heterodimers. The mechanical strengths and lifetimes of the dimeric interactions increase dramatically when the neutrophils are stimulated by the chemokine IL-8 or are bound with an allosterically activating (anti-CD18) monoclonal antibody, demonstrating the major impact of cell signaling on LFA-1.     

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ∼17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.     

Determining Protein-Induced DNA Bending in Force-Extension Experiments: Theoretical Analysis

Computer simulations were used to investigate the possibility of determining protein-induced DNA bend angles by measuring the extension of a single DNA molecule. Analysis of the equilibrium sets of DNA conformations showed that shortening of DNA extension by a single protein-induced DNA bend can be as large as 35 nm. The shortening has a maximum value at the extending force of ∼0.1 pN. At this force, the DNA extension experiences very large fluctuations that dramatically complicate the measurement. Using Brownian dynamics simulation of a DNA molecule extended by force, we were able to estimate the observation time needed to obtain the desired accuracy of the extension measurement. Also, the simulation revealed large fluctuations of the force, acting on the attached magnetic bead from the stretched DNA molecule.     

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

Background

Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.

Methods

To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (F_unb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin.

Results

F_unb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the F_unb (0.06 ± 0.01 pN). Inspection of the traces of the F_unb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.

Conclusions

CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.

General significance

This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Spatiotemporal analysis of cell response to a rigidity gradient: a quantitative study using multiple optical tweezers

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN · μm⁻¹ range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.     

PicoNewton-millisecond force steps reveal the transition kinetics and mechanism of the double-stranded DNA elongation

We study the kinetics of the overstretching transition in λ-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5–2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ∼25 basepairs. This mechanism increases the free energy for the elementary reaction to ∼94 k_BT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.