A mutation in switch I alters the load-dependent kinetics of myosin Va.

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (Pi) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated Pi on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of Pi-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM Pi, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of Pi from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the Pi-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of Pi-rebinding.

Steroid-Resistant Nephrotic Syndrome-Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism.

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Functional Role of Class III Myosins in Hair Cells.

Cytoskeletal motors produce force and motion using the energy from ATP hydrolysis and function in a variety of mechanical roles in cells including muscle contraction, cargo transport, and cell division. Actin-based myosin motors have been shown to play crucial roles in the development and function of the stereocilia of auditory and vestibular inner ear hair cells. Hair cells can contain hundreds of stereocilia, which rely on myosin motors to elongate, organize, and stabilize their structure. Mutations in many stereocilia-associated myosins have been shown to cause hearing loss in both humans and animal models suggesting that each myosin isoform has a specific function in these unique parallel actin bundle-based protrusions. Here we review what is known about the classes of myosins that function in the stereocilia, with a special focus on class III myosins that harbor point mutations associated with delayed onset hearing loss. Much has been learned about the role of the two class III myosin isoforms, MYO3A and MYO3B, in maintaining the precise stereocilia lengths required for normal hearing. We propose a model for how class III myosins play a key role in regulating stereocilia lengths and demonstrate how their motor and regulatory properties are particularly well suited for this function. We conclude that ongoing studies on class III myosins and other stereocilia-associated myosins are extremely important and may lead to novel therapeutic strategies for the treatment of hearing loss due to stereocilia degeneration.

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Converter domain mutations in myosin alter structural kinetics and motor function.

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ß-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Getting site-specific with actomyosin inhibitors.

Actin and myosin play important roles in many devastating diseases and thus are attractive targets for small-molecule therapy. In this issue of JBC, Guhathakurta et al. have developed a high-throughput screening assay to find small molecules that interfere with the actomyosin interaction. They utilized time-resolved FRET (TR-FRET) and a unique donor-acceptor pair (filamentous actin and a peptide that binds near the myosin-binding site on actin) to find novel molecules that interfere with the actomyosin ATPase and alter the structure of actin filaments. These findings demonstrate the power and potential of high-throughput TR-FRET in monitoring molecular interactions.

Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca(2+) Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils.

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

The actin-bundling protein TRIOBP-4 and -5 promotes the motility of pancreatic cancer cells.

TRIOBP isoforms 4 and 5 (TRIOBP-4/-5) are an actin-bundling protein associated with hearing loss. Here, we showed that TRIOBP-4/-5 was up-regulated in human pancreatic carcinoma cells. Knockdown of TRIOBP-4/-5 led to a loss of filopodia and a decrease in cell motility. Confocal microscopy showed that re-expression of GFP-TRIOBP-4 or -5 restored the filopodial formation in TRIOBP-4/-5-deficient PANC-1 cells. Finally, TRIOBP-4/-5 was shown to be overexpressed in human pancreatic cancer tissues. These results demonstrate a novel role of TRIOBP-4/-5 that promotes the motility of pancreatic cancer cells via regulating actin cytoskeleton reorganization in the filopodia of the cells.

Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments.

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30-36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

Actin structure-dependent stepping of myosin 5a and 10 during processive movement.

How myosin 10, an unconventional myosin, walks processively along actin is still controversial. Here, we used single molecule fluorescence techniques, TIRF and FIONA, to study the motility and the stepping mechanism of dimerized myosin 10 heavy-meromyosin-like fragment on both single actin filaments and two-dimensional F-actin rafts cross-linked by fascin or α-actinin. As a control, we also tracked and analyzed the stepping behavior of the well characterized processive motor myosin 5a. We have shown that myosin 10 moves processively along both single actin filaments and F-actin rafts with a step size of 31 nm. Moreover, myosin 10 moves more processively on fascin-F-actin rafts than on α-actinin-F-actin rafts, whereas myosin 5a shows no such selectivity. Finally, on fascin-F-actin rafts, myosin 10 has more frequent side steps to adjacent actin filaments than myosin 5a in the F-actin rafts. Together, these results reveal further single molecule features of myosin 10 on various actin structures, which may help to understand its cellular functions.

R1 motif is the major actin-binding domain of TRIOBP-4.

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.