Tensile Stress on Microtubules Facilitates Dynein-Driven Cargo Transport.

Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.

A Synthetic Minimal Beating Axoneme.

Cilia and flagella are beating rod-like organelles that enable the directional movement of microorganisms in fluids and fluid transport along the surface of biological organisms or inside organs. The molecular motor axonemal dynein drives their beating by interacting with microtubules. Constructing synthetic beating systems with axonemal dynein capable of mimicking ciliary beating still represents a major challenge. Here, the bottom-up engineering of a sustained beating synthoneme consisting of a pair of microtubules connected by a series of periodic arrays of approximately eight axonemal dyneins is reported. A model leads to the understanding of the motion through the cooperative, cyclic association-dissociation of the molecular motor from the microtubules. The synthoneme represents a bottom-up self-organized bio-molecular machine at the nanoscale with cilia-like properties.

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists.

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

Collective motility of dynein linear arrays built on DNA nanotubes.

Dynein motor proteins usually work as a group in vesicle transport, mitosis, and ciliary/flagellar beating inside cells. Despite the obvious importance of the functions of dynein, the effect of inter-dynein interactions on collective motility remains poorly understood due to the difficulty in building large dynein ensembles with defined geometry. Here, we describe a method to build dynein ensembles to investigate the collective motility of dynein on microtubules. Using electron microscopy, we show that tens to hundreds of cytoplasmic dynein monomers were anchored along a 4- or 10-helix DNA nanotube with an average periodicity of 19 or 44 nm (a programmed periodicity of 14 or 28 nm, respectively). They drove the sliding movement of DNA nanotubes along microtubules at a velocity of 170-620 nm/s. Reducing the stiffness of DNA nanotubes made the nanotube movement discontinuous and considerably slower. Decreasing the spacing between motors simply slowed down the nanotube movement. This slowdown was independent of the number of motors involved but heavily dependent on motor-motor distance. This suggests that steric hindrance or mechanical coupling between dynein molecules was responsible for the slowdown. Furthermore, we observed cyclical buckling of DNA nanotubes on microtubules, reminiscent of ciliary/flagellar beating. These results highlight the importance of the geometric arrangement of dynein motors on their collective motility.

Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble.

In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules.

Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing ß-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.

Small teams of myosin Vc motors coordinate their stepping for efficient cargo transport on actin bundles.

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to move continuously on single actin filaments. Single molecules of myoVc cannot take multiple hand-over-hand steps from one actin-binding site to the next without dissociating, in stark contrast to the well studied myosin Va (myoVa) isoform. At low salt, single myoVc motors can, however, move processively on actin bundles, and at physiologic ionic strength, even teams of myoVc motors require actin bundles to sustain continuous motion. Here, we linked defined numbers of myoVc or myoVa molecules to DNA nanostructures as synthetic cargos. Using total internal reflectance fluorescence microscopy, we compared the stepping behavior of myoVc versus myoVa ensembles and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVc and myoVa teams increased with motor number, but only multiple myoVc motors showed a run-length enhancement on actin bundles compared with actin filaments. By resolving the stepping behavior of individual myoVc motors with a quantum dot bound to the motor domain, we found that coupling of two myoVc motors significantly decreased the futile back and side steps that were frequently observed for single myoVc motors. Changes in the inter-motor distance between two coupled myoVc motors affected stepping dynamics, suggesting that mechanical tension coordinates the stepping behavior of two myoVc motors for efficient directional motion. Our study provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

Large-scale vortex lattice emerging from collectively moving microtubules.

Spontaneous collective motion, as in some flocks of bird and schools of fish, is an example of an emergent phenomenon. Such phenomena are at present of great interest and physicists have put forward a number of theoretical results that so far lack experimental verification. In animal behaviour studies, large-scale data collection is now technologically possible, but data are still scarce and arise from observations rather than controlled experiments. Multicellular biological systems, such as bacterial colonies or tissues, allow more control, but may have many hidden variables and interactions, hindering proper tests of theoretical ideas. However, in systems on the subcellular scale such tests may be possible, particularly in in vitro experiments with only few purified components. Motility assays, in which protein filaments are driven by molecular motors grafted to a substrate in the presence of ATP, can show collective motion for high densities of motors and attached filaments. This was demonstrated recently for the actomyosin system, but a complete understanding of the mechanisms at work is still lacking. Here we report experiments in which microtubules are propelled by surface-bound dyneins. In this system it is possible to study the local interaction: we find that colliding microtubules align with each other with high probability. At high densities, this alignment results in self-organization of the microtubules, which are on average 15 µm long, into vortices with diameters of around 400 µm. Inside the vortices, the microtubules circulate both clockwise and anticlockwise. On longer timescales, the vortices form a lattice structure. The emergence of these structures, as verified by a mathematical model, is the result of the smooth, reptation-like motion of single microtubules in combination with local interactions (the nematic alignment due to collisions)--there is no need for long-range interactions. Apart from its potential relevance to cortical arrays in plant cells and other biological situations, our study provides evidence for the existence of previously unsuspected universality classes of collective motion phenomena.

Spontaneous Formation of a Globally Connected Contractile Network in a Microtubule-Motor System.

Microtubule (MT) networks play key roles in cell division, intracellular transport, and cell motility. These functions of MT networks occur through interactions between MTs and various associated proteins, notably motor proteins that bundle and slide MTs. Our objective in this study was to address the question of how motors determine the nature of MT networks. We conducted in vitro assays using homotetrameric kinesin Eg5, a motor protein involved in the formation and maintenance of the mitotic spindle. The mixing of Eg5 and MTs produced a range of spatiotemporal dynamics depending on the motor/filament ratio. Low motor/filament ratios produced globally connected static MT networks with sparsely distributed contractile active nodes (motor-accumulating points with radially extending MTs). Increasing the motor/filament ratio facilitated the linking of contractile active nodes and led to a global contraction of the network. When the motor/filament ratio was further increased, densely distributed active nodes formed local clusters and segmented the network into pieces with their strong contractile forces. Altering the properties of the motor through the use of chimeric Eg5, which has kinesin-1 heads, resulted in the generation of many isolated asters. These results suggest that the spatial distribution of contractile active nodes determines the dynamics of MT-motor networks. We then developed a coarse-grained model of MT-motor networks and identified two essential features for reproducing the experimentally observed patterns: an accumulation of motors that form the active nodes necessary to generate contractile forces, and a nonlinear dependency of contractile force on motor densities. Our model also enabled us to characterize the mechanical properties of the contractile network. Our study provides insight into how local motor-MT interactions generate the spatiotemporal dynamics of macroscopic network structures.

Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors.

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 µm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

X-Ray Fiber Diffraction Recordings from Oriented Demembranated Chlamydomonas Flagellar Axonemes.

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the α-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

Calaxin drives sperm chemotaxis by Ca²âº-mediated direct modulation of a dynein motor.

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca(2+) influx through specific Ca(2+) channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca(2+) is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca(2+)-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca(2+) concentrations. This study describes the missing link between chemoattractant-mediated Ca(2+) signaling and motor-driven microtubule sliding during sperm chemotaxis.

Slow axonemal dynein e facilitates the motility of faster dynein c.

We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s(-1) and kcat,MT = 1.09 s(-1), respectively) were lower than those of dynein c (kcat = 1.75 s(-1) and kcat,MT = 2.03 s(-1), respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 µm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 µm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 µm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c's power stroke.

Formation of highly twisted ribbons in a carboxymethylcellulase gene-disrupted strain of a cellulose-producing bacterium.

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Calcium-induced mechanical change in the neck domain alters the activity of plant myosin XI.

Plant myosin XI functions as a motor that generates cytoplasmic streaming in plant cells. Although cytoplasmic streaming is known to be regulated by intracellular Ca(2+) concentration, the molecular mechanism underlying this control is not fully understood. Here, we investigated the mechanism of regulation of myosin XI by Ca(2+) at the molecular level. Actin filaments were easily detached from myosin XI in an in vitro motility assay at high Ca(2+) concentration (pCa 4) concomitant with the detachment of calmodulin light chains from the neck domains. Electron microscopic observations showed that myosin XI at pCa 4 shortened the neck domain by 30%. Single-molecule analysis revealed that the step size of myosin XI at pCa 4 was shortened to 27 nm under low load and to 22 nm under high load compared with 35 nm independent of the load for intact myosin XI. These results indicate that modulation of the mechanical properties of the neck domain is a key factor for achieving the Ca(2+)-induced regulation of cytoplasmic streaming.

Myosin Mg-ATPase of molluscan muscles is slightly activated by F-actin under catch state in vitro.

Molluscan muscle twitchin, a titin/connectin-related giant protein, regulates interactions between actin and myosin filaments at low Ca(2+) concentrations. When it is dephosphorylated, actin filaments tightly bind to myosin filaments, resulting in the catch state known as the state of high passive tension with very low energy consumption. Yet when twitchin is phosphorylated actin filaments detach from the myosin filaments, resulting in relaxation of the catch. Here, steady-state Mg-ATPase activities of purified myosin were measured under various conditions: without twitchin, with dephosphorylated twitchin, or with phosphorylated twitchin; with or without phalloidin-stabilized F-actin; and at various Ca(2+) concentrations. At low Ca(2+) concentration, Mg-ATPase was activated by F-actin only in the presence of dephosphorylated twitchin (catch state). The activation was about two orders lower than that fully activated by Ca(2+) and F-actin. In the absence of F-actin, twitchin and its phosphorylation state did not affect Mg-ATPase activities in any of the conditions we tested. Based on these results, we propose a molecular mechanism for the catch, where twitchin alone does not interact with the myosin catalytic motor domain but its complex with F-actin does, forming the bridge between actin and myosin filaments and the myosin slowly hydrolyzes Mg-ATP in the catch state.

Self-emergent vortex flow of microtubule and kinesin in cell-sized droplets under water/water phase separation.

By facilitating a water/water phase separation (w/wPS), crowded biopolymers in cells form droplets that contribute to the spatial localization of biological components and their biochemical reactions. However, their influence on mechanical processes driven by protein motors has not been well studied. Here, we show that the w/wPS droplet spontaneously entraps kinesins as well as microtubules (MTs) and generates a micrometre-scale vortex flow inside the droplet. Active droplets with a size of 10-100 µm are generated through w/wPS of dextran and polyethylene glycol mixed with MTs, molecular-engineered chimeric four-headed kinesins and ATP after mechanical mixing. MTs and kinesin rapidly created contractile network accumulated at the interface of the droplet and gradually generated vortical flow, which can drive translational motion of a droplet. Our work reveals that the interface of w/wPS contributes not only to chemical processes but also produces mechanical motion by assembling species of protein motors in a functioning manner.

X-ray diffraction recording from single axonemes of eukaryotic flagella.

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 µm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-µm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 µm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.

Recent progress in dynein structure and mechanism.

Dynein is a minus-end-directed microtubule motor crucial to diverse cellular processes. The unwieldy size of the molecule and the difficulty of expressing and purifying mutants have hampered mechanistic studies of dynein. Recent progress sheds light on key unsolved questions concerning how the molecule is really organized, what conformational changes accompany ATP hydrolysis and whether two or three motor domains are coordinated in their motion.