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.

] i ) in sperm significantly alters swimming direction during chemotaxis (3, 4). Recent studies of human sperm suggest that progesteronemediated Ca 2+ influx through a Ca 2+ channel, CatSper, plays essential role in motility changes, such as hyperactivation and chemotaxis (5, 6), although both phenomena are argued to be separated (7). However, Ca 2+ is a primary factor regulating symmetry of flagellar waveform (8, 9). Chemotactic movements are achieved by continuous changes in waveform symmetry of sperm flagella and subsequent changes in swimming direction to access the egg (10). Ca 2+ -dependent regulation of flagellar beating is reportedly governed by Ca 2+ -binding proteins that likely regulate axonemal dynein (8, 9). These proteins have not been identified, however, and the molecular mechanism of Ca 2+ -dependent control of flagellar asymmetry in sperm chemotaxis remains uncharacterized.Marine invertebrates are excellent models to study sperm motility because their sperm show clear motility changes and are produced in quantities sufficient for biochemical analysis. In the ascidian, Ciona intestinalis, a transient flagellar [Ca 2+ ] i burst is induced by a chemoattractant called sperm activating and attracting factor (SAAF), which triggers rapid sperm-turning followed by straight swimming toward eggs (4, 11). In a search for candidates that regulate Ca 2+ -dependent flagellar movement, we recently identified an axonemal Ca 2+ -binding protein, calaxin, which binds to outer-arm dynein in sperm flagella of C. intestinalis (12). Calaxin is highly conserved in metazoa, including mouse and human. In the present study, using Ciona sperm we show that calaxin is essential for Ca 2+ -dependent modulation of sperm movement necessary for chemotaxis toward the egg. We use in vitro motility assays to demonstrate that calaxin directly suppresses microtubule sliding driven by outer-arm dynein. Results and DiscussionTo test the function of calaxin in regulation of sperm motility in chemotaxis, we used an inhibitor of neuronal calcium sensor family proteins, repaglinide, which specifically binds to calaxin in sperm flagella (Fig. S1) (13). We first asked whether calaxin plays a critical role in sperm chemotaxis. During chemotactic movements, sperm show a unique turning movement associated with a flagellar change to an asymmetric waveform, followed by a straight-ahead movement (11). We observed sperm chemotactic movement toward a glass capillary filled with SAAF in the absence and presence of repaglinide (Fig. 1A). Sperm in control artificial sea water (ASW) with 0.5% (vol/vol) solvent (DMSO) showed very strong chemotaxis toward the glass capillary. However, sperm in the ASW containing 150 μM repaglinide did not exhibit the unique turn movement and showed less-effective chemotaxis (Fig. 1A). Linear equation chemotaxis index (LECI) (11) analysis quantitatively showed significantly decreased chemotactic property promoted by repaglinide at >100 μM (Fig. 1B). Sperm-swimming velocity showed no dramatic change following repagli...

Fluorescence resonance energy transfer showed that troponin-I changes the position on an actin filament corresponding to three states (relaxed, closed, and open) of the thin filament (Hai et al. (2002) J. Biochem. 131, 407-418). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to position 1, 133, or 181 of troponin-I and Cys-374 of actin on reconstituted thin filaments was measured to follow the transition between three states of the thin filament. When the free Ca(2+) concentration was increased, the transition from relaxed to closed states occurred with a rate constant of approximately 500 s(-1). For the reverse transition, the rate constant was approximately 60 s(-1). When myosin subfragment-1 was dissociated from thin filaments in the presence of Ca(2+) by rapid mixing with ATP, the transition from open to closed states occurred with a single rate constant of approximately 300 s(-1). Light-scattering measurements showed that the ATP-induced myosin subfragment-1 dissociation occurred with a rate constant of approximately 900 s(-1). In the absence of Ca(2+), the transition from open to relaxed states occurred with two rate constants of approximately 400 and approximately 80 s(-1). These transition rates are fast enough to allow the spatial rearrangement of thin filaments to be involved in the regulation mechanism of muscle contraction.

Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca 2؉ concentration was rapidly changed, these domains moved with rates of ϳ450 and ϳ85 s ؊1 at pH 7.0 on Ca 2؉ up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of ϳ400 s ؊1 in the presence and absence of Ca 2؉ . The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s ؊1 . When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739 -10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.

In vitro motility assays, where protein motors (attached to a surface) move protein filaments, have been used for investigating protein motors' functions. In recent decades, these assays are extended to explore potential applications of motor proteins as biological motors in nano-bio-machine development. Recent attempts include fabricating micrometer-scale tracks on the surface to confine and guide the flow of bio-filaments as a power transfer medium driven by the motor proteins. Understanding the interaction between bio-filaments and fabricated tracks as well as the mutual interaction between bio-filaments is of importance to the design of potential nano-bio-machines. In this study, we investigate the behaviors of a microtubule driven by axonemal dynein at the collision against another microtubule and micro-fabricated walls, respectively. Based on experimental observations, we propose a model to study possible mechanisms for the microtubule-microtubule and microtubule-wall interactions, which involve bumping force, bending moment and torque generation.

Creatures have evolved extremely intelligent and complex adaptive systems for conducting their movements. They are protein motors with typical sizes of a few tens of nanometers. Protein motors include three major protein families, myosin, kinesin and dynein, which participate in a wide range of cellular processes, using energy from the hydrolysis of adenosinetriphosphate ATP. To harness these protein motors to power nanometer-scale devices, we have investigated effective and non-destructive methods for immobilizing protein motors on surfaces and to arrange the output of these motors, e.g. force and movement, to be in a defined direction. We found NEB-22 to be useful for retaining the abilities of protein motors to support the movement of protein filaments. We fabricated various patterns of tracks of NEB-22 on coverslips and protein motors were introduced and immobilized on glass surface. The trajectories of protein polymers were confined to these tracks. Simple patterns readily biased and guide polymer movement confining it to be unidirectional. In addition, having used dynein c purified from Chlamydomonas flagellar axoneme, we showed that microtubules driven by surface-bound dynein were self-organized into dynamic streams through collisions between the microtubules and their subsequent joining.

No abstract