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.

Programmable molecular transport achieved by engineering protein motors to move on DNA nanotubes.

Intracellular transport is the basis of microscale logistics within cells and is powered by biomolecular motors. Mimicking transport for in vitro applications has been widely studied; however, the inflexibility in track design and control has hindered practical applications. Here, we developed protein-based motors that move on DNA nanotubes by combining a biomolecular motor dynein and DNA binding proteins. The new motors and DNA-based nanoarchitectures enabled us to arrange the binding sites on the track, locally control the direction of movement, and achieve multiplexed cargo transport by different motors. The integration of these technologies realized microscale cargo sorters and integrators that automatically transport molecules as programmed in DNA sequences on a branched DNA nanotube. Our system should provide a versatile, controllable platform for future applications.

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.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating.

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)-dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

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.

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.

Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics.

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Creating biomolecular motors based on dynein and actin-binding proteins.

Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors-combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins-robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

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.

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.

Changes in vitreous temperature during intravitreal surgery.

PURPOSE: To determine how different intraoperative surgical procedures affect the midvitreous temperature. METHODS: The vitreous temperatures were monitored continuously with an intravitreal thermocouple in 87 eyes of 81 cases undergoing vitrectomy. Thirty-three eyes had diabetic retinopathy (DR), 35 eyes had an epiretinal membrane, and 19 eyes had an idiopathic macular hole. In eyes with DR, the correlation between the number of photocoagulations (PCs) and the change in temperature was analyzed. The temperature was also recorded before and after combined phacoemulsification and aspiration (PEA) and vitrectomy in 10 eyes. RESULTS: The average midvitreal temperature before the vitrectomy was 33.0 ± 1.3°C, 30.7 ± 1.7°C after core vitrectomy, 32.9 ± 1.3°C after membrane peeling, and 29.2 ± 1.4°C after peripheral vitrectomy. The temperature before PC was 29.8 ± 1.3°C, and it increased to 31.5 ± 1.9°C post-PC. The differences in the temperatures between consecutive procedures were significant (P < 0.01, respectively, Wilcoxon signed-rank test). The difference in the temperatures of the same procedures among the different diseases was not significant except after membrane peeling. A significant correlation was detected between the number of PCs and the duration of the PCs, and between the duration of PCs and the change in vitreous temperature after PC (r = 0.719, P = 0.0010, and r = 0.800, P = 0.0002, respectively, Spearman's rank correlation coefficient test). The temperature after PEA decreased significantly by 2.3°C. CONCLUSIONS: Our results showed that vitreous temperatures vary during different vitrectomy procedures.

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.

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.

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.

ATP-driven remodeling of the linker domain in the dynein motor.

Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis.

F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities.

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.

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.

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.

Molecular organization and force-generating mechanism of dynein.

Dynein, which is a minus-end-directed microtubule motor, is crucial to a range of cellular processes. The mass of its motor domain is about 10 times that of kinesin, the other microtubule motor. Its large size and the difficulty of expressing and purifying mutants have hampered progress in dynein research. Recently, however, electron microscopy, X-ray crystallography and single-molecule nanometry have shed light on several key unsolved questions concerning how the dynein molecule is organized, what conformational changes in the molecule accompany ATP hydrolysis, and whether two or three motor domains are coordinated in the movements of dynein. This minireview describes our current knowledge of the molecular organization and the force-generating mechanism of dynein, with emphasis on findings from electron microscopy and single-molecule nanometry.