Microtubule Dumbbells to Assess the Effect of Force Geometry on Single Kinesin Motors.

The cytoskeletal motors myosin, kinesin, and dynein and their corresponding tracks, actin and microtubules, are force generating ATPases responsible for motility and morphological changes at the intracellular, cellular, and tissue levels. The pioneering application of optical tweezers to measure the force-producing properties of cytoskeletal motors has provided an unparalleled understanding of their mechanochemistry. The mechanosensitivity of processive, microtubule-based motors has largely been studied in the optical trap using the "single-bead" assay, where a bead-attached motor is held adjacent to a cytoskeletal filament as it processively steps along it. However, because of the geometrical constraints in the conventional single-bead assay, the motor-filament bond is not only loaded parallel to the long axis of the filament, but also perpendicular to the long axis of the filament. This perpendicular force, which is inherent in the conventional single-bead assay, accelerates the motor-filament detachment and has not been carefully considered in prior experiments. An alternative approach is the "three-bead" assay, which was developed for the study of non-processive myosin motors. The vertical force component is minimized in this assay, and the total opposing force is mainly parallel to the microtubule. Experiments with kinesin show that microtubule attachment durations can be highly variable and last for up to tenfold longer times in the three-bead assay, compared to the single-bead assay. Thus, the ability of kinesin to bear mechanical load and remain attached to microtubules depends on the forces in more than one dimension. In this chapter, we provide detailed methods for preparing the proteins, buffers, flow chambers, and bead-filament assemblies for performing the three-bead assay with microtubules and their motors.

Myosin with hypertrophic cardiac mutation R712L has a decreased working stroke which is rescued by omecamtiv mecarbil.

Hypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout ß-cardiac myosin, including in the motor domain, are associated with HCM. A ß-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near-normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal ß-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM.

Modulation of Kinesin's Load-Bearing Capacity by Force Geometry and the Microtubule Track.

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport.

High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing.

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes.

KIF16B is a highly processive kinesin-3 family member that participates in the trafficking and tubulation of early endosomes along microtubules. KIF16B attaches to lipid cargoes via a PX motif at its C-terminus, which has nanomolar affinity for bilayers containing phosphatidylinositol-3-phosphate (PI[3]P). As the PX domain has been proposed to be a primary mechanical anchor for the KIF16B-cargo attachment, we measured the adhesion forces and detachment kinetics of the PX domain as it interacts with membranes containing 2% PI(3)P and 98% phosphatidylcholine. Using optical tweezers, we found that the adhesion strength of a single PX domain ranged between 19 and 54 pN at loading rates between 80 and 1500 pN/s. These forces are substantially larger than the interaction of the adhesion of a pleckstrin homology domain with phosphatidylinositol 4,5-bisphosphate. This increased adhesion is the result of the membrane insertion of hydrophobic residues adjacent to the PI(3)P binding site, in addition to electrostatic interactions with PI(3)P. Attachment lifetimes under load decrease monotonically with force, indicating slip-bond behavior. However, the lifetime of membrane attachment under load appears to be well matched to the duration of processive motility of the KIF16B motor, indicating the PX domain is a suitable mechanical anchor for intracellular transport.

Measuring the Kinetic and Mechanical Properties of Non-processive Myosins Using Optical Tweezers.

The myosin superfamily of molecular motors utilizes energy from ATP hydrolysis to generate force and motility along actin filaments in a diverse array of cellular processes. These motors are structurally, kinetically, and mechanically tuned to their specific molecular roles in the cell. Optical trapping techniques have played a central role in elucidating the mechanisms by which myosins generate force and in exposing the remarkable diversity of myosin functions. Here, we present thorough methods for measuring and analyzing interactions between actin and non-processive myosins using optical trapping techniques.

Force Generation by Membrane-Associated Myosin-I.

Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions.

Mechanochemical tuning of myosin-I by the N-terminal region.

Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post-power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms.

Inherent force-dependent properties of ß-cardiac myosin contribute to the force-velocity relationship of cardiac muscle.

The heart adjusts its power output to meet specific physiological needs through the coordination of several mechanisms, including force-induced changes in contractility of the molecular motor, the ß-cardiac myosin (ßCM). Despite its importance in driving and regulating cardiac power output, the effect of force on the contractility of a single ßCM has not been measured. Using single molecule optical-trapping techniques, we found that ßCM has a two-step working stroke. Forces that resist the power stroke slow the myosin-driven contraction by slowing the rate of ADP release, which is the kinetic step that limits fiber shortening. The kinetic properties of ßCM are affected by load, suggesting that the properties of myosin contribute to the force-velocity relationship in intact muscle and play an important role in the regulation of cardiac power output.

A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning.

Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor's lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.

Method for measuring single-molecule adhesion forces and attachment lifetimes of protein-membrane interactions.

Many proteins that reside in the cytoplasm bind directly to cell membranes and play roles in signaling, adhesion, metabolism, cell structure, and cell motility. Several of these membrane-binding proteins, especially cytoskeletal proteins, have mechanical functions that result in the transmission of forces to the plasma membrane and organelle-membranes. Despite the importance of these interactions, remarkably little is known about the mechanical properties of the bonds between membranes and proteins. In this chapter, we describe a single-molecule, optical-trapping method for the measurement of protein-membrane adhesion forces and force-dependent attachment-lifetimes.

Resolving two-dimensional kinetics of the integrin αIIbß3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbß3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbß3 molecules and forced dissociation of an αIIbß3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbß3 pre-exists in at least two states that differ in their zero force on-rates (k(on1) = 1.4 × 10(-4) and k(on2) = 2.3 × 10(-4) µm(2)/s), off-rates (k(off1) = 2.42 and k(off2) = 0.60 s(-1)), and dissociation constants (K(d)(1) = 1.7 × 10(4) and K(d)(2) = 2.6 × 10(3) µm(-2)). The integrin activator Mn(2+) changed the on-rates and affinities (K(d)(1) = 5 × 10(4) and K(d)(2) = 0.3 × 10(3) µm(-2)) but did not affect the off-rates. The strength of αIIbß3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbß3-fibrinogen complex characterized by a faster on-rate. Upon Mn(2+)-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbß3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbß3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.

Myosin IC generates power over a range of loads via a new tension-sensing mechanism.

Myosin IC (myo1c), a widely expressed motor protein that links the actin cytoskeleton to cell membranes, has been associated with numerous cellular processes, including insulin-stimulated transport of GLUT4, mechanosensation in sensory hair cells, endocytosis, transcription of DNA in the nucleus, exocytosis, and membrane trafficking. The molecular role of myo1c in these processes has not been defined, so to better understand myo1c function, we utilized ensemble kinetic and single-molecule techniques to probe myo1c's biochemical and mechanical properties. Utilizing a myo1c construct containing the motor and regulatory domains, we found the force dependence of the actin-attachment lifetime to have two distinct regimes: a force-independent regime at forces < 1 pN, and a highly force-dependent regime at higher loads. In this force-dependent regime, forces that resist the working stroke increase the actin-attachment lifetime. Unexpectedly, the primary force-sensitive transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. This force-sensing behavior is unique amongst characterized myosins and clearly demonstrates mechanochemical diversity within the myosin family. Based on these results, we propose that myo1c functions as a slow transporter rather than a tension-sensitive anchor.

Force-dependent detachment of kinesin-2 biases track switching at cytoskeletal filament intersections.

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.

Calcium regulation of myosin-I tension sensing.

Myo1b is a myosin that is exquisitely sensitive to tension. Its actin-attachment lifetime increases > 50-fold when its working stroke is opposed by 1 pN of force. The long attachment lifetime of myo1b under load raises the question: how are actin attachments that last >50 s in the presence of force regulated? Like most myosins, forces are transmitted to the myo1b motor through a light-chain binding domain that is structurally stabilized by calmodulin, a calcium-binding protein. Thus, we examined the effect of calcium on myo1b motility using ensemble and single-molecule techniques. Calcium accelerates key biochemical transitions on the ATPase pathway, decreases the working-stroke displacement, and greatly reduces the ability of myo1b to sense tension. Thus, calcium provides an effective mechanism for inhibiting motility and terminating long-duration attachments.

Kinetic schemes for post-synchronized single molecule dynamics.

Recordings from single molecule experiments can be aggregated to determine average kinetic properties of the system under observation. The kinetics after a synchronized reaction step can be interpreted using all of the standard tools developed for ensemble perturbation experiments. The kinetics leading up to a synchronized event, determined by the lifetimes of the preceding states; however, are not as obvious if the reaction has reversible steps or branches. Here we describe a general procedure for dealing with these situations.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments.

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Dissociation of bimolecular αIIbß3-fibrinogen complex under a constant tensile force.

The regulated ability of integrin αIIbß3 to bind fibrinogen plays a crucial role in platelet aggregation, adhesion, and hemostasis. Employing an optical-trap-based electronic force clamp, we studied the thermodynamics and kinetics of αIIbß3-fibrinogen bond formation and dissociation under constant unbinding forces, mimicking the forces of physiologic blood shear on a thrombus. The distribution of bond lifetimes was bimodal, indicating that the αIIbß3-fibrinogen complex exists in two bound states with different mechanical stability. The αIIbß3 antagonist, abciximab, inhibited binding without affecting the unbinding kinetics, whereas Mn²(+) biased the αIIbß3-fibrinogen complex to the strong bound state with reduced off-rate. The average bond lifetimes decreased exponentially with increasing pulling force from ∼5 pN to 50 pN, suggesting that in this force range the αIIbß3-fibrinogen interactions are classical slip bonds. We found no evidence for catch bonds, which is consistent with the known lack of shear-enhanced platelet adhesion on fibrinogen-coated surfaces. Taken together, these data provide important quantitative and qualitative characteristics of αIIbß3-fibrinogen binding and unbinding that underlie the dynamics of platelet adhesion and aggregation in blood flow.

Single-molecule adhesion forces and attachment lifetimes of myosin-I phosphoinositide interactions.

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3-2.5 pN), with rates exceeding 360 s(-1) at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.