Peroxisomal localization of a myosin XI isoform in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains 13 myosin XI isoforms. Here we prepared a specific antibody against a peptide that mimics a unique C-terminal region from the myosin XI isoform, MYA2. The resulting antibody was used to demonstrate that MYA2 in Arabidopsis protein extracts co-sedimented with actin filaments and dissociated from the filaments with ATP treatment. Immunolocalization studies showed that MYA2 co-localized predominantly with actin filaments in clustered punctuate dots in leaf epidermal cells, root hair cells and suspension-cultured cells. In a transgenic plant in which peroxisomes are labeled with green fluorescent protein, some MYA2 signals were localized on peroxisomes in an actin-dependent manner. We propose that the peroxisome is one of the cargos translocated by MYA2 on actin filaments.     

Measurement of enzymatic and motile activities of Arabidopsis myosins by using Arabidopsis actins

There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.     

Maize myosins: diversity, localization, and function

This first analysis of monocotyledon myosin genes showed that at least five genes, one of which was probably spliced to yield two isoforms, were expressed in maize (Zea mays L.). The complete coding sequence of ZMM1 was determined, as were most of the sequences of two other myosin cDNAs (ZMM2 and ZMM3). ZMM1 and ZMM2 belonged to myosin class XI while ZMM3 was in class VIII. ZMM1 was abundantly expressed in leaves, roots, coleoptiles, and stems. ZMM3 showed a similar distribution but was expressed poorly in pollen. ZMM2 was predominantly expressed in seeds and may be part of a suite of cytoskeletal proteins in reproductive tissues. Phylogenetic analysis suggested that the origin of myosin classes VIII and XI predated that of angiosperms. Immunofluorescence studies using M11L1, a myosin XI antibody specific for the exposed loop 1 head region of myosin, indicated that myosin XI occurred in the cytoplasm of all root tip cells. The highest concentration of myosin XI was in the differentiating epidermal cells. In dividing cells, myosin XI was present near the cytokinetic apparatus at approximately the same concentration seen in other portions of the cytoplasm. Western blot analysis of subcellular fractions indicated that myosin XI was concentrated in mitochondria and low-density membranes.     

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.     

Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations

Background

Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in high-velocity cytoplasmic streaming in various plant cells from algae to angiosperms. The velocities at which they process are ten times faster than its closest class V homologues.

Results

To provide sequence determinants and structural rationale for the molecular mechanism of this fast pace myosin, we have compared the sequences from myosin class V and XI through Evolutionary Trace (ET) analysis. The current study identifies class-specific residues of myosin XI spread over the actin binding site, ATP binding site and light chain binding neck region. Sequences for ET analysis were accumulated from six plant genomes, using literature based text search and sequence searches, followed by triple validation viz. CDD search, string-based searches and phylogenetic clustering. We have identified nine myosin XI genes in sorghum and seven in grape by sequence searches. Both the plants possess one gene product each belonging to myosin type VIII as well. During this process, we have re-defined the gene boundaries for three sorghum myosin XI genes using fgenesh program.

Conclusion

Molecular modelling and subsequent analysis of putative interactions involving these class-specific residues suggest a structural basis for the molecular mechanism behind high velocity of plant myosin XI. We propose a model of a more flexible switch I region that contributes to faster ADP release leading to high velocity movement of the algal myosin XI.     

Organelle targeting of myosin XI is mediated by two globular tail subdomains with separate cargo binding sites

Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding to organelles may occur via the globular tail domain in both types of motors, even though sequence similarity is low. To address this hypothesis, we developed a structure homology model for the globular tail of MYA1, a myosin XI from Arabidopsis, based on the known structure of yeast myosin V (Myo2p) globular tail. This model suggested an interaction between two subdomains of the globular tail which was verified by yeast two-hybrid assay and by in vivo bimolecular fluorescence complementation (BiFC). Interface mapping demonstrated that this subdomain interaction depends critically on the C terminus of helix H6 as well as three specific residues in helices H3 and H15, consistent with the structural prediction. The reconstituted globular tails of several Arabidopsis myosin XIs in BiFC assays targeted to peroxisomes in plant cells, identifying this domain as sufficient for cargo binding. Unlike myosin V, either subdomain of myosin XI alone was targeting-competent and responsible for association with different organelles. In addition, our data suggest that organelle binding is regulated by an allosteric interaction between two tail subdomains. We conclude that the globular tail of myosin XI shares a similar structure with that of myosin V, but has evolved plant-specific cargo binding mechanisms.     

Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.     

Muscular myosin isoforms of Taenia solium (Cestoda)

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.     

Identification and molecular characterization of myosin gene family in Oryza sativa genome

Myosins play an important role in various developmental processes in plants. We have identified 14 myosin genes in rice (Oryza sativa cv. Nipponbare) genome using sequence information available in public databases. Phylogenetic analysis of these sequences with other plant and non-plant myosins revealed that two of the predicted sequences belonged to class VIII and the others to class XI. All of these genes were distributed on seven chromosomes in the rice genome. Domain searches on these sequences indicated that a typical rice myosin consisted of Myosin_N, head domain, neck (IQ motifs), tail, and dilute (DIL) domain. Based on the sequence information obtained from predicted myosins, we isolated and sequenced two full-length cDNAs, OsMyoVIIIA and OsMyoXIE, representing each of the two classes of myosins. These two cDNAs isolated from different organs existed in isoforms due to differential splicing and showed minor differences from the predicted myosin in exon organization. Out of 14 myosin genes 11 were expressed in three major organs: leaves, panicles, and roots, among which three myosins exhibited different expression levels. On the other hand, three of the total myosin sequences showed organ-specific expression. The existence of different myosin genes and their isoforms in different organs or tissues indicates the diversity of myosin functions in rice.     

Differential localization of Acanthamoeba myosin I isoforms

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.     

Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region

Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.     

Expression, purification, and refolding of recombinant collagen alpha1(XI) amino terminal domain splice variants

The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.     

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.     

Myosin polymorphism in urodelan amphibian dorsal skeletal muscle

• 1.1. Myosin isoforms were analyzed in the dorsal skeletal muscle of four urodelan amphibian species using a modified pyrophosphate gel electrophoresis which allowed better discrimination than classical methods.
• 2.2. The three fast and the intermediate isomyosins were characterized by a specific heavy chain, respectively HCf and HCi, associated with different combinations of the fast light chains LC1f, LC2f and LC3f.
• 3.3. Slow myosin was characterized by one (P. waltlii, T. palmatus, S. maculata) or two (T. alpestris) isoforms, combining a specific slow myosin heavy chain (HCs) with slow light chains only in the case of P. waltlii, or with slow and fast light chains in the other species.

     

Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.     

Fast skeletal myosin isoforms in thermally acclimated carp.

Fast skeletal myosins were isolated from carp acclimated to 10 and 30 degrees C, and their structural and enzymatic properties were compared. Myosins in 0.5 M KCl were subjected to limited proteolysis by using various proteases including alpha-chymotrypsin, trypsin, and papain, and different SDS-PAGE patterns were seen for the 10- and 30 degrees C-acclimated myosins in all cases. Myosin subfragment-1 (S1) prepared from the 10 degrees C-acclimated myosin by alpha-chymotryptic digestion in 0.12 M NaCl showed higher acto-S1 Mg(2+)-ATPase activity and lower thermostability than S1 from the warm-acclimated myosin. The peptide maps and ATP-induced spectral changes of tryptophan fluorescence also showed an obvious difference between the two types of S1. Temperature acclimation further caused changes in the rod region of myosin, since the apparent sizes of light meromyosin were different from each other for the two types of myosin. Myosin from carp acclimated to 20 degrees C showed intermediate properties between those of the 10- and 30 degrees C-acclimated myosins. Myosin isoforms might be expressed in a temperature-dependent manner to compensate for the effect of seasonal environmental temperature variation on swimming ability.     

The evolutionary relationship of avian and mammalian myosin heavy-chain genes

Summary Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms forms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat α- and β-cardiac myosin heavy-chain sequences suggest that geneconversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.     

Phylogenetic Analysis of New Plant Myosin Sequences

We have sampled a large number of plant taxa, ranging from brown algae to angiosperms, for the presence of myosin sequences. Using phylogenetic analysis, we show that all but two of the new plant myosin sequences fall into two of three preexisting myosin classes. We identified two outlying sequences, which do not fall into any preexisting myosin class. Additionally, all genomic sequences encoding class XI myosins contain an intron in the region studied, suggesting that this genomic region has been conserved over at least 1 billion years of plant evolution. With these data, we can rapidly and consistently classify partial myosin sequences from plants. Our data show that plant myosins do not have clear orthologues in other kingdoms, providing interesting insights into the diversification of myosins.