Optical Trapping Assay Research Articles

Optical trapping assays to study force-dependent mobility of the diffusing microtubule-associated proteins

Optical trapping assays to study force-dependent mobility of the diffusing microtubule-associated proteins

Theoretical analysis of cargo transport by catch bonded motors in optical trapping assays.

Dynein motors exhibit catch bonding, where the unbinding rate of the motors from microtubule filaments decreases with increasing opposing load. The implications of this catch bond on the transport properties of dynein-driven cargo are yet to be fully understood. In this context, optical trapping assays constitute an important means of accurately measuring the forces generated by molecular motor proteins. We investigate, using theory and stochastic simulations, the transport properties of cargo transported by catch bonded dynein molecular motors - both singly and in teams - in a harmonic potential, which mimics the variable force experienced by cargo in an optical trap. We estimate the biologically relevant measures of first passage time - the time during which the cargo remains bound to the microtubule and detachment force - the force at which the cargo unbinds from the microtubule, using both two-dimensional and one-dimensional force balance frameworks. Our results suggest that even for cargo transported by a single motor, catch bonding may play a role depending on the force scale which marks the onset of the catch bond. By comparing with experimental measurements on single dynein-driven transport, we estimate realistic bounds of this catch bond force scale. Generically, catch bonding results in increased persistent motion, and can also generate non-monotonic behaviour of first passage times. For cargo transported by multiple motors, emergent collective effects due to catch bonding can result in non-trivial re-entrant phenomena wherein average first passage times and detachment forces exhibit non-monotonic behaviour as a function of the stall force and the motor velocity.

Cardiac myosin's mechanics and load dependent kinetics are not altered by the thin-filament proteins.

The force necessary to power human heart contraction is generated by beta cardiac myosin pulling on regulated thin filaments. Myosin's force generation is a tightly regulated process, and its disruption causes diseases including genetic cardiomyopathies. Deciphering the details of force generation at the molecular scale is critical for our understanding of both cardiac physiology and disease. While excellent studies have elucidated the mechanics and kinetics underlying the interactions between cardiac myosin and actin at the single molecule scale, the majority of these studies have been conducted in the absence of regulatory proteins. In the case of several other myosin isoforms, regulatory proteins alter the mechanics and load-dependent kinetics of the myosin working stroke, and it is not clear what role, if any they play in tuning the cardiac myosin working stroke and load-dependent kinetics. Here, we used a single molecule optical trapping assay to measure myosin's mechanics in the presence and absence of reconstituted thin-filaments (RTFs). We observed that regulatory proteins do not affect the myosin working stroke or its substeps. Interestingly, at low ATP, we observed that regulatory proteins slow the rate of actomyosin dissociation, and we showed using stopped flow kinetics that this is due to tuning of the rate of ATP-induced dissociation. The difference in the dissociation rate disappears at physiologically relevant ATP concentrations. We also used an isometric force clamp to investigate the load dependence of the myosin working stroke at physiologically relevant ATP concentrations, and this was also unchanged with RTFs. Taken together, these results show that cardiac myosin's mechanics are not altered by regulatory proteins at physiologically relevant ATP concentrations, and they provide important insights into the molecular mechanisms underlying heart contraction.

Dissecting actin-microtubule crosstalk with a novel multi-filament optical trapping assay.

The dynamic mechanical properties of the cytoskeleton are typically not the sum from those of its constituent proteins, and optical trapping can be used to probe such emergent mechanics. Previous optical trapping experiments tend to focus on the mechanics of individual proteins within the same cytoskeletal family. However, the cytoskeleton is a hierarchical ordered system, and the resulting emergent mechanics are difficult to identify or predict from studying proteins at the single molecule level. Building upon these reductionist assays, we seek to develop a more structurally relevant in vitro assay that incorporates multiple cytoskeletal filaments in addition to accessory proteins. To better understand how seemingly disparate cytoskeletal proteins and filaments work together to facilitate cellular-level dynamics, we constructed an in vitro layered bundle assay, comprising proteins and filaments from both the microtubule and actin cytoskeleton, and measured system mechanics using optical tweezers. In particular, the crosslinker protein anillin and the motor protein myosin II were assayed. Anillin can interact with both microtubules and actin and has recently been demonstrated to have the ability to generate pN-level forces as a crosslinker. The interplay of such crosslinking with myosin, whose motility dynamics change when going from single molecules to ensembles, will reveal how entropic crosslinkers, dynamic motor proteins, and filaments of different stiffness facilitate communication at the molecular scale and throughout the local cytoskeletal environment.

Lon protease degrades stable substrates slowly but with enhanced processivity, redefining the model of a strong and robust AAA+ protease.

AAA+ proteases function in all domains of life using an ATP-dependent unfoldase/polypeptide-translocase motor. Lon is a highly conserved AAA+ protease especially important for degrading oxidatively-damaged proteins. To address if Lon is a weak vs. robust unfoldase, as its substrates are often poorly folded, and elucidate whether its mechanism of translocation is similar to the previously-characterized AAA+ proteases, ClpXP and ClpAP, which have a distinct subunit assembly/architecture, we used a modified single-bead optical trapping assay. We find that Lon: (i) unfolds stable proteins using substantially longer pre-unfolding dwell times compared to ClpXP/ClpAP but (ii) has a higher probability of fully degrading a multi-domain substrate than the other enzymes. (iii) Although a slower translocase, Lon's fundamental step size is shared by ClpXP/ClpAP and its step-size distribution mirrors that of ClpAP. (iv) Finally, both ClpXP and Lon hydrolyze ∼1 ATP per translocation step. Thus, the mechanism of the ATP hydrolysis-driven ‘power stroke’ appears largely conserved between three distinct AAA+ proteases, regardless of their speed, processivity, or whether the ATPase and peptidase modules are physically linked (Lon) or assembled from distinct polypeptides (ClpXP and ClpAP). Lon therefore likely uses a common set of operating principles but is ‘geared differently’ than other AAA+ proteases.

Studying Dynein Mechanochemistry with an Optical Trap.

Molecular motors generate force and mechanical work to perform some of the most energy-demanding cellular processes, such as whole cell motility and cell division. These motors experience resistance from the viscoelastic environment of the surrounding cytoplasm, and opposing forces that can originate from other motors bound to cytoskeleton. Optical trapping is the most widely used method to measure the force-generating and force-responsecharacteristics of motor proteins. Here we present the methodologies of three different optical trapping assays we use to measure how forces originating from external factors affect the microtubule-detachment rate and velocity of dynein. We also briefly discuss the remaining challenges and future directions of optical trapping studies of dyneins and other microtubule-based motors.

Myosin II Adjusts Motility Properties and Regulates Force Production Based on Motor Environment.

Myosin II has been investigated with optical trapping, but single motor-filament assay arrangements are not reflective of the complex cellular environment. To understand how myosin interactions propagate up in scale to accomplish system force generation, we devised a novel actomyosin ensemble optical trapping assay that reflects the hierarchy and compliancy of a physiological environment and is modular for interrogating force effectors. Hierarchical actomyosin bundles were formed in vitro. Fluorescent template and cargo actin filaments (AF) were assembled in a flow cell and bundled by myosin. Beads were added in the presence of ATP to bind the cargo AF and activate myosin force generation to be measured by optical tweezers. Three force profiles resulted across a range of myosin concentrations: high force with a ramp-plateau, moderate force with sawtooth movement, and baseline. The three force profiles, as well as high force output, were recovered even at low solution concentration, suggesting that myosins self-optimize within AFs. Individual myosin steps were detected in the ensemble traces, indicating motors are taking one step at a time while others remain engaged in order to sustain productive force generation. Motor communication and system compliancy are significant contributors to force output. Environmental conditions, motors taking individual steps to sustain force, the ability to backslip, and non-linear concentration dependence of force indicate that the actomyosin system contains a force-feedback mechanism that senses the local cytoskeletal environment and communicates to the individual motors whether to be in a high or low duty ratio mode. The online version contains supplementary material available at 10.1007/s12195-022-00731-1.

The membrane-actin linker ezrin acts as a sliding anchor.

Protein linkages to filamentous (F)–actin provide the cell membrane with mechanical stability and support intricate membrane architectures. However, the actin cytoskeleton is highly dynamic and undergoes rapid changes in shape during cell motility and other processes. The molecular mechanisms that generate a mechanically robust yet fluid connection between the membrane and actin cytoskeleton remain poorly understood. Here, we adapted a single-molecule optical trap assay to examine how the prototypical membrane-actin linker ezrin acts to anchor F-actin to the cell membrane. We find that ezrin forms a complex that slides along F-actin over micrometer distances while resisting detachment by forces oriented perpendicular to the filament axis. The ubiquity of ezrin and analogous proteins suggests that sliding anchors such as ezrin may constitute an important but overlooked element in the construction of the actin cytoskeleton.

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Measuring force generation within reconstituted microtubule bundle assemblies using optical tweezers.

Kinesins and microtubule associated proteins (MAPs) are critical to sustain life, facilitating cargo transport, cell division, and motility. To interrogate the mechanistic underpinnings of their function, these microtubule-based motors and proteins have been studied extensively at the single molecule level. However, a long-standing issue in the single molecule biophysics field has been how to investigate motors and associated proteins within a physiologically relevant environment in vitro. While the one motor/one filament orientation of a traditional optical trapping assay has revolutionized our knowledge of motor protein mechanics, this reductionist geometry does not reflect the structural hierarchy in which many motors work within the cellular environment. Here, we review approaches that combine the precision of optical tweezers with reconstituted ensemble systems of microtubules, MAPs, and kinesins to understand how each of these unique elements work together to perform large scale cellular tasks, such as but not limited to building the mitotic spindle. Not only did these studies develop novel techniques for investigating motor proteins in vitro, but they also illuminate ensemble filament and motor synergy that helps bridge the mechanistic knowledge gap between previous single molecule and cell level studies.

Cholesterol in Cargo Membrane Amplifies inhibitory Effect of Map Tau On Kinesin-1

Motor protein-based transport underlies all eukaryotic cell function and survival; dysfunctions are implicated in many diseases including neurodegeneration. Whereas motor proteins have been extensively studied both in vivo and in vitro, many important questions remain. Chief among these is whether and how the composition of the cargo membrane impacts transport. To address this question, we combined advances in membrane biophysics with established single-molecule optical-trapping assays to characterize the transport of membrane-enclosed cargos in vitro.

Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.

MAP7 Targets Intracellular Transport towards the Microtubule Plus End by Recruiting Kinesin-1 to Microtubules

Microtubule-associated proteins (MAPs) play well-characterized roles in regulating microtubule polymerization dynamics and organization. In addition, MAPs modulate the interactions between motor proteins and microtubules. By differentially regulating kinesin and dynein motors, MAPs direct trafficking towards the microtubule plus or minus end. MAP7 (ensconsin, E-MAP-115) is a ubiquitous microtubule-associated protein that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also mediates the interaction of kinesin-1 with microtubules and in some systems is required for kinesin-1 function. We expressed and purified full-length kinesin-1 and MAP7 in Sf9 cells. MAP7 recruits kinesin-1 to microtubules, increasing the frequency of both diffusive and processive runs. We performed optical trapping assays on beads transported by single and teams of kinesin-1 motors. We find that MAP7 increases the relative binding rate of single kinesin-1 motors by ∼25% and teams of kinesin-1 motors by ∼50%. To examine the role of MAP7 in regulating bidirectional transport, we isolated late phagosomes along with their native set of kinesin-1, kinesin-2, dynein motors. The fraction of motility towards the plus end shifts from 55% to 78% in the presence of 10 nM MAP7. The relative binding rate of kinesin motors increases by ∼30% while the dynein binding increases by ∼10%. Collectively, our results indicate that MAP7 enhances kinesin binding such that kinesin teams resist opposing loads and stay engaged on the microtubules for a longer period of time before detaching. MAP7 targets cargoes to the microtubule plus end in contrast to tau which reduces kinesin processivity to shift transport towards the microtubule minus end. Thus, each MAP has specific effects on motor function. Through mediating the interaction between motors and the microtubule lattice, MAPs tune the balance of plus- and minus-end directed transport.

Transcription Factor Regulation of RNA Polymerase's Torque Generation Capacity

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN⋅nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.

Transcription factor regulation of RNA polymerase’s torque generation capacity

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN⋅nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.

Processivity of dimeric kinesin-1 molecular motors.

Kinesin‐1 is a homodimeric motor protein that can move along microtubule filaments by hydrolyzing ATP with a high processivity. How the two motor domains are coordinated to achieve such high processivity is not clear. To address this issue, we computationally studied the run length of the dimer with our proposed model. The computational data quantitatively reproduced the puzzling experimental data, including the dramatically asymmetric character of the run length with respect to the direction of external load acting on the coiled‐coil stalk, the enhancement of the run length by addition of phosphate, and the contrary features of the run length for different types of kinesin‐1 with extensions of their neck linkers compared with those without extension of the neck linker. The computational data on other aspects of the movement dynamics such as velocity and durations of one‐head‐bound and two‐head‐bound states in a mechanochemical coupling cycle were also in quantitative agreement with the available experimental data. Moreover, predicted results are provided on dependence of the run length upon external load acting on one head of the dimer, which can be easily tested in the future using single‐molecule optical trapping assays.

Bio-Functionalized Core-Shell Microparticles for High Force Optical Trapping

Optical traps are widely used to study the mechanics of biomolecules and biopolymers at the single-molecule level due to their piconewton and nanometer resolution. Recently, Jannasch et al. developed core-shell particles with optical properties that enhance trapping ability and are promising candidates for high-force experiments. To fully harness these particles in biophysical assays, a straightforward method for functionalizing these particles with biocompatible handles is required. We developed a robust synthetic strategy for producing titania microparticles with an anatase core that can be covalently functionalized with proteins and nucleic acids. This was accomplished by adding a silane-thiol chemical group to the shell surface. These particles display up to a four-fold increase in trap stiffness compared to conventional plastic beads featured in optical tweezers experiments. The core-shell beads were also utilized in inherently high force biophysical assays such as amyloid fiber tethering and actin rupturing to demonstrate their use and range of applications. We anticipate that our synthetic approach will permit wider use of these improved beads in optical trapping applications requiring currently inaccessible high forces, such as with stable protein dynamics, as well as in combined coincident optical trapping and fluorescence assays where low laser power but strong trapping ability is imperative.

Vinculin Forms a Directionally Asymmetric Catch Bond with F-Actin

Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin–F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (–) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin–F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

Real-time observation of polymerase-promoter contact remodeling during transcription initiation

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (−10 to −35) and with the −10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein's molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.