Optical Trapping Measurements Research Articles

Progress in Optical Trapping and Spectroscopic Measurements of Airborne Particles

Progress in Optical Trapping and Spectroscopic Measurements of Airborne Particles

Introduction to Optical Tweezers: Background, System Designs, and Applications.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, allowing for accurate measurement of the forces applied to these objects. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes the technique well suited for the study of biological processes from the single-cell down to the single-molecule level. In this chapter, we aim to provide an introduction to the use of optical tweezers for single-molecule analyses. We start from the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance. Finally, we will provide an overview of the broad applications in context of biological research, with the emphasis on the measurement modes, experimental assays, and possible combinations with fluorescence microscopy techniques.

Multi-level Force-dependent Allosteric Enhancement of αE-catenin Binding to F-actin by Vinculin

Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell–cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell–cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (−) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (−) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

Switch-like control of helicase processivity by single-stranded DNA binding protein.

Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. Ferroplasma acidarmanus xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent 'processivity switch' in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.

FRET and Optical Trapping Measurements Reveal Relationship between Phosphate Release and the Power Stroke in Myosin V

Myosin V is able to processively walk along actin filaments by coordinating its mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. Our previous FRET measurements of the power stroke in myosin V containing a single IQ domain (MV 1IQ) revealed a fast rotation of the lever arm prior to the phosphate release step and a slower rotation of the lever that occurs during the transition between actomyosin.ADP states. Here we introduce a mutation in the active site (S217A) that is proposed to slow entry of phosphate into the exit tunnel. The mutation causes both fast lever arm rotation and phosphate release to occur with the same rate constant (slowing the maximum rate 10-fold), suggesting entry of phosphate into the exit tunnel gates the lever arm swing. In single molecule laser trapping studies we found the step size of S217A was similar to WT (4nm). Furthermore, raising the Pi concentration in the laser trap assay, which should increase the population of the actomyosin.ADP.Pi state, also did not affect the step size of S217A MV 1IQ. Interestingly, the attached time increased dramatically in the presence of 15 mM Pi. We also found that Pi can inhibit actin gliding in the vitro motility assay in both S217A and WT MV 1IQ in a dose-dependent manner, which is consistent with the optical trapping studies, while Pi did not impact steady-state ATPase activity. Our results suggest a model in which attachment to actin and the power stroke (lever arm swing) occur rapidly before phosphate is released in myosin V. In addition, Pi rebinding to the actomyosin.ADP state may be capable of reversing the power stroke without altering the actin binding interface.

Stiffness of Cargo-Motor Linkage Tunes Myosin VI Motility and Response to Load.

We examine the effect of cargo-motor linkage stiffness on the mechanobiological properties of the molecular motor myosin VI. We use the programmability of DNA nanostructures to modulate cargo-motor linkage stiffness and combine it with high-precision optical trapping measurements to measure the effect of linkage stiffness on the motile properties of myosin VI. Our results reveal that a stiff cargo-motor linkage leads to shorter step sizes and load-induced anchoring of myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. Our findings suggest a novel regulatory mechanism for tuning the dual cellular roles of the anchor and transporter ascribed to myosin VI.

Probing the Basis of α-Synuclein Aggregation by Comparing Simulations to Single-Molecule Experiments

Intrinsically disordered proteins often play an important role in protein aggregation. However, it is challenging to determine the structures and interactions that drive the early stages of aggregation because they are transient and obscured in a heterogeneous mixture of disordered states. Even computational methods are limited because the lack of ordered structure makes it difficult to ensure that the relevant conformations are sampled. We address these challenges by integrating atomistic simulations with high-resolution single-molecule measurements reported previously, using the measurements to help discern which parts of the disordered ensemble of structures in the simulations are most probable while using the simulations to identify residues and interactions that are important for oligomer stability. This approach was applied to α-synuclein, an intrinsically disordered protein that aggregates in the context of Parkinson’s disease. We simulated single-molecule pulling experiments on dimers, the minimal oligomer, and compared them to force spectroscopy measurements. Force-extension curves were simulated starting from a set of 66 structures with substantial structured content selected from the ensemble of dimer structures generated at zero force via Monte Carlo simulations. The pattern of contour length changes as the structures unfolded through intermediate states was compared to the results from optical trapping measurements on the same dimer to discern likely structures occurring in the measurements. Simulated pulling curves were generally consistent with experimental data but with a larger number of transient intermediates. We identified an ensemble of β-rich dimer structures consistent with the experimental data from which dimer interfaces could be deduced. These results suggest specific druggable targets in the structural motifs of α-synuclein that may help prevent the earliest steps of oligomerization.

Transformation of the Nonprocessive Fast Skeletal Myosin II into a Processive Motor.

Myosin family motors play diverse cellular roles. Precise insights into how the light chains contribute to the functional variabilities among myosin motors, however, remain unresolved. Here, it is demonstrated that the fast skeletal muscle myosin II isoform myosin heavy chain (MHC-IID) can be transformed into a processive motor, by simply replacing the native regulatory light chain MLC2f with the regulatory light chain variant MLC2v from the slow muscle myosin II. Single molecule kinetic analyses and optical trapping measurements of the hybrid motor reveal marked changes such as increased association rate of myosin toward adenosine triphosphate (ATP) and actin by more than twofold. The direct consequence of high adenosine diphosphate (ADP) affinity and increased actin rebinding is the altered overall actomyosin association time during the cross-bridge cycle. The data indicate that the MLC2v influences the duty ratio in the hybrid motor, suggestive of promoting interhead communication and enabling processive movement. This finding establishes that the regulatory light chain fine-tunes the motor's mechanical output that may have important implications under physiological conditions. Furthermore, the success of this approach paves the way to engineer motors from a known motor protein element to assemble highly specialized biohybrid machines for potential applications in nano-biomedicine and engineering.

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams.

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus-end directed kinesin and minus-end directed dynein motors. Microtubules are decorated by microtubule-associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single-molecule assays indicate that kinesin-1 is more strongly inhibited than kinesin-2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin-1, kinesin-2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus-end in a dose-dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin-1, kinesin-2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor-specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus- and minus-end directed transport.

Introduction to Optical Tweezers: Background, System Designs, and Commercial Solutions.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, while forces on the trapped objects can be accurately measured and exerted. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes them excellently suited to study biological processes from the single-cell down to the single-molecule level. In this chapter, we will provide an introduction on the use of optical tweezers in single-molecule approaches. We will introduce the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next we describe the components of an optical tweezers setup and their experimental relevance in single-molecule approaches. Finally, we provide a concise overview of commercial optical tweezers systems. Commercial systems are becoming increasingly available and provide access to single-molecule optical tweezers experiments without the need for a thorough background in physics.

The RNA helicase Mtr4p is a duplex-sensing translocase.

The conserved Saccharomyces cerevisiae Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3'-to-5' translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p molecule performs consecutive unwinding steps. We further show that RNA unwinding by Mtr4p requires interaction with upstream RNA duplex. Inclusion of Mtr4p within the TRAMP complex increases the rate constant for unwinding initiation but does not change the characteristics of Mtr4p's helicase mechanism. Our data indicate that Mtr4p utilizes a previously unknown unwinding mode that combines aspects of canonical translocating helicases and non-canonical duplex-sensing helicases, thereby restricting directional translocation to duplex regions.

Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution.

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

Quantifying Instrumental Artifacts in Folding Kinetics Measured by Single-Molecule Force Spectroscopy

Force spectroscopy is commonly used to measure the kinetics of processes occurring in single biological molecules. These measurements involve attaching the molecule of interest to micron-sized or larger force probes via compliant linkers. Recent theoretical work has described how the properties of the probes and linkers can alter the observed kinetics from the intrinsic behavior of the molecule in isolation. We applied this theory to estimate the errors in measurements of folding made using optical tweezers. Errors in the folding rates arising from instrument artifacts were only ∼20% for constant-force measurements of DNA hairpins with typical choices of linker length and probe size. Measurements of transition paths using a constant trap position at high trap stiffness were also found to be in the low-artifact limit. These results indicate that typical optical trap measurements of kinetics reflect the dynamics of the molecule fairly well, and suggest practical limitations on experimental design to ensure reliable kinetic measurements.

RNA Unwinding by the Helicase Mtr4p and the TRAMP Complex Investigated via High-Resolution Optical Trapping

We present single-molecule high-resolution optical trapping measurements of RNA unwinding by the Ski2-like nuclear helicase Mtr4p both alone and within the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex. RNA helicases such as Mtr4p play key roles in RNA metabolism. Mtr4p is thought to assist RNA degradation by the exosome presumably by unwinding RNA structures. In addition, the TRAMP complex appends short 3′ poly(A) tails to RNA meant for degradation by the exosome. Mtr4p within the TRAMP complex plays a crucial role in regulating the polyadenylation activity, limiting the poly(A) tail length. The unwinding activity of Mtr4p is required and indeed within the TRAMP complex, Mtr4p unwinds duplex RNA 10x faster than when alone. However, the dynamic details of the RNA unwinding activity and interaction of Mtr4p and TRAMP and indeed the Ski2-like family of RNA helicases are unknown. We have performed high-resolution RNA hairpin unwinding experiments for both Mtr4p and TRAMP. We find that Mtr4p unwinds RNA duplexes in a single or multiple bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4p remains stably bound after unwinding the duplex, preventing re-annealing. TRAMP unwinding experiments reveal a faster molecular recognition by the Mtr4p helicase when in complex as well as enhanced RNA duplex unwinding dynamics. Combined high-resolution trapping and fluorescence experiments underway may reveal how the competition within TRAMP between the 3′-to-5′ Mtr4p helicase and the 5′-to-3′ Trf4p polymerase is resolved dynamically.

Selective Adsorption of Functionalized Nanoparticles to Patterned Polymer Brush Surfaces and Its Probing with an Optical Trap

The site-specific attachment of nanoparticles is of interest for biomaterials or biosensor applications. Polymer brushes can be used to regulate this adsorption, so the conditions for selective adsorption of phosphonate-functionalized nanoparticles onto micropatterned polymer brushes with different functional groups are optimized. By choosing the strong polyelectrolytes poly(3-sulfopropyl methacrylate), poly(sulfobetaine methacrylate), and poly[2-(methacryloyloxy)ethyl trimethylammonium chloride], it is possible to direct the adsorption of nanoparticles to specific regions of the patterned substrates. A pH-dependent adsorption can be achieved by using the polycarboxylate brush poly(methacrylic acid) (PMAA) as substrate coating. On PMAA brushes, the nanoparticles switch from attachment to the brush regions to attachment to the grooves of a patterned substrate on changing the pH from 3 to 7. In this manner, patterned substrates are realized that assemble nanoparticles in pattern grooves, in polymer brush areas, or substrates that resist the deposition of the nanoparticles. The nanoparticle deposition can be directed in a pH-dependent manner on a weak polyelectrolyte, or is solely charge-dependent on strong polyelectrolytes. These results are correlated with surface potential measurements and show that an optical trap is a versatile method to directly probe interactions between nanoparticles and polymer brushes. A model for these interactions is proposed based on the optical trap measurements.

Teamwork in microtubule motors

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.

Kinetics and Thermodynamics of Nucleosome Winding and Unwinding

Nucleosome post-translational modifications (PTMs), histone variants, and cofactors can activate and repress transcription of the DNA they package. Modified and variant nucleosomes are thought to regulate transcription by two mechanisms acting independently or in concert: “Tuning” the DNA binding affinity, or recruiting additional cofactors. To truly understand the regulation of nucleosome packaging processes, it is essential to deconvolute the effects of cofactor recruitment from the effects of changes in binding affinity. To distinguish these two mechanisms, we use single molecule optical trap measurements of nucleosomes under force to measure the kinetics of transitions between nucleosome states. Using a system of known phenotypic response (the sin mutant nucleosomes) we find that nucleosome variations affect the transition rates between states of nucleosome unwinding. From these rates, we are able to determine the free energy difference induced by a nucleosome variation, thus correlating a phenotypic response with rates and energy and giving mechanistic meaning to chromatin “loosening”. Our measurement of the energy serves as a benchmark against which the kinetics and free energies of nucleosomes containing histones with PTMs, variants, or cofactors may be compared to in future studies.

Practical axial optical trapping

We describe a new method for calibrating optical trapping measurements in which tension is applied in the direction of the laser beam to a molecule tethered between a surface and an optically trapped bead. Specifically, we present a generally-applicable procedure for converting from the measured scattering intensity and the measured stage displacement to applied tension and bead-coverslip separation, using measurements of the light intensity scattered from an untethered, trapped bead. Our calibration accounts for a number of effects, including aberrations and the interference of forward-reflected bead-scattered light with the trapping beam. To demonstrate the accuracy of our method, we show measurements of the DNA force-versus-extension relation using a range of laser intensities, and show that these measurements match the expected extensible wormlike-chain (WLC) behavior. Finally, we also demonstrate a force-clamp, in which the tension in a tether is held fixed while the extension varies as a result of molecular events.