Reexamining the origin of the directionality of myosin V.

The nature of the conversion of chemical energy to directional motion in myosin V is examined by careful simulations that include two complementary methods: direct Langevin Dynamics (LD) simulations with a scaled-down potential that provided a detailed time-resolved mechanism, and kinetic equations solution for the ensemble long-time propagation (based on information collected for segments of the landscape using LD simulations and experimental information). It is found that the directionality is due to the rate-limiting ADP release step rather than the potential energy of the lever arm angle. We show that the energy of the power stroke and the barriers involved in it are of minor consequence to the selectivity of forward over backward steps and instead suggest that the selective release of ADP from a postrigor myosin motor head promotes highly selective and processive myosin V. Our model is supported by different computational methods-LD simulations, Monte Carlo simulations, and kinetic equations solution-as well as by structure-based binding energy calculations.

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.

Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va.

Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems--the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32-125 motors per µm(2)), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track.

Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin-myosin Va interaction.

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan-a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan-a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan-a melanocytes were co-cultured with SP-1 keratinocytes or stimulated by α-MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.

Age-related changes in myosin-V myenteric neurons, CGRP and VIP immunoreactivity in the ileum of rats supplemented with ascorbic acid.

We examined the effects of ascorbic acid supplementation on myosin-V, calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) immunoractivities in the myenteric neurons in aging rats. Male rats were divided into groups: young 90-day-old rats (E90), 345-day-old control rats (E345), 428-day-old control rats (E428), 90- to 345-day-old rats treated with ascorbic acid (1 g/L) (EA345), and 90- to 428-day-old rats treated with ascorbic acid (1g/L) (EA428). The quantitative results showed that aging reduced the number of myosin-V-immunoreactive neurons compared with young animals (E90). Ascorbic acid supplementation in the EA345 and EA428 groups increased the average area of myosin-V neurons by 24.6% and 24.1% compared with the E345 and E428 groups, respectively. When all groups were compared, we observed significant differences for the CGRP- and VIP-immunoractive varicosities of nerve fibers from myenteric neurons. Ascorbic acid supplementation had a neurotrophic effect on all neurons studied, suggesting a neuroprotective role.

Myenteric neurons and intestinal mucosa of diabetic rats after ascorbic acid supplementation.

AIM: To investigate the effect of ascorbic acid (AA) dietary supplementation on myenteric neurons and epithelial cell proliferation of the jejunum of adult rats with chronic diabetes mellitus. METHODS: Thirty rats at 90 d of age were divided into three groups: Non-diabetic, diabetic and diabetic treated with AA (DA) (1 g/L). After 120 d of treatment with AA the animals were killed. The myenteric neurons were stained for myosin-V and analyzed quantitatively in an area of 11.2 mm(2)/animal. We further measured the cellular area of 500 neurons per group. We also determined the metaphasic index (MI) of the jejunum mucosa layer of about 2500 cells in the intestinal crypts, as well as the dimensions of 30 villi and 30 crypts/animal. The data area was analyzed using the Olympus BX40 microscope. RESULTS: There was an increase of 14% in the neuronal density (792.6 +/- 46.52 vs 680.6 +/- 30.27) and 4.4% in the cellular area (303.4 +/- 5.19 vs 291.1 +/- 6.0) respectively of the diabetic group treated with AA when compared to control diabetic animals. There were no significant differences in MI parameters, villi height or crypt depths among the groups. CONCLUSION: Supplementation with AA in the diabetic animal promoted moderate neuroprotection. There was no observation of alteration of the cellular proliferation of the jejunum mucosa layer of rats with chronic diabetes mellitus with or without supplementation with AA.

Vitamin E supplementation in rats with experimental diabetes mellitus: analysis of myosin-V and nNOS immunoreactive myenteric neurons from terminal ileum.

The effect of vitamin E (1 g/kg body weight) supplementation on myosin-V and neuronal nitric oxide synthase (nNOS) immunoreactive myenteric neurons from the ileum of diabetic rats was investigated in the present study. Forty animals were divided into the following groups: normoglycemics (N), normoglycemics treated with vitamin E (NE), diabetics (D), and diabetics treated with vitamin E (DE). Quantitative and morphometric analyses were performed. The area of the tertiary plexus was also determined. Diabetes produced a 24% reduction in the number of myosin-V neurons in group D compared with group N, an effect that was accompanied by an increase in the tertiary plexus area (P < 0.05). Neuronal density was 27% higher in group NE than group N (P < 0.05). Nitrergic neuronal density was not altered as a consequence of either diabetes or vitamin E treatment. Myosin-V and nNOS immunoreactive neuronal cell body area increased significantly in group NE. The area of myosin-V and nNOS myenteric neurons also increased in group D. Vitamin E treatment (group DE) increased only the size of nitrergic neurons. The present results suggest that vitamin E elicited a neuroprotective and neurotrophic effect on the natural aging process, but with regard to diabetes, vitamin E supplementation exerted a neurotrophic effect only on nitrergic neurons.

'Should I stay or should I go?': myosin V function in organelle trafficking.

Actin- and microtubule-based motors can propel different cargos along filaments. Within cells, they control the distribution of membrane-bound compartments by performing complementary tasks. Organelles make long journeys along microtubules, with class V myosins ensuring their capture and their dispersal in actin-rich regions. Myosin Va is recruited on to diverse organelles, such as melanosomes and secretory vesicles, by a mechanism involving Rab GTPases. The role of myosin Va in the recruitment of secretory vesicles at the plasma membrane reveals that the cortical actin network cannot merely be seen as a physical barrier hindering vesicle access to release sites. In neurons, myosin Va controls the targeting of IP(3) (inositol 1,4,5-trisphosphate)-sensitive Ca(2+) stores to dendritic spines and the transport of mRNAs. These defects probably account for the severe neurological symptoms observed in Griscelli syndrome due to mutations in the MYO5A gene.

Drebrin attenuates the interaction between actin and myosin-V.

Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

Diabetic rats supplemented with L-glutamine: a study of immunoreactive myosin-V myenteric neurons and the proximal colonic mucosa.

We studied the neuronal density and size of myenteric neurons and the epithelial cell proliferation and crypt depth of the proximal colon in diabetic Wistar rats after supplementing them with L-glutamine (1%). The animals were divided into five groups: untreated normoglycemic (UN), L-glutamine-treated normoglycemic (NG), untreated diabetic (UD), and L-glutamine-treated diabetics 4 days (DG4) and 45 days (DG45) days after the onset of diabetes. We observed a reduction of 52.7% and 50.44% in the neuronal density of the proximal colon of the UD group compared to the UN and NG groups, respectively (P<0.05). The neuronal density found for the DG4 (32.8%) and DG45 (28.6%) groups was higher than that of the UD group (P>0.05). There were no significant differences (P>0.05) when the data relative to the area of the myenteric neuron cell bodies, metaphasic index, and crypt depth in the proximal colon were compared among experimental groups.

Nonmuscle myosins II-B and Va are components of detergent-resistant membrane skeletons derived from mouse forebrain.

Myosins are actin-based molecular motors that may have specialized trafficking and contractile functions in cytoskeletal compartments that lack microtubules. The postsynaptic excitatory synapse is one such specialization, yet little is known about the spatial organization of myosin motor proteins in the mature brain. We used a proteomics approach to determine if class II and class V myosin isoforms are associated with Triton X-100-resistant membranes isolated from mouse forebrain. Two nonmuscle myosin isoforms (II-B and Va), were identified as components of lipid raft fractions that also contained typical membrane skeletal proteins such as non-erythrocyte spectrins, actin, alpha-actinin-2 and tubulin subunits. Other raft-associated proteins included lipid raft markers, proteins involved in cell adhesion and membrane dynamics, receptors and channels including glutamate receptor subunits, scaffolding and regulatory proteins. Myosin II-B and Va were also present in standard postsynaptic density (PSD) fractions, however retention of myosin II-B was strongly influenced by ATP status. If homogenates were supplemented with ATP, myosin II-B could be extracted from PSD I whereas myosin Va and other postsynaptic proteins were resistant to extraction. In summary, both myosin isoforms are components of a raft-associated membrane skeleton and are likely detected in standard PSD fractions as a result of their intrinsic ability to form actomyosin. Myosin II-B, however, is more loosely associated with PSD fractions than myosin Va, which appears to be a core PSD protein.

Regulation and recycling of myosin V.

Recently there has been considerable progress in our understanding of regulation for unconventional myosin-V through elucidation of the structure of its inactive conformation and the factors that affect stability of this conformation. The inactive conformation is a folded compact structure characterized by interactions between the myosin head and the C-terminal cargo binding domain. Concentrations of Ca2+ greater than 10 microM disrupt folding. The 3-D structure determined by cryoelectron tomography of 2-D arrays in one study and electron micrographs of isolated molecules reported in another reveal similar features, but suggest different F-actin affinities for the inactive conformation. This has raised the question of how inactive myosin-V is recycled to other sites for additional rounds of cargo transport.

Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features.

A 2.5-A resolution structure of calcium-free calmodulin (CaM) bound to the first two IQ motifs of the murine myosin V heavy chain reveals an unusual CaM conformation. The C-terminal lobe of each CaM adopts a semi-open conformation that grips the first part of the IQ motif (IQxxxR), whereas the N-terminal lobe adopts a closed conformation that interacts more weakly with the second part of the motif (GxxxR). Variable residues in the IQ motif play a critical role in determining the precise structure of the bound CaM, such that even the consensus residues of different motifs show unique interactions with CaM. This complex serves as a model for the lever arm region of many classes of unconventional myosins, as well as other IQ motif-containing proteins such as neuromodulin and IQGAPs.

Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic.

Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N(6)-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments.

A structural state of the myosin V motor without bound nucleotide.

The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 A structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

Neck length and processivity of myosin V.

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 microm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.

Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

Regulatory implications of a novel mode of interaction of calmodulin with a double IQ-motif target sequence from murine dilute myosin V.

Apo-Calmodulin acts as the light chain for unconventional myosin V, and treatment with Ca(2+) can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca(2+) on the stoichiometry and affinity of interactions of calmodulin and its two domains with two myosin-V peptides (IQ3 and IQ4) have therefore been quantified in vitro, using fluorescence and near- and far-UV CD. The results with separate domains show their differential affinity in interactions with the IQ motif, with the apo-N domain interacting surprisingly weakly. Contrary to expectations, the effect of Ca(2+) on the interactions of either peptide with either isolated domain is to increase affinity, reducing the K(d) at physiological ionic strengths by >200-fold to approximately 75 nM for the N domain, and approximately 10-fold to approximately 15 nM for the C domain. Under suitable conditions, intact (holo- or apo-) calmodulin can bind up to two IQ-target sequences. Interactions of apo- and holo-calmodulin with the double-length, concatenated sequence (IQ34) can result in complex stoichiometries. Strikingly, holo-calmodulin forms a high-affinity 1:1 complex with IQ34 in a novel mode of interaction, as a "bridged" structure wherein two calmodulin domains interact with adjacent IQ motifs. This apparently imposes a steric requirement for the alpha-helical target sequence to be discontinuous, possibly in the central region, and a model structure is illustrated. Such a mode of interaction could account for the Ca(2+)-dependent regulation of myosin V in vitro motility, by changing the structure of the regulatory complex, and paradoxically causing calmodulin dissociation through a change in stoichiometry, rather than a Ca(2+)-dependent reduction in affinity.

Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin.

Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2 domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A.Slac2-a.myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432-12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B both in vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400-590) and Slac2-c (amino acids 670-856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery.