The unique enzymatic and mechanistic properties of plant myosins (original) (raw)
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Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity
The EMBO Journal, 2003
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 puri®ed 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 con®rmed 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 mm/s, the fastest known processive motion. Processivity was con®rmed by actin landing rate assays. Mean maximal force was~0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules ®tted 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.
Arabidopsis Myosin XI: A Motor Rules the Tracks 1(C)(W)(OPEN)
2014
Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change.
Arabidopsis myosin XI: a motor rules the tracks
Plant physiology, 2014
Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin fil...
2009
Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.
Different subcellular localizations and functions of Arabidopsis myosin VIII
BMC Plant Biology, 2008
Background Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. Results In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, i...
The Plant Journal, 1999
Myosins are a large superfamily of motor proteins which, in association with actin, are involved in intracellular motile processes. In addition to the conventional myosins involved in muscle contractility, there is, in animal cells, a wide range of unconventional myosins implicated in membrane-associated processes, such as vesicle transport and membrane dynamics. In plant cells, however, very little is known about myosins. We have raised an antibody to the recombinant tail region of Arabidopsis thaliana myosin 1 (a class VIII myosin) and used it in immuno¯uorescence and EM studies on root cells from cress and maize. The plant myosin VIII is found to be concentrated at newly formed cross walls at the stage in which the phragmoplast cytoskeleton has depolymerized and the new cell plate is beginning to mature. These walls are rich in plasmodesmata and we show that they are the regions where the longitudinal actin cables appear to attach. Myosin VIII appears to be localized in these plasmodesmata and we suggest that this protein is involved in maturation of the cell plate and the re-establishment of cytoplasmic actin cables at sites of intercellular communication.
How myosin motors power cellular functions - an exciting journey from structure to function
FEBS Journal, 2012
Molecular motors such as myosins are allosteric enzymes that power essential motility functions in the cell and structural biology is an important tool to decipher how these motors work. Force is produced by myosins upon the actin-driven conformational changes that control the sequential release of the hydrolysis products of ATP (Pi followed by ADP). These conformational changes are amplified by a "lever arm" that includes the region of the motor known as the converter and the adjacent elongated light chain binding region. Analysis of four structural states of the motor provides a detailed understanding of the rearrangements and pathways of communication in the motor necessary for detachment from the actin track and repriming of the motor. However, the important part of the cycle in which force is produced remains enigmatic and awaits new high resolution structures. The value of a structural approach is particularly evident from the clues that have been provided from the structural states of the reverse myosin VI motor. Crystallographic structures have revealed that rearrangements within the converter subdomain occur which explains why this myosin can produce a large stroke in the opposite direction of all other myosins despite a very short lever arm. By providing detailed understanding of the motor rearrangements, structural biology will continue to reveal essential information to solve current enigma such as how actin promotes force production, how motors are tuned for specific cellular roles, or how motor/cargo interactions regulate myosin function in the cell. Tension sensing and tension generation are at the heart of essential cellular functions such as intra-cellular transport and cell division. The molecular motors of the myosin superfamily are major players in both of these processes. These actin-activated ATPases convert chemical energy into mechanical work. The acto-myosin complex originally called "myosin" was first discovered as a major component of muscle by Kühne in 1864 [1]. In addition to the filament-forming myosin II class, the myosin superfamily includes at least 34 distinct "unconventional" classes of motors [2]. These motors play essential roles as molecular cargo transporters in the cell, as dynamic tethers or mediators of membranecytoskeleton adhesion and they can power actin filament contraction or possibly disassembly of cytoskeleton tracks among many other functions [3-7]. The coordinated action of many motors allows them to play important roles in cell migration; in the formation and maintenance of cell extensions such as filopodia, microvilli and stereocilia; and in the trafficking of cellular components. Muscle contraction and cytokinesis are also powered by different types of myosin II filaments that slide along and contract actin
Molecular Plant, 2009
The actin cytoskeleton plays a role in mobility of many different organelles in plant cells, including chloroplasts, mitochondria, Golgi, and peroxisomes. While progress has been made in identifying the myosin motors involved in trafficking of various plant organelles, not all of the cargoes mobilized by different members of the myosin XI family have yet been identified. The involvement of myosins in chloroplast positioning and mitochondrial movement was demonstrated by expression of a virus-induced gene silencing (VIGS) construct in tobacco. When VIGS with two different conserved sequences from a myosin XI motor was performed in plants with either GFP-labeled plastids or mitochondria, chloroplast positioning in the dark was abnormal, and mitochondrial movement ceased. Because these and prior observations have implicated a role for myosins and the actin cytoskeleton in plastid and stromule movement, we searched for myosin tail domains that could associate with plastids and stromules. While a yellow fluorescent protein (YFP) fusion with the entire tail region of myosin XI-F was usually found only in the cytoplasm, we observed that an Arabidopsis or Nicotiana benthamiana YFP::myosin XI-F tail domain homologous to the yeast myo2p vacuole-binding domain associated with plastids and stromules after transient expression in N. benthamiana. Taken together, these observations implicate myosin motor proteins in dynamics of plastids and stromules.
Biochemical and Biophysical Research Communications, 2003
The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 lm/s at 30°C and a CCM motor domain with an artificial lever arm consisting of two a-actinin repeats moved actin filaments at 16.2 lm/s. Both constructs displayed high actin-activated ATPase activities ($500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.
Evolutionary traces decode molecular mechanism behind fast pace of myosin XI
BMC Structural Biology, 2011
Background: Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in highvelocity 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 redefined 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.