An Automated Two-dimensional Optical Force Clamp for Single Molecule Studies

Biophysical Journal. Jul, 2002  |  Pubmed ID: 12080136

We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.

Probing the Kinesin Reaction Cycle with a 2D Optical Force Clamp

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2003  |  Pubmed ID: 12591957

With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADPP(i) release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.

Combined Optical Trapping and Single-molecule Fluorescence

Journal of Biology. 2003  |  Pubmed ID: 12733997

Two of the mainstay techniques in single-molecule research are optical trapping and single-molecule fluorescence. Previous attempts to combine these techniques in a single experiment - and on a single macromolecule of interest - have met with little success, because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore. Instead, the two techniques have been employed sequentially, or spatially separated by distances of several micrometers within the sample, imposing experimental restrictions that limit the utility of the combined method. Here, we report the development of an instrument capable of true, simultaneous, spatially coincident optical trapping and single-molecule fluorescence.

Resource Letter: LBOT-1: Laser-based Optical Tweezers

American Journal of Physics. Mar, 2003  |  Pubmed ID: 16971965

This Resource Letter provides a guide to the literature on optical tweezers, also known as laser-based, gradient-force optical traps. Journal articles and books are cited for the following main topics: general papers on optical tweezers, trapping instrument design, optical detection methods, optical trapping theory, mechanical measurements, single molecule studies, and sections on biological motors, cellular measurements and additional applications of optical tweezers.

Simultaneous, Coincident Optical Trapping and Single-molecule Fluorescence

Nature Methods. Nov, 2004  |  Pubmed ID: 15782176

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

Interlaced Optical Force-fluorescence Measurements for Single Molecule Biophysics

Biophysical Journal. Aug, 2006  |  Pubmed ID: 16648165

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence, does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-basepair DNA segment labeled with Cy3.

Detecting Force-induced Molecular Transitions with Fluorescence Resonant Energy Transfer

Angewandte Chemie (International Ed. in English). 2007  |  Pubmed ID: 17279589

Force of an Actin Spring

Biophysical Journal. May, 2007  |  Pubmed ID: 17351007

Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine--a biological spring. During the acrosome reaction, a 60-microm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2 degrees /subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope.

Single M13 Bacteriophage Tethering and Stretching

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2007  |  Pubmed ID: 17360403

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution. With this system, we report the development of a SM assay based on M13 bacteriophage. We also report the quantitative characterization of the biopolymer's elasticity by using an optical trap with nanometer-scale position resolution. Expanding the fluctuating rod limit of the wormlike chain to incorporate enthalpic polymer stretching yielded a model capable of accurately capturing the full range of extensions. Fits of the force-extension measurements gave a mean persistence length of approximately 1,265 nm, lending SM support for a shorter filamentous bacteriophage persistence length than previously thought. Furthermore, a predicted stretching modulus roughly two times that of dsDNA, coupled with the system's linkage versatility and load-bearing capability, makes the M13 template an attractive candidate for use in tethered bead architectures.

Active Particle Control Through Silicon Using Conventional Optical Trapping Techniques

Lab on a Chip. Dec, 2007  |  Pubmed ID: 18030409

Functional integration of optical trapping techniques with silicon surfaces and environments can be realized with minimal modification of conventional optical trapping instruments offering a method to manipulate, track and position cells or non-biological particles over silicon substrates. This technique supports control and measurement advances including the optical control of silicon-based microfluidic devices and precision single molecule measurement of biological interactions at the semiconductor interface. Using a trapping laser in the near infra-red and a reflective imaging arrangement enables object control and measurement capabilities comparable to trapping through a classical glass substrate. The transmission efficiency of the silicon substrate affords the only reduction in trap stiffness. We implement conventional trap calibration, positioning, and object tracking over silicon surfaces. We demonstrate control of multiple objects including cells and complex non-spherical objects on silicon wafers and fabricated surfaces.

Force Generation in Kinesin Hinges on Cover-neck Bundle Formation

Structure (London, England : 1993). Jan, 2008  |  Pubmed ID: 18184584

In kinesin motors, a fundamental question concerns the mechanism by which ATP binding generates the force required for walking. Analysis of available structures combined with molecular dynamics simulations demonstrates that the conformational change of the neck linker involves the nine-residue-long N-terminal region, the cover strand, as an element that is essential for force generation. Upon ATP binding, it forms a beta sheet with the neck linker, the cover-neck bundle, which induces the forward motion of the neck linker, followed by a latch-type binding to the motor head. The estimated stall force and anisotropic response to external loads calculated from the model agree with force-clamp measurements. The proposed mechanism for force generation by the cover-neck bundle formation appears to apply to several kinesin families. It also elucidates the design principle of kinesin as the smallest known processive motor.

Measuring Molecular Rupture Forces Between Single Actin Filaments and Actin-binding Proteins

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2008  |  Pubmed ID: 18591676

Actin-binding proteins (ABPs) regulate the assembly of actin filaments (F-actin) into networks and bundles that provide the structural integrity of the cell. Two of these ABPs, filamin and alpha-actinin, have been extensively used to model the mechanical properties of actin networks grown in vitro; however, there is a lack in the understanding of how the molecular interactions between ABPs and F-actin regulate the dynamic properties of the cytoskeleton. Here, we present a native-like assay geometry to test the rupture force of a complex formed by an ABP linking two quasiparallel actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin and alpha-actinin. For filamin/actin and alpha-actinin/actin, we measured similar rupture forces of 40-80 pN for loading rates between 4 and 50 pN/s. Both ABP unfolding and conformational transition events were observed, demonstrating that both are important and may be a significant mechanism for the temporal regulation of the mechanical properties of the actin cytoskeleton. With this modular, single-molecule assay, a wide range of ABP/actin interactions can be studied to better understand cytoskeletal and cell dynamics.

Kinesin's Cover-neck Bundle Folds Forward to Generate Force

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2008  |  Pubmed ID: 19047639

Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a beta-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-terminal "dangling end," unlike the neck linker and nucleotide-binding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.

Mechanical Design of Translocating Motor Proteins

Cell Biochemistry and Biophysics. 2009  |  Pubmed ID: 19452133

Translocating motors generate force and move along a biofilament track to achieve diverse functions including gene transcription, translation, intracellular cargo transport, protein degradation, and muscle contraction. Advances in single molecule manipulation experiments, structural biology, and computational analysis are making it possible to consider common mechanical design principles of these diverse families of motors. Here, we propose a mechanical parts list that include track, energy conversion machinery, and moving parts. Energy is supplied not just by burning of a fuel molecule, but there are other sources or sinks of free energy, by binding and release of a fuel or products, or similarly between the motor and the track. Dynamic conformational changes of the motor domain can be regarded as controlling the flow of free energy to and from the surrounding heat reservoir. Multiple motor domains are organized in distinct ways to achieve motility under imposed physical constraints. Transcending amino acid sequence and structure, physically and functionally similar mechanical parts may have evolved as nature's design strategy for these molecular engines.

Single-molecule Denaturation and Degradation of Proteins by the AAA+ ClpXP Protease

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2009  |  Pubmed ID: 19892734

ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function.

The Alphabeta T Cell Receptor is an Anisotropic Mechanosensor

The Journal of Biological Chemistry. Nov, 2009  |  Pubmed ID: 19755427

Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric alphabeta, CD3 epsilon gamma, CD3 epsilon delta, and CD3 zeta zeta subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3 epsilon lobe that they approach diagonally, adjacent to the lever-like C beta FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3 epsilon and CD3 gamma in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force ( approximately 50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.

Passive and Active Microrheology for Cross-linked F-actin Networks in Vitro

Acta Biomaterialia. Apr, 2010  |  Pubmed ID: 19883801

Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.

Molecular Origin of Strain Softening in Cross-linked F-actin Networks

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Jul, 2010  |  Pubmed ID: 20866660

Two types of measurement are presented that relate molecular events to macroscopic behavior of F-actin networks. First, shear modulus is measured by oscillating an embedded microbead. Second, a microbead is translated at constant rate and transitions in the resisting force are observed. The loading rate dependence of the force at the transitions is similar to that of the molecular unbinding force, suggesting that they share a common origin. Reversibility tests of shear modulus provide further evidence that strain softening of F-actin networks is caused by force-induced rupture of cross-links.

Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

PloS One. 2010  |  Pubmed ID: 21217821

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

Motor Proteins: Kinesin Drives with an Underhead Cam

Current Biology : CB. May, 2010  |  Pubmed ID: 20462483

A high-resolution cryo-EM structure of kinesin bound to its microtubule track allows for near-atomistic visualization of nucleotide-dependent conformational changes in this motor protein.

Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils

Nature Structural & Molecular Biology. Dec, 2010  |  Pubmed ID: 21113168

Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length.

Quantification of Urinary Stone Volume: Attenuation Threshold-based CT Method--a Technical Note

Radiology. Mar, 2011  |  Pubmed ID: 21339353

To compare two threshold-based computed tomographic (CT) methods for the quantification of urinary stone volume; to assess their accuracy and precision at varying tube voltages, tube currents, and section thicknesses in a phantom; and to determine interobserver agreement with each of these methods in a pilot clinical study.

Single-molecule Protein Unfolding and Translocation by an ATP-fueled Proteolytic Machine

Cell. Apr, 2011  |  Pubmed ID: 21496645

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Physical Properties of Polymorphic Yeast Prion Amyloid Fibers

Biophysical Journal. Jul, 2011  |  Pubmed ID: 21767497

Amyloid fibers play important roles in many human diseases and natural biological processes and have immense potential as novel nanomaterials. We explore the physical properties of polymorphic amyloid fibers formed by yeast prion protein Sup35. Amyloid fibers that conferred distinct prion phenotypes ([PSI(+)]), strong (S) versus weak (W) nonsense suppression, displayed different physical properties. Both S[PSI(+)] and W[PSI(+)] fibers contained structural inhomogeneities, specifically local regions of static curvature in S[PSI(+)] fibers and kinks and self-cross-linking in W[PSI(+)] fibers. Force-extension experiments with optical tweezers revealed persistence lengths of 1.5 μm and 3.3 μm and axial stiffness of 5600 pN and 9100 pN for S[PSI(+)] and W[PSI(+)] fibers, respectively. Thermal fluctuation analysis confirmed the twofold difference in persistence length between S[PSI(+)] and W[PSI(+)] fibers and revealed a torsional stiffness of kinks and cross-links of ~100-200 pN·nm/rad.

Adhesion Through Single Peptide Aptamers

The Journal of Physical Chemistry. A. Apr, 2011  |  Pubmed ID: 20795685

Aptamer and antibody mediated adhesion is central to biological function and is valuable in the engineering of "lab on a chip" devices. Single molecule force spectroscopy using optical tweezers enables direct nonequilibrium measurement of these noncovalent interactions for three peptide aptamers selected for glass, polystyrene, and carbon nanotubes. A comprehensive examination of the strong attachment between antifluorescein 4-4-20 and fluorescein was also carried out using the same assay. Bond lifetime, barrier width, and free energy of activation are extracted from unbinding histogram data using three single molecule pulling models. The evaluated aptamers appear to adhere stronger than the fluorescein antibody under no- and low-load conditions, yet weaker than antibodies at loads above ∼25 pN. Comparison to force spectroscopy data of other biological linkages shows the diversity of load dependent binding and provides insight into linkages used in biological processes and those designed for engineered systems.

A Microfluidic System with Optical Laser Tweezers to Study Mechanotransduction and Focal Adhesion Recruitment

Lab on a Chip. Feb, 2011  |  Pubmed ID: 21152510

We present a new method to locally apply mechanical tensile and compressive force on single cells based on integration of a microfluidic device with an optical laser tweezers. This system can locate a single cell within customized wells exposing a square-like membrane segment to a functionalized bead. Beads are coated with extracellular matrix (ECM) proteins of interest (e.g. fibronectin) to activate specific membrane receptors (e.g. integrins). The functionalized beads are trapped and manipulated by optical tweezers to apply mechanical load on the ECM-integrin-cytoskeleton linkage. Activation of the receptor is visualized by accumulation of expressed fluorescent proteins. This platform facilitates isolation of single cells and excitation by tensile/compressive forces applied directly to the focal adhesion via specific membrane receptors. Protein assembly or recruitment in a focal adhesion can then be monitored and identified using fluorescent imaging. This platform is used to study the recruitment of vinculin upon the application of external tensile force to single endothelial cells. Vinculin appears to be recruited above the forced bead as an elliptical cloud, centered 2.1 ± 0.5 μm from the 2 μm bead center. The mechanical stiffness of the membrane patch inferred from this measurement is 42.9 ± 6.4 pN μm(-1) for a 5 μm × 5 μm membrane segment. This method provides a foundation for further studies of mechanotransduction and tensile stiffness of single cells.