Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.     

Glycine 699 is pivotal for the motor activity of skeletal muscle myosin

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.     

Cross-bridge action: present views, prospects, and unknowns

When the sliding filament hypothesis was proposed in 1953-1954, existing evidence showed that (1) contributions to tension were given by active sites uniformly distributed within each zone of filament overlap and (2) each site functioned cyclically. These sites were identified by electron microscopy as cross-bridges between the two filaments, formed of the heads of myosin molecules projecting from a thick filament and attaching to a thin filament. The angle of these cross-bridges was found to be different at rest and in rigor, suggesting that the event causing relative motion of the filaments was a change of the angle of the cross-bridges. At first, it seemed likely that the whole cross-bridge rotated about its attachment to actin, but when the atomic structures of actin and myosin were obtained by X-ray crystallography, a possible hinge was found between the "catalytic domain" which attaches to the actin filament and the "light-chain domain" which appears to act as a lever arm. Two attitudes of the lever arm are now well established, the transition between them being driven by a conformational change coupled to some step in the hydrolysis of ATP, but several recent observations suggest that this is not the whole story: a third attitude has been shown by X-ray crystallography; a non-muscle myosin has been shown to produce its working stroke in two steps; and there are suggestions that an additional displacement of the filaments is produced by a change in the attitude of the catalytic domain on the thin filament.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Cooperativity of myosin molecules through strain-dependent chemistry

There is mounting evidence that the myosin head domain contains a lever arm which amplifies small structural changes that occur at the nucleotide-binding site. The mechanical work associated with movement of the lever affects the rates at which the products of ATP hydrolysis are released. During muscle contraction, this strain-dependent chemistry leads to cooperativity of the myosin molecules within a thick filament. Two aspects of cooperative action are discussed, in the context of a simple stochastic model. (i) A modest motion of the lever arm on ADP release can serve to regulate the fraction of myosin bound to the thin filament, in order to recruit more heads at higher loads. (ii) If the lever swings through a large angle when phosphate is released, the chemical cycles of the myosin molecules can be synchronized at high loads. This leads to stepwise sliding of the filaments and suggests that the isometric condition is not a steady state.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay

Popular views of force generation in muscle indicate that a lever arm in the myosin head initiates displacement of the thin filament. However, this lever arm is attached to the thick filament backbone by a flexible combination of coiled coils and hinges in the myosin subfragment-2 (S2); therefore, efficient force generation depends on tension development in this linking structure. Herein, a single molecule assay is developed to examine the flexibility of the intact S2 relative to that of the myosin head. Fluorescently labeled myosin rod is polymerized onto a single myosin molecule that is bound to actin, and the resulting Brownian motion of the rod is analyzed at video rates by digital image processing. Complete rotations of the rod suggest significant amounts of random coil in the linking structure. The close similarity of twist rates for double-headed and single-headed myosin indicates that most of the flexibility originates at or beyond the first pitch of coiled coil in S2 and most likely at the hinge connecting S2 and the light meromyosin. The myosin head has a smaller but still detectable impact on this flexibility, since the addition of ADP to the rigor crossbridge produces differential effects on the torsional characteristics of double-headed versus single-headed myosin.     

Myosin VI walks "wiggly" on actin with large and variable tilting

Myosin VI is an unconventional motor protein with unusual motility properties such as its direction of motion and path on actin and a large stride relative to its short lever arms. To understand these features, the rotational dynamics of the lever arm were studied by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy during processive motility of myosin VI along actin. The axial angle is distributed in two peaks, consistent with the hand-over-hand model. The changes in lever arm angles during discrete steps suggest that it exhibits large and variable tilting in the plane of actin and to the sides. These motions imply that, in addition to the previously suggested flexible tail domain, there is a compliant region between the motor domain and lever arm that allows myosin VI to accommodate the helical position of binding sites while taking variable step sizes along the actin filament.     

Myosin S2 Origins Track Evolution of Strong Binding on Actin by Azimuthal Rolling of Motor Domain

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin’s α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.     

Structure and function of the essential light chain of myosin

The review summarizes the recent data on the structure and function of the essential light chain of myosin. It is known that the essential light chain of myosin stabilizes the lever arm. Consistent with the model of the shift of the dynamic population of conformations, the conformational flexibility of the essential light chain is emphasized, which opens the way to determining its new functions. It is proposed that the interaction between the C-terminal domain of the essential light chain and the N-terminal subdomain of the heavy chain of myosin may be involved in the coupling of ATP hydrolysis and rotation of the lever arm. The recent data indicate that the isoforms of the essential light chain with the additional N-terminal peptide are capable of interacting with actin and src-homologous domain 3 of myosin. The structural aspects of these interactions and the modulatory role of the isoforms of the essential light chain of myosin are discussed.     

How myosin motors power cellular functions: an exciting journey from structure to function: based on a lecture delivered at the 34th FEBS Congress in Prague, Czech Republic, July 2009

Molecular motors such as myosins are allosteric enzymes that power essential motility functions in the cell. Structural biology is an important tool for deciphering how these motors work. Myosins produce force upon the actin-driven conformational changes controlling the sequential release of the hydrolysis products of ATP (Pi followed by ADP). These conformational changes are amplified by a 'lever arm', which includes the region of the motor known as the converter and the adjacent elongated light chain binding region. Analysis of four structural states of the motor provides a detailed understanding of the rearrangements and pathways of communication in the motor that are necessary for detachment from the actin track and repriming of the motor. However, the important part of the cycle in which force is produced remains enigmatic and awaits new high-resolution structures. The value of a structural approach is particularly evident from clues provided by the structural states of the reverse myosin VI motor. Crystallographic structures have revealed that rearrangements within the converter subdomain occur, which explains why this myosin can produce a large stroke in the opposite direction to all other myosins, despite a very short lever arm. By providing a detailed understanding of the motor rearrangements, structural biology will continue to reveal essential information and help solve current enigma, such as how actin promotes force production, how motors are tuned for specific cellular roles or how motor/cargo interactions regulate the function of myosin in the cell.     

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.     

Myosin II isoforms in smooth muscle: heterogeneity and function

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC(20) has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.     

Electron Tomography of Cryofixed,Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Background

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology

We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.

Conclusion

We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.     

The structural basis of muscle contraction

The myosin cross-bridge exists in two conformations, which differ in the orientation of a long lever arm. Since the lever arm undergoes a 60 degree rotation between the two conformations, which would lead to a displacement of the myosin filament of about 11 nm, the transition between these two states has been associated with the elementary 'power stroke' of muscle. Moreover, this rotation is coupled with changes in the active site (CLOSED to OPEN), which probably enable phosphate release. The transition CLOSED to OPEN appears to be brought about by actin binding. However, kinetics shows that the binding of myosin to actin is a two-step process which affects both ATP and ADP affinity and vice versa. The structural basis of these effects is only partially explained by the presently known conformers of myosin. Therefore, additional states of the myosin cross-bridge should exist. Indeed, cryoelectron microscopy has revealed other angles of the lever arm induced by ADP binding to a smooth muscle actin-myosin complex.     

The regulatory domain of the myosin head behaves as a rigid lever.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.     

Myosin motors: missing structures and hidden springs

High-resolution structures of the motor domain of myosin II and lower resolution actin-myosin structures have led to the "swinging lever arm" model for myosin force generation. The available kinetic data are not all easily reconciled with this model and understanding the final details of the myosin motor mechanism must await actin-myosin co-crystals. The observation that myosin can populate multiple states in the absence of actin has nonetheless led to significant insights. The currently known myosin structures correspond to defined kinetic states that bind weakly (K(d)>microM) to actin. It is possible that the myosin lever arm could complete its swing before strong binding to actin and force generation--a process that would correspond, in the absence of load, to a Brownian ratchet. We further suggest that, under load, internal springs within the myosin head could decouple force generation and lever arm movement.     

Kinetics and thermodynamics of the rate-limiting conformational change in the actomyosin V mechanochemical cycle

We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain-sensitive step in many muscle and non-muscle myosins. We also explored essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time-resolved FRET analysis demonstrates a temperature-dependent reversible conformational change in the nucleotide-binding pocket (NBP). Our kinetic results demonstrate that the NBP goes from a closed to an open conformation prior to the release of ADP, while the actin-binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating that this is the rate-limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates that the transition from the open to closed NBP conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the NBP and actin-binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open NBP conformation may be altered by lever arm position.     

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.     

Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.