Cell cytoskeleton and tether extraction

We perform a detailed investigation of the force × deformation curve in tether extraction from 3T3 cells by optical tweezers. Contrary to conventional wisdom about tethers extracted from cells, we find that actin filaments are present within them, so that a revised theory of tether pulling from cells is called for. We also measure steady and maximum tether force values significantly higher than previously published ones for 3T3 cells. Possible explanations for these differences are investigated. Further experimental support of the theory of force barriers for membrane tube extension is obtained. The potential of studies on tether pulling force × deformation for retrieving information on membrane-cytoskeleton interaction is emphasized.     

Effects of chlorpromazine on mechanical properties of the outer hair cell plasma membrane

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.     

Coalescence of membrane tethers: experiments, theory, and applications

Tethers are nanocylinders of lipid bilayer membrane, arising in situations ranging from micromanipulation experiments on synthetic vesicles to the formation of dynamic tubular networks in the Golgi apparatus. Relying on the extensive theoretical and experimental works aimed to understand the physics of individual tethers formation, we addressed the problem of the interaction between two nanotubes. By using a combination of micropipette manipulation and optical tweezers, we quantitatively studied the process of coalescence that occurred when the separation distance between both vesicle-tether junctions became smaller than a threshold length. Our experiments, which were supported by an original theoretical analysis, demonstrated that the measurements of the tether force and angle between tethers at coalescence directly yield the bending rigidity, kappa, and the membrane tension, sigma, of the vesicles. Contrary to other methods used to probe the bending rigidity of vesicles, the proposed approach permits a direct measurement of kappa without requiring any control of the membrane tension. Finally, after validation of the method and proposal of possible applications, we experimentally investigated the dynamics of the coalescence process.     

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.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Visco-elastic membrane tethers extracted from Escherichia coli by optical tweezers

Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10-12 pN/μm describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6-7 pN/μm, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion.     

Step length measurement--theory and simulation for tethered bead constant-force single molecule assay

Linear molecular motors translocate along polymeric tracks using discrete steps. The step length is usually measured using constant-force single molecule experiments in which the polymer is tethered to a force-clamped microsphere. During the enzymatic cycle the motor shortens the tether contour length. Experimental conditions influence the achievable step length resolution, and ideally experiments should be conducted with high clamp-force using slow motors linked to small beads via stiff short tethers. We focus on the limitations that the polymer-track flexibility, the thermal motion of the microsphere, and the motor kinetics pose for step-length measurement in a typical optical tweezers experiment. An expression for the signal/noise ratio in a constant-force, worm-like chain tethered particle, single-molecule experiment is developed. The signal/noise ratio is related to the Fourier transform of the pairwise distance distribution, commonly used to determine step length from a time-series. Monte Carlo simulations verify the proposed theory for experimental parameter values typically encountered with molecular motors (polymerases and helicases) translocating along single- or double-stranded nucleic acids. The predictions are consistent with recent experimental results for double-stranded DNA tethers. Our results map favorable experimental conditions for observing single motor steps on various substrates but indicate that principal resolution limits are set by thermal fluctuations.     

Eukaryotic membrane tethers revisited using magnetic tweezers

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.     

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers.

Many cell phenomena involve major morphological changes, particularly in mitosis and the process of cell migration. For cells or neuronal growth cones to migrate, they must extend the leading edge of the plasma membrane as a lamellipodium or filopodium. During extension of filopodia, membrane must move across the surface creating shear and flow. Intracellular biochemical processes driving extension must work against the membrane mechanical properties, but the forces required to extend growth cones have not been measured. In this paper, laser optical tweezers and a nanometer-level analysis system were used to measure the neuronal growth cone membrane mechanical properties through the extension of filopodia-like tethers with IgG-coated beads. Although the probability of a bead attaching to the membrane was constant irrespective of treatment; the probability of forming a tether with a constant force increased dramatically with cytochalasin B or D and dimethylsulfoxide (DMSO). These are treatments that alter the organization of the actin cytoskeleton. The force required to hold a tether at zero velocity (F0) was greater than forces generated by single molecular motors, kinesin and myosin; and F0 decreased with cytochalasin B or D and DMSO in correlation with the changes in the probability of tether formation. The force of the tether on the bead increased linearly with the velocity of tether elongation. From the dependency of tether force on velocity of tether formation, we calculated a parameter related to membrane viscosity, which decreased with cytochalasin B or D, ATP depletion, nocodazole, and DMSO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improving signal/noise resolution in single-molecule experiments using molecular constructs with short handles

We investigate unfolding/folding force kinetics in DNA hairpins exhibiting two and three states with newly designed short dsDNA handles (29 bp) using optical tweezers. We show how the higher stiffness of the molecular setup moderately enhances the signal/noise ratio (SNR) in hopping experiments as compared to conventional long-handled constructs (≅700 bp). The shorter construct results in a signal of higher SNR and slower folding/unfolding kinetics, thereby facilitating the detection of otherwise fast structural transitions. A novel analysis, as far as we are aware, of the elastic properties of the molecular setup, based on high-bandwidth measurements of force fluctuations along the folded branch, reveals that the highest SNR that can be achieved with short handles is potentially limited by the marked reduction of the effective persistence length and stretch modulus of the short linker complex.     

Characteristics of a membrane reservoir buffering membrane tension.

When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.     

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.     

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.     

Characterization of Photoactivated Singlet Oxygen Damage in Single-Molecule Optical Trap Experiments

Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols.     

Magnetic forces and DNA mechanics in multiplexed magnetic tweezers

Magnetic tweezers (MT) are a powerful tool for the study of DNA-enzyme interactions. Both the magnet-based manipulation and the camera-based detection used in MT are well suited for multiplexed measurements. Here, we systematically address challenges related to scaling of multiplexed magnetic tweezers (MMT) towards high levels of parallelization where large numbers of molecules (say 10³) are addressed in the same amount of time required by a single-molecule measurement. We apply offline analysis of recorded images and show that this approach provides a scalable solution for parallel tracking of the xyz-positions of many beads simultaneously. We employ a large field-of-view imaging system to address many DNA-bead tethers in parallel. We model the 3D magnetic field generated by the magnets and derive the magnetic force experienced by DNA-bead tethers across the large field of view from first principles. We furthermore experimentally demonstrate that a DNA-bead tether subject to a rotating magnetic field describes a bicircular, Limaçon rotation pattern and that an analysis of this pattern simultaneously yields information about the force angle and the position of attachment of the DNA on the bead. Finally, we apply MMT in the high-throughput investigation of the distribution of the induced magnetic moment, the position of attachment of DNA on the beads, and DNA flexibility. The methods described herein pave the way to kilo-molecule level magnetic tweezers experiments.     

DNA,Flexibly Flexible

Investigators have constructed dsDNA molecules with several different base modifications and have characterized their bending and twisting flexibilities using atomic force microscopy, DNA ring closure, and single-molecule force spectroscopy with optical tweezers. The three methods provide persistence length measurements that agree semiquantitatively, and they show that the persistence length is surprisingly similar for all of the modified DNAs. The circular dichroism spectra of modified DNAs differ substantially. Simple explanations based on base stacking strength, polymer charge, or groove occupancy by functional groups cannot explain the results, which will guide further high-resolution theory and experiments.     

Mechanical equilibrium of thick, hollow, liquid membrane cylinders.

The mechanical equilibrium of bilayer membrane cylinders is analyzed. The analysis is motivated by the observation that mechanically formed membrane strands (tethers) can support significant axial loads and that the tether radius varies inversely with the axial force. Previously, thin shell theory has been used to analyze the tether formation process, but this approach is inadequate for describing and predicting the equilibrium state of the tether itself. In the present work the membrane is modeled as two adjacent, thick, anisotropic liquid shells. The analysis predicts an inverse relationship between axial force and tether radius, which is consistent with experimental observation. The area expansivity modulus and bending stiffness of the tether membrane are calculated using previously measured values of tether radii. These calculated values are consistent with values of membrane properties measured previously. Application of the analysis to precise measurements of the relationship between tether radius and axial force will provide a novel method for determining the mechanical properties of biomembrane.     

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.     

Influence of thermally driven surface undulations on tethers formed from bilayer membranes

Tether formation is a powerful method to study the mechanical properties of soft lipid bilayer membranes. The force required to maintain a tether at a given length depends upon both membrane elastic properties and tension. In this report, we develop a theoretical analysis that considers the contribution of thermally driven surface undulations and the corresponding entropically driven tensions on the conformation of tethers formed from unaspirated lipid vesicles. In this model, thermal undulations of the vesicle surface provide the excess area required for tether formation. Energy minimization demonstrates the dependence of equilibrium tether conformation on membrane tension and provides an analytical relationship between tether force and radius. If the contributions of nonlocal bending are not considered, an analytical relationship between tether force and length can also be obtained. The predictions of the model are compared to recently reported experimental data, and a value for the initial vesicle tension is obtained. Since most analyses of tether formation from cells and unaspirated vesicles neglect the contributions of nonlocal bending, the appropriateness of this assumption is analyzed. The effect of surface microvesiculations on the tether force-length relation is also considered.