Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.
exocytosis; Saccharomyces; molecular motors; myosin; cell polarity
We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (Vmax) by ~60% compared to wild-type myosin, but there is no change in apparent actin affinity (Km). While ATP or AMP-PNP binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence ~15% or ~8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ~35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from one-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in two-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In one-week-old adults, flight capability is lost. Thus altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.
myosin; muscle; Drosophila; ATPase; myofibril
Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins.
We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1), myosin XI-6 (At MYA2), myosin XI-8 (At XI-B), myosin XI-15 (At XI-I), myosin XI-16 (At XI-J) and myosin XI-17 (At XI-K) were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2), previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria.
6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and/or organelles when transiently expressed as YFP fusions in plant cells. Although comparable constructs lacking the motor domain result in a dominant negative effect on organelle motility in animal systems, the plant organelles remained motile. YFP-myosin tail fusions provide specific labeling for vesicles of unknown composition, whose identity can be investigated in future studies.
Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.
Myosin-V is a processive molecular motor that moves membrane vesicles along actin tracks. In the simple model for motor and cargo motion investigated here, an elastic connection between motor and cargo transiently absorbs the abrupt mechanical transitions of the motor, and allows smooth relaxation of the cargo to a new position. We use a stochastic description to model motor stepping, with kinetics that depends on the instantaneous force exerted on the motor through the elastic connection. Tether relaxation is modelled as a continuous process, in which the rate is determined by the viscous drag of the cargo and the stiffness profile of the connection. Quantitative combined stochastic–continuous simulation of the dynamics of this system shows that bulky loads can impose a highly regular gait on the motor. If the characteristics of the elastic connection are similar to those of the myosin-II coiled-coil domain, the myosin-V motor, tether and cargo form a true escapement, in which the motor only escapes from its current position after one or more force thresholds have been crossed. Multiple thresholds limit the variation in tether length to values below that of the total step size.
myosin-V; modelling; force-dependent kinetics; non-Markov process; stochastic; hybrid simulation
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.
Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.
Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.
Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.
We have solved a 2.4Å structure of a truncated version of the reverse direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulins. The structure reveals only minor differences in the motor domain as compared to plus-end directed myosins, with the exception of two unique inserts. The first insert is near the nucleotide-binding pocket, and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a previously unseen binding motif within the insert. This serves to redirect the effective “lever arm” of myosin VI, which includes a second calmodulin bound to an “IQ motif,” towards the pointed (−) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to fully explain the movements of myosin VI.
The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis.
We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar) F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin.
The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of organelles such as peroxisomes. Potential roles of MYA2 may also exist in the cell nucleus. Whether the low quality of the F-actin-labeling by MYA2-head6IQ compared to other F-actin-binding proteins (ABPs) signifies a weak association of the myosin with actin filaments remains to be proven by other means than in vivo. Clues for the mode of contact between the myosin molecules and F-actin so far cannot be drawn from sequence-related data.
Myosin V (myoV), a processive cargo transporter, has arguably been the most well-studied unconventional myosin of the past decade. Considerable structural information is available for the motor domain, the IQ motifs with bound calmodulin or light chains, and the cargo-binding globular tail, all of which have been crystallized. The repertoire of adapter proteins that link myoV to a particular cargo is becoming better understood, enabling cellular transport processes to be dissected. MyoV is processive, meaning that it takes many steps on actin filaments without dissociating. Its extended lever arm results in long 36 nm steps, making it ideal for single molecule studies of processive movement. In addition, electron microscopy revealed the structure of the inactive, folded conformation of myoV when it is not transporting cargo. This review provides a background on myoV, and highlights recent discoveries that show why myoV will continue to be an active focus of investigation.
myosin V; motor protein; IQ motif; cargo-binding; processivity; calmodulin
The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the “DIL” domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL–YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs.
Arabidopsis; myosin XI; yeast; myo2p; DIL domain; dominant-negative; fluorescent protein; vesicles
Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p–Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.
membrane transport; Myo2p; Vac17p; yeast; vacuole
Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism.
An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain.
Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function.
Kinesin is the founding member of a superfamily of microtubule-based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation1,2. It has two identical ~960-amino-acid chains containing an amino-terminal globular motor domain, a central α-helical region that enables dimer formation through a coiled-coil, and a carboxy-terminal tail domain that binds light chains and possibly an organelle receptor1. The kinesin motor domain of ~340 amino acids, which can produce movement in vitro3, is much smaller than that of myosin (~850 amino acids) and dynein (1,000 amino acids), and is the smallest known molecular motor. Here, we report the crystal structure of the human kinesin motor domain with bound ADP determined to 1.8-Å resolution by X-ray crystallography. The motor consists primarily of a single α/β arrowhead-shaped domain with dimensions of 70 × 45 × 45 Å. Unexpectedly, it has a striking structural similarity to the core of the catalytic domain of the actin-based motor myosin. Although kinesin and myosin have virtually no amino-acid sequence identity, and exhibit distinct enzymatic4–6 and motile7–10 properties, our results suggest that these two classes of mechanochemical enzymes evolved from a common ancestor and share a similar force-generating strategy.
Myosins are ATP-driven linear molecular motors that work as cellular force
generators, transporters, and force sensors. These functions are driven by
large-scale nucleotide-dependent conformational changes, termed
“strokes”; the “power stroke” is the force-generating
swinging of the myosin light chain–binding “neck” domain
relative to the motor domain “head” while bound to actin; the
“recovery stroke” is the necessary initial motion that primes, or
“cocks,” myosin while detached from actin. Myosin Va is a processive
dimer that steps unidirectionally along actin following a “hand over
hand” mechanism in which the trailing head detaches and steps forward
∼72 nm. Despite large rotational Brownian motion of the detached head about
a free joint adjoining the two necks, unidirectional stepping is achieved, in
part by the power stroke of the attached head that moves the joint forward.
However, the power stroke alone cannot fully account for preferential forward
site binding since the orientation and angle stability of the detached head,
which is determined by the properties of the recovery stroke, dictate actin
binding site accessibility. Here, we directly observe the recovery stroke
dynamics and fluctuations of myosin Va using a novel, transient caged
ATP-controlling system that maintains constant ATP levels through stepwise
UV-pulse sequences of varying intensity. We immobilized the neck of monomeric
myosin Va on a surface and observed real time motions of bead(s) attached
site-specifically to the head. ATP induces a transient swing of the neck to the
post-recovery stroke conformation, where it remains for ∼40 s, until ATP
hydrolysis products are released. Angle distributions indicate that the
post-recovery stroke conformation is stabilized by ≥5
kBT of energy. The high kinetic
and energetic stability of the post-recovery stroke conformation favors
preferential binding of the detached head to a forward site 72 nm away. Thus,
the recovery stroke contributes to unidirectional stepping of myosin Va.
Myosin Va is a “two-legged” ATP-dependent linear molecular motor that
transports cellular organelles by “stepping” along actin filaments
in a processive manner analogous to human walking, the two “feet”
alternating between forward and backward positions. During stepping, the lifted
leg undergoes rotational Brownian movements around a free joint at the
leg–leg junction. Although these movements are random, the lifted foot
lands preferentially on forward sites and rarely steps backward. This
directional bias arises in part from the forward movement of the junction
bending the “ankle” of the attached leg. Here, we show that the
lifted foot also plays a role in the direction of stepping by controlling the
orientation of its actin-binding site (the “sole”), which dictates
the accessibility of potential stepping positions. We observed the ATP-dependent
foot orientation and its stabilizing on individual myosin Va molecules in real
time under an optical microscope; we show that the lifted foot of walking myosin
Va is oriented in a “toe-down” conformation so that binding to a
forward site on actin is preferred largely over backward or adjacent sites.
Thus, the great kinetic and energetic stability of the myosin Va lifted foot
conformation contributes to unidirectional stepping along actin filaments.
Myosin-Va transports intracellular cargos along actin filaments in cells1.
This processive two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis2. The ability of myosin-Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded3-10. Mechanical studies at the multiple and single molecule level suggest that there is tight coupling (i.e. one ATP is hydrolyzed per power stroke), but this has not been directly demonstrated4,5,11. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin-Va and directly visualized the binding and dissociation of single fluorescently-labelled nucleotide molecules while simultaneously observing the stepping motion of the fluorescently labelled myosin-V as it moves along an actin filament. Here we show that preferential ADP dissociation from the trail head of myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin-V molecule retains at least one nucleotide (ADP in the lead head position) while moving. Thus we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with nanometer precision.
Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks.
The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins.
The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree.
Dictyostelium contains thirteen myosins together with 6–8 MLCs (myosin light chain) to assist in a variety of actin-based processes in the cell. Although they are homologous to myosins of higher eukaryotes, the myosins of Dictyostelium should be considered with care as models for specific functions of vertebrate myosins.
The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual ~ 180° lever arm swing and achieves its large step size using special structural features in its tail domain.
Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of
central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this
molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain
myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport
complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings.
Coimmunoprecipitation assays further indicate that
Ca2+ disrupts the in vitro binding of synaptobrevin II to
synaptophysin in the presence but not in the absence of
Mg2+. We conclude that hydrophilic forces reversibly
couple the myosin V tail to a biochemically defined
class of organelles in brain nerve terminals.
► First description of the production and detailed functional characterization of a human myosin-6 motor. ► Identification of halogenated phenols as inhibitors of myosin-6 activity. ► In vitro and in vivo characterization of the effect of tri-iodophenole on myosin-6 function.
Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion.
Actin myosin; Kinetics; Inhibition; Allostery
Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility.
Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database.
Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms.
MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell.
Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.
Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.
As in many eukaryotic cells, fission yeast cytokinesis depends on
the assembly of an actin ring. We cloned
myp2+, a myosin-II in
Schizosaccharomyces pombe, conditionally required for
cytokinesis. myp2+, the second myosin-II
identified in S. pombe, does not completely overlap in
function with myo2+. The catalytic domain of
Myp2p is highly homologous to known myosin-IIs, and phylogenetic
analysis places Myp2p in the myosin-II family. The Myp2p sequence
contains well-conserved ATP- and actin-binding motifs, as well as two
IQ motifs. However, the tail sequence is unusual, since it is predicted
to form two long coiled-coils separated by a stretch of sequence
containing 19 prolines. Disruption of myp2+
is not lethal but under nutrient limiting conditions cells lacking
myp2+ function are multiseptated, elongated,
and branched, indicative of a defect in cytokinesis. The presence of
salt enhances these morphological defects. Additionally,
Δmyp2 cells are cold sensitive in high salt, failing
to form colonies at 17°C. Thus, myp2+ is
required under conditions of stress, possibly linking extracellular
growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p
localizes to a ring in the middle of late mitotic cells, consistent
with a role in cytokinesis. Additionally, we constructed double mutants
of Δmyp2 with temperature-sensitive mutant strains
defective in cytokinesis. We observed synthetic lethal interactions
between Δmyp2 and three alleles of
cdc11ts, as well as more modest synthetic interactions
with cdc14ts and cdc16ts, implicating
myp2+ function for efficient cytokinesis
under normal conditions.