Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k ₊=0.3–1.4 s⁻¹ and k _off=0.05–0.25 s⁻¹, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm²) raised k ₊ in a diffusion-dependent manner, leaving k _off relatively unchanged. Increasing the temperature (23–37 °C) raised both k ₊ and k _off, allowing calculation of the activation energy of the chemical reaction (around 20 k _B T). Increasing the compressive force on the cell during contact (up to 60 pN) raised k ₊ in a logarithmic manner, probably through an increase in the contact area, whereas k _off was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond. Received: 2 November 1999 / Revised version: 6 March 2000 / Accepted: 19 April 2000     

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

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.     

Facile tethering of stable and unstable proteins for optical tweezers experiments

Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach “molecular handles” to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models.     

Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

Accurately predicting binding affinity constant (K_A) is highly required to determine the binding energetics of the driving forces in drug–DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K_A for PD153035, where K_A is determined from the changes in B-form contour length (L) of PD153035–DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K_A = 1.18(±0.09) × 10⁴ (1/M) at 23 ± 0.5 °C and the minimum distance between adjacent bound PD153035 ≈ 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.     

Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.     

The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer.

Muscle contraction is brought about by the cyclical interaction of myosin with actin coupled to the breakdown of ATP. The current view of the mechanism is that the bound actomyosin complex (or "cross-bridge") produces force and movement by a change in conformation. This process is known as the "working stroke." We have measured the stiffness and working stroke of a single cross-bridge (kappa xb, dxb, respectively) with an optical tweezers transducer. Measurements were made with the "three bead" geometry devised by Finer et al. (1994), in which two beads, supported in optical traps, are used to hold an actin filament in the vicinity of a myosin molecule, which is immobilized on the surface of a third bead. The movements and forces produced by actomyosin interactions were measured by detecting the position of both trapped beads. We measured, and corrected for, series compliance in the system, which otherwise introduces large errors. First, we used video image analysis to measure the long-range, force-extension property of the actin-to-bead connection (kappa con), which is the main source of "end compliance." We found that force-extension diagrams were nonlinear and rather variable between preparations, i.e., end compliance depended not only upon the starting tension, but also upon the F-actin-bead pair used. Second, we measured kappa xb and kappa con during a single cross-bridge attachment by driving one optical tweezer with a sinusoidal oscillation while measuring the position of both beads. In this way, the bead held in the driven optical tweezer applied force to the cross-bridge, and the motion of the other bead measured cross-bridge movement. Under our experimental conditions (at approximately 2 pN of pretension), connection stiffness (kappa con) was 0.26 +/- 0.16 pN nm-1. We found that rabbit heavy meromyosin produced a working stroke of 5.5 nm, and cross-bridge stiffness (kappa xb) was 0.69 +/- 0.47 pN nm-1.     

Distinct membrane mechanical properties of human mesenchymal stem cells determined using laser optical tweezers

The therapeutic efficacy of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is determined by their unique biological, mechanical, and physicochemical characteristics, which are yet to be fully explored. Cell membrane mechanics, for example, has been shown to critically influence MSC differentiation. In this study, we used laser optical tweezers to measure the membrane mechanics of human MSCs and terminally differentiated fibroblasts by extracting tethers from the outer cell membrane. The average tether lengths were 10.6+/-1.1 microm (hMSC) and 3.0+/-0.5 microm (fibroblasts). The tether extraction force did not increase during tether formation, which suggests existence of a membrane reservoir intended to buffer membrane tension fluctuations. Cytoskeleton disruption resulted in a fourfold tether length increase in fibroblasts but had no effect in hMSCs, indicating weak association between the cell membrane and hMSC actin cytoskeleton. Cholesterol depletion, known to decrease lipid bilayer stiffness, caused an increase in the tether length both in fibroblasts and hMSCs, as does the treatment of cells with DMSO. We postulate that whereas fibroblasts use both the membrane rigidity and membrane-cytoskeleton association to regulate their membrane reservoir, hMSC cytoskeleton has only a minor impact on stem cell membrane mechanics.     

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.     

Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments

Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.     

Unzipping DNA with optical tweezers: high sequence sensitivity and force flips

Force measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system.     

DNA as a metrology standard for length and force measurements with optical tweezers

Optical tweezers have broad applications in studies of structures and processes in molecular and cellular biophysics. Use of optical tweezers for quantitative molecular-scale measurement requires careful calibration in physical units. Here we show that DNA molecules may be used as metrology standards for force and length measurements. Analysis of DNA molecules of two specific lengths allows simultaneous determination of all essential measurement parameters. We validate this biological-calibration method experimentally and with simulated data, and show that precisions in determining length scale factor ( approximately 0.2%), length offset ( approximately 0.03%), force scale factor ( approximately 2%), and compliance of the traps ( approximately 3%) are limited only by current measurement variation, much of which arises from polydispersity of the microspheres ( approximately 2%). We find this procedure to be simpler and more convenient than previous methods, and suggest that it provides an easily replicated standard that can insure uniformity of measurements made in different laboratories.     

Stretching short biopolymers using optical tweezers

Optical tweezers is a useful technique to study single molecules. It has been applied to stretch biopolymers in several approaches. However, these approaches may not be appropriate to measure short biopolymers. In the present study, the optical signals of beads in different situations were studied. A new method was developed for stretching short biopolymers, as short as 300 nm in length. This new method was successfully applied to determine the stiffness of procollagen I molecules.     

Adherence of Staphylococcus aureus fibronectin binding protein A mutants: an investigation using optical tweezers

Bacterial adhesion to extracellular matrix proteins plays a major role in infections of host tissue and medical devices. In some species of gram-positive cocci, this adhesion is mediated by specific molecules present on the bacterial cell surface. We have used optical tweezers to dynamically measure the adhesive force between an individual Staphylococcus aureus bacterium and a fibronectin-coated surface. A bacterium was optically trapped and brought in contact with a 10-microm diameter polystyrene microsphere coated with fibronectin. The force required to detach the cell from the microsphere was measured by tracking the displacement signals of the trapped cell on a quadrant photodiode throughout the detachment process for a series of S. aureus strains expressing fibronectin-binding proteins with various degrees of mutation. The single-bond rupture forces ranged between 15 and 26 pN depending on the extent of mutation. No binding was observed in the strain with the highest degree of mutation. These results confirm that multiple regions of the S. aureus fibronectin adhesin participate in the binding process and provide further insight into the role of these regions in the adhesive process.     

A general method for manipulating DNA sequences from any organism with optical tweezers

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein–DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from ~10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino–carboxyl linkage, which allows for reliable DNA overstretching, is also described.     

Experimentally manipulating fungi with optical tweezers

A short review of the use of optical tweezers in fungal cell biological research is provided. First, we describe how optical tweezers work. Second, we review how they have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Third, we indicate how optically trapped microbeads can be used for the localized delivery of chemicals or mechanical stimulation to cells, as well as permitting measurements of the growth forces generated by germ tubes. Finally, the effects of optical trapping on fungal cell viability and growth are assessed. Parts of this review were presented at the Mycological Society of Japan (MSJ) / British Mycological Society (BMS) Joint Symposium, “The new generation mycologists in Japan and the UK” held in Chiba, Japan on June 3, 2006.     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.     

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.     

Sorting out bacterial viability with optical tweezers

We have developed a method, using laser, optical tweezers and direct microscopic analysis of reproductive potential and membrane integrity, to assess single-cell viability in a stationary-phase Escherichia coli population. It is demonstrated here that a reduction in cell integrity, determined by using the fluorescent nucleic acid stain propidium iodide, correlated well with a reduction in cell proliferating potential during the stationary-phase period studied. Moreover, the same cells that exhibited reduced integrity were found to be the ones that failed to divide upon nutrient addition. A small but significant number of the intact cells (496 of 7,466 [6.6%]) failed to replicate. In other words, we did not find evidence for the existence of a large population of intact but nonculturable cells during the stationary-phase period studied but it is clear that reproductive ability can be lost prior to the loss of membrane integrity. In addition, about 1% of the stationary-phase cells were able to divide only once upon nutrient addition, and in a few cases, only one of the two cells produced by division was able to divide a second time, indicating that localized cell deterioration, inherited by only one of the daughters, may occur. The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary-phase-induced bacterial death and population heterogeneity is discussed.