Myosin genes in Tetrahymena

This report presents an initial comparison of motor, neck, and tail domains of myosin genes in Tetrahymena thermophila. An unrooted phylogenetic tree drawn from alignment of predicted amino acid translations determined the relationship among 13 myosins in Tetrahymena and their relationship to the myosin superfamily. The myosins in Tetrahymena did not align with any of the previously named myosin classes. Twelve of the Tetrahymena myosins form a new class designated as XX. The other Tetrahymena myosin is divergent from the twelve. Surprisingly, none of the myosins in Tetrahymena aligned with either class I, class II, or class V myosins. Apparent absence of a class II myosin is an indication that cytokinesis in Tetrahymena either utilizes an unconventional myosin or does not require a myosin motor.     

Identification and phylogenetic analysis of Drosophila melanogaster myosins

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.     

Isolation of myosin XI genes from the Closterium peracerosum-strigosum-littorale complex and analysis of their expression during sexual reproduction

Myosins comprise a large superfamily of molecular motors that generate mechanical force in ATP-dependent interactions with actin filaments. On the basis of their conserved motor domain sequences, myosins can be divided into at least 17 classes, 3 of which (VIII, XI, XIII) are found in plants. Although full sequences of myosins are available from several species of green plants, little is known about the functions of these proteins. Additionally, sequence information for algal myosin is incomplete, and little attention has been given to the molecular evolution of myosin from green plants. In the present study, the Closterium peracerosum-strigosum-littorale complex was used as a model system for investigating a unicellular basal charophycean alga. This organism has been well studied with respect to sexual reproduction between its two mating types. Three types of partial sequences belonging to class XI myosins were obtained using degenerate primers designed to amplify motor domain sequences. Real-time polymerase chain reaction analysis of the respective myosin genes during various stages of the algal life cycle showed that one of the genes was more highly expressed during sexual reproduction, and that expression was cell-cycle-dependent in vegetatively grown cells.     

Unique sequences and predicted functions of myosins in Tetrahymena thermophila

Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.     

Myosins: a diverse superfamily

Myosins constitute a large superfamily of actin-dependent molecular motors. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosins which form filaments in muscle and non-muscle cells form class II. There has been extensive characterization of these myosins and much is known about their function. With the exception of class I and class V myosins, little is known about the structure, enzymatic properties, intracellular localization and physiology of most unconventional myosin classes. This review will focus on myosins from class IV, VI, VII, VIII, X, XI, XII, XIII, XIV and XV. In addition, the function of myosin II in non-muscle cells will also be discussed.     

Myosin XI-K Is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana

A prominent feature of plant cells is the rapid, incessant movement of the organelles traditionally defined as cytoplasmic streaming and attributed to actomyosin motility. We sequenced six complete Nicotiana benthamiana cDNAs that encode class XI and class VIII myosins. Phylogenetic analysis indicates that these two classes of myosins diverged prior to the radiation of green algae and land plants from a common ancestor and that the common ancestor of land plants likely possessed at least seven myosins. We further report here that movement of Golgi stacks, mitochondria, and peroxisomes in the leaf cells of N. benthamiana is mediated mainly by myosin XI-K. Suppression of myosin XI-K function using dominant negative inhibition or RNA interference dramatically reduced movement of each of these organelles. When similar approaches were used to inhibit functions of myosin XI-2 or XI-F, only moderate to marginal effects were observed. Organelle trafficking was virtually unaffected in response to inhibition of each of the three class VIII myosins. Interestingly, none of the tested six myosins appears to be involved in light-induced movements of chloroplasts. Taken together, these data strongly suggest that myosin XI-K has a major role in trafficking of Golgi stacks, mitochondria, and peroxisomes, whereas myosins XI-2 and XI-F might perform accessory functions in this process. In addition, our analysis of thousands of individual organelles revealed independent movement patterns for Golgi stacks, mitochondria, and peroxisomes, indicating that the notion of coordinated cytoplasmic streaming is not generally applicable to higher plants.     

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.     

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

Background

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.

Results

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.

Conclusions

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.     

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.     

Genetic analysis of myosin assembly inCaenorhabditis elegans

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.     

Class II myosins in nematodes-- genetic relationships, fundamental and applied implications

Myosins are represented by a wide range of different classes of molecule, of which the most extensively studied are the class II myosins which drive muscle contraction and cell organization; the functional unit of class II myosins comprises two myosin heavy chains (MHCs). This minireview gives an update on class II MHCs of nematodes and describes a comparative analysis of MHC genes from nematodes and other organismal groups. Genetic analyses of sequence data for the four functional domains of MHCs (i.e., the SH3-like N-terminal, head, neck and tail domains) reveal a delineation between both the nematode and non-nematode myosins and between muscle and non-muscle myosins. The distinctiveness of the MHCs of nematodes suggests functional and tissue specialization. The elucidation of the functional roles of myosins and other molecules in specific signaling pathways in nematodes has the potential to lead to new intervention strategies for parasites via the specific disruption or interruption of key developmental processes, having biotechnological implications in the longer term.     

Regulation of myosin 5a and myosin 7a

The myosin superfamily is diverse in its structure, kinetic mechanisms and cellular function. The enzymatic activities of most myosins are regulated by some means such as Ca2+ ion binding, phosphorylation or binding of other proteins. In the present review, we discuss the structural basis for the regulation of mammalian myosin 5a and Drosophila myosin 7a. We show that, although both myosins have a folded inactive state in which domains in the myosin tail interact with the motor domain, the details of the regulation of these two myosins differ greatly.     

果蝇非常规肌球蛋白的结构与功能

肌球蛋白是一类重要的分子马达,可以将ATP水解产生的能量转化成动能,沿由肌动蛋白组成的细丝运动。肌球蛋白构成一个大的基因家族,在许多细胞活动中起着重要作用,包括肌肉收缩、胞内转运、听觉、视觉等。果蝇基因组有13种肌球蛋白基因,包括2种常规肌球蛋白和11种非常规肌球蛋白。本文综述了近年来果蝇非常规肌球蛋白的研究进展。     

Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis

Multigene families encoding class XI myosins are conserved in higher plants, however, little information is available on specific functions of these ubiquitous molecular motors. We isolated gene knockout mutants for all 13 class XI myosins present in Arabidopsis (Arabidopsis thaliana) genome. Inactivation of 11 myosin genes resulted in no discernible phenotypes under the normal growth conditions. In contrast, the knockouts of the remaining two myosin genes, XI-2 (formerly MYA2) and XI-K, exhibited similar defects in root hair elongation suggesting that the myosin-driven motility plays a significant role in a polar tip growth. Strikingly, inactivation of each of these myosins also reduced trafficking of Golgi stacks, peroxisomes, and mitochondria in root hairs and in leaf epidermal cells. These results indicate that myosins XI-K and XI-2 play major and overlapping roles in the cell dynamics in Arabidopsis and highlight the redundant nature of myosin function in plants.     

Arabidopsis thaliana myosin XIK is involved in root hair as well as trichome morphogenesis on stems and leaves

Myosins form a large superfamily of molecular motors that move along actin filaments. The functions of myosins in plant cells are thought to be related to various processes: cell division, movement of mitochondria and chloroplasts, cytoplasmic streaming, rearrangement of transvacuolar strands, and statolith positioning. Class VIII and XI myosins are represented in the Arabidopsis thaliana genome by 4 and 13 potential genes, respectively. The roles of individual class XI myosins and their cellular targets in A. thaliana are still unclear. In this work we implemented a reverse genetic approach to analyse the loss-of-function mutants of XIK, a representative of class XI myosins in A. thaliana. Three different T-DNA insertion mutants in the myosin XIK gene showed similar phenotypes: impaired growth of root hair cells, twisted shape of stem trichomes, and irregular size, branch positioning, and branch expansion of leaf trichomes. Morphometric analysis of mutant seedlings showed that the average length of root hairs was reduced up to 50% in comparison with wild-type root hairs, suggesting an involvement of the class XI myosin XIK in tip growth. On leaves, the proportion of trichomes with short branches was doubleed in mutant plants, and the mutant trichomes possessed a mildly twisted shape. Therefore, we concluded that myosin XIK is involved also in the elongation of stalks and branches of trichomes.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4)

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.     

A possible mechanism for determining the directionality of myosin molecular motors

There is a large superfamily of myosins, which play various fundamental roles in cellular motility. In this superfamily, most of myosins, including myosins II and V, move to the barbed end of an actin filament, whereas myosin VI was found to move in the opposite direction to the pointed end. Although myosin VI has structural differences compared with the other myosins, the mechanism for the reversal of the directionality has not been satisfactorily explained by conventional theories for myosin motility, including the widely accepted lever-arm hypothesis. In this paper, a simple mechanism for determining the directionality is proposed. The mechanism assumes that the driving force for the power stroke is caused by elastic energy stored within a myosin molecule at the joint between the head and the neck. The elastic energy originates from the attractive force between myosin and actin, and accumulates during the docking process. The energy of ATP is used to reduce the attractive force between myosin and actin and to facilitate the dissociation of these molecules. Therefore, it is not directly engaged in the power stroke. With this mechanism, the directionality of the myosin motility is simply determined by the direction of the neck with respect to the head in the dissociated configuration. This structural difference is actually observed in myosin VI. The same mechanism also explains the behavior of a backward moving engineered myosin. Computer simulations demonstrated the feasibility of this working mechanism.     

Overlapping distribution of the 130- and 110-kDa myosin I isoforms on rat liver membranes

The biochemical and mechanochemical properties and localization of myosin I suggest the involvement of these small members of the myosin superfamily in some aspects of intracellular motility in higher cells. We have determined by quantitative immunoblotting with isoform-specific antibodies that the 130-kDa myosin I (myr 1 gene product) and 110-kDa myosin I (myr 2 gene product) account for 0.5 and 0.4%, respectively, of total rat liver protein. Immunoblot analyses reveal that the 130- and 110-kDa myosins I are found in several purified subcellular fractions from rat liver. The membrane-associated 130-kDa myosin I is found at the highest concentration in the plasma membrane (28 ng/microg plasma membrane protein) followed by the endoplasmic reticulum-like mitochondria-associated membrane fraction (MAM; 10 ng/microg MAM protein), whereas the 110-kDa myosin I is found at the highest concentration in Golgi (50 ng/?g Golgi protein) followed by plasma membrane (20 ng/microg) and MAM (7 ng/microg). Our analyses indicate that myosin I is peripherally associated with Golgi and MAM and its presence in these fractions is not a consequence of myosin I bound to contaminating actin filaments. Although found in relatively low concentrations in microsomes, because of the abundance of microsomes, in liver most of the membrane-associated myosin I is associated with microsomes. Neither myosin I isoform is detected in purified mitochondria. This is the first quantitative analysis addressing the cellular distribution of these mammalian class I myosins.