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1.
Kon T  Oyama T  Shimo-Kon R  Imamula K  Shima T  Sutoh K  Kurisu G 《Nature》2012,484(7394):345-350
Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8?? resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.  相似文献   

2.
M Irving  V Lombardi  G Piazzesi  M A Ferenczi 《Nature》1992,357(6374):156-158
Motor proteins such as myosin, dynein and kinesin use the free energy of ATP hydrolysis to produce force or motion, but despite recent progress their molecular mechanism is unknown. The best characterized system is the myosin motor which moves actin filaments in muscle. When an active muscle fibre is rapidly shortened the force first decreases, then partially recovers over the next few milliseconds. This elementary force-generating process is thought to be due to a structural 'working stroke' in the myosin head domain, although structural studies have not provided definitive support for this. X-ray diffraction has shown that shortening steps produce a large decrease in the intensity of the 14.5 nm reflection arising from the axial repeat of the myosin heads along the filaments. This was interpreted as a structural change at the end of the working stroke, but the techniques then available did not allow temporal resolution of the elementary force-generating process itself. Using improved measurement techniques, we show here that myosin heads move by about 10 nm with the same time course as the elementary force-generating process.  相似文献   

3.
Mallik R  Carter BC  Lex SA  King SJ  Gross SP 《Nature》2004,427(6975):649-652
Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.  相似文献   

4.
H Higuchi  Y E Goldman 《Nature》1991,352(6333):352-354
Muscle contraction is generally thought to be driven by tilting of the 19-nm-long myosin head, part of the thick filament, while attached to actin, part of the thin filament. This motion would produce about 12 nm of filament sliding. Recent estimates of the sliding distance per ATP molecule hydrolysed by actomyosin in vitro vary widely from 8 nm to greater than or equal to 200 nm. The latter value is incompatible with a power stroke incorporating a single tilting motion of the head. We have measured the isotonic sliding distance per ATP molecule hydrolysed during the interaction between myosin and actin in skinned muscle fibres. We directly estimated the proportion of simultaneously attached actomyosin complexes and their ATP use. We report here that at low loads the interaction distance is at least 40 nm. This distance corresponds to the length of the power stroke plus the filament sliding while actomyosin crossbridges bear negative drag forces. If the power stroke is 12 nm, then our results indicate the drag distance to be at least 28 nm. Our results could also be explained by multiple power strokes per ATP molecule hydrolysed.  相似文献   

5.
H Sakakibara  H Kojima  Y Sakai  E Katayama  K Oiwa 《Nature》1999,400(6744):586-590
Axonemal dyneins are force-generating ATPases that produce movement of eukaryotic cilia and flagella. Several studies indicate that inner-arm dyneins mainly produce bending moments in flagella and that these motors have inherent oscillations in force and motility. Processive motors such as kinesins have high duty ratios of attached to total ATPase cycle (attached plus detached) times compared to sliding motors such as myosin. Here we provide evidence that subspecies-c, a single-headed axonemal inner-arm dynein, is processive but has a low duty ratio. Ultrastructurally it is similar to other dyneins, with a single globular head, long stem and a slender stalk that attaches to microtubules. In vitro studies of microtubules sliding over surfaces coated with subspecies-c at low densities (measured by single-molecule fluorescence) show that a single molecule is sufficient to move a microtubule more than 1 microm at 0.7 microm s(-1). When many motors interact the velocity is 5.1 microm s(-1), fitting a duty ratio of 0.14. Using optical trap nanometry, we show that beads carrying a single subspecies-c motor move processively along the microtubules in 8-nm steps but slip backwards under high loads. These results indicate that dynein subspecies-c functions in a very different way from conventional motor proteins, and has properties that could produce self-oscillation in vivo.  相似文献   

6.
High-frequency nanometre-scale vibration in 'quiescent' flagellar axonemes   总被引:2,自引:0,他引:2  
S Kamimura  R Kamiya 《Nature》1989,340(6233):476-478
The movement of cilia and flagella is based on the interaction between dynein arms and microtubules coupled with ATP hydrolysis. Although it is established that dynein arms cause adjacent microtubules to slide, little is known about the elementary process underlying the force production. To look more closely at the mechano-chemical conversion mechanism, we recently developed an optical method for measuring a nanometre-scale displacement with a time-resolution better than 1 ms. We now report the detection of high frequency (approximately 300 Hz) vibration of sub-nanometre amplitude in non-beating flagellar axonemes. This vibration could reflect the movement of individual activated dynein arms.  相似文献   

7.
Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.  相似文献   

8.
Mancoff FB  Rizzo ND  Engel BN  Tehrani S 《Nature》2005,437(7057):393-395
Spin-transfer in nanometre-scale magnetic devices results from the torque on a ferromagnet owing to its interaction with a spin-polarized current and the electrons' spin angular momentum. Experiments have detected either a reversal or high-frequency (GHz) steady-state precession of the magnetization in giant magnetoresistance spin valves and magnetic tunnel junctions with current densities of more than 10(7) A cm(-2). Spin-transfer devices may enable high-density, low-power magnetic random access memory or direct-current-driven nanometre-sized microwave oscillators. Here we show that the magnetization oscillations induced by spin-transfer in two 80-nm-diameter giant-magnetoresistance point contacts in close proximity to each other can phase-lock into a single resonance over a frequency range from approximately <10 to >24 GHz for contact spacings of less than about approximately 200 nm. The output power from these contact pairs with small spacing is approximately twice the total power from more widely spaced (approximately 400 nm and greater) contact pairs that undergo separate resonances, indicating that the closely spaced pairs are phase-locked with zero phase shift. Phase-locking may enable control of large arrays of coupled spin-transfer devices with increased power output for microwave oscillator applications.  相似文献   

9.
Rapid regeneration of the actin-myosin power stroke in contracting muscle.   总被引:1,自引:0,他引:1  
V Lombardi  G Piazzesi  M Linari 《Nature》1992,355(6361):638-641
At the molecular level, muscle contraction is the result of cyclic interaction between myosin crossbridges, which extend from the thick filament, and the thin filament, which consists mainly of actin. The energy for work done by a single crossbridge during a cycle of attachment, generation of force, shortening and detachment is believed to be coupled to the hydrolysis of one molecule of ATP. The distance the actin filament slides relative to the myosin filament in one crossbridge cycle has been estimated as 12 nm by step-length perturbation studies on single fibres from frog muscle. The 'mechanical' power stroke of the attached crossbridge can therefore be defined as 12-nm shortening with a force profile like that shown by the quick recovery of force following a length perturbation. According to this definition, power strokes cannot be repeated faster than the overall ATPase rate. Here, however, we show that the power stroke can be regenerated much faster than expected from the ATPase rate. This contradiction can be resolved if, in the shortening muscle, the free energy of ATP hydrolysis is used in several actin-myosin interactions consisting of elementary power strokes each of 5-10 nm.  相似文献   

10.
Thermoelectric materials interconvert thermal gradients and electric fields for power generation or for refrigeration. Thermoelectrics currently find only niche applications because of their limited efficiency, which is measured by the dimensionless parameter ZT-a function of the Seebeck coefficient or thermoelectric power, and of the electrical and thermal conductivities. Maximizing ZT is challenging because optimizing one physical parameter often adversely affects another. Several groups have achieved significant improvements in ZT through multi-component nanostructured thermoelectrics, such as Bi(2)Te(3)/Sb(2)Te(3) thin-film superlattices, or embedded PbSeTe quantum dot superlattices. Here we report efficient thermoelectric performance from the single-component system of silicon nanowires for cross-sectional areas of 10 nm x 20 nm and 20 nm x 20 nm. By varying the nanowire size and impurity doping levels, ZT values representing an approximately 100-fold improvement over bulk Si are achieved over a broad temperature range, including ZT approximately 1 at 200 K. Independent measurements of the Seebeck coefficient, the electrical conductivity and the thermal conductivity, combined with theory, indicate that the improved efficiency originates from phonon effects. These results are expected to apply to other classes of semiconductor nanomaterials.  相似文献   

11.
Cytoplasmic dynein is localized to kinetochores during mitosis   总被引:90,自引:0,他引:90  
Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.  相似文献   

12.
Identification of globular mechanochemical heads of kinesin   总被引:37,自引:0,他引:37  
J M Scholey  J Heuser  J T Yang  L S Goldstein 《Nature》1989,338(6213):355-357
Kinesin is a mechanoenzyme which uses energy liberated from ATP hydrolysis to transport particles towards the 'plus ends' of microtubules. The enzyme consists of two polypeptide heavy chains of relative molecular mass (Mr) approximately 110,000-140,000 (110K-140K) plus copurifying light chains; these polypeptides are arranged in a structure consisting of two globular heads attached to a fibrous stalk which terminates in a 'feathered' tail. Here we report that a function-disrupting monoclonal antikinesin, which binds to the 45K fragment of the kinesin heavy chain, recognizes an epitope located towards the N-terminal end of the heavy chain, and decorates the two globular heads lying at one end of the intact molecules (one antibody per head). The results show that the two heavy chains of native kinesin are arranged in parallel, and that the 45K fragments, which display nucleotide-sensitive interactions with microtubules, represent mechanochemical 'heads' located at the N-terminal regions of the heavy chains. Thus, it is likely that the kinesin heads are analogous to the subfragment-1 domains of myosin.  相似文献   

13.
Sakamoto T  Webb MR  Forgacs E  White HD  Sellers JR 《Nature》2008,455(7209):128-132
Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.  相似文献   

14.
借助金相、扫描电等手术对低扭矩脆断的BJ212汽车轮壳螺栓的金相组织特征,宏观和向量观断口形貌等进行了研究,结果表明,这种头部薄的螺栓,渗层和淬透层过使头我脆断抗力降低,在头部与杆的根部,由于淬火应力不均匀导致根部产生拉应力,均是导致螺栓低扭矩断裂的原因。  相似文献   

15.
Gupta V  Poss KD 《Nature》2012,484(7395):479-484
As vertebrate embryos develop to adulthood, their organs undergo marked changes in size and tissue architecture. The heart acquires muscle mass and matures structurally to fulfil increasing circulatory needs, a process that is incompletely understood. Here we used multicolour clonal analysis to define the contributions of individual cardiomyocytes as the zebrafish heart undergoes morphogenesis from a primitive embryonic structure into its complex adult form. We find that the single-cardiomyocyte-thick wall of the juvenile ventricle forms by lateral expansion of several dozen cardiomyocytes into muscle patches of variable sizes and shapes. As juvenile zebrafish mature into adults, this structure becomes fully enveloped by a new lineage of cortical muscle. Adult cortical muscle originates from a small number of cardiomyocytes--an average of approximately eight per animal--that display clonal dominance reminiscent of stem cell populations. Cortical cardiomyocytes initially emerge from internal myofibres that in rare events breach the juvenile ventricular wall, and then expand over the surface. Our results illuminate the dynamic proliferative behaviours that generate adult cardiac structure, revealing clonal dominance as a key mechanism that shapes a vertebrate organ.  相似文献   

16.
The role of barren stalk1 in the architecture of maize   总被引:4,自引:0,他引:4  
The architecture of higher plants is established through the activity of lateral meristems--small groups of stem cells formed during vegetative and reproductive development. Lateral meristems generate branches and inflorescence structures, which define the overall form of a plant, and are largely responsible for the evolution of different plant architectures. Here, we report the isolation of the barren stalk1 gene, which encodes a non-canonical basic helix-loop-helix protein required for the initiation of all aerial lateral meristems in maize. barren stalk1 represents one of the earliest genes involved in the patterning of maize inflorescences, and, together with the teosinte branched1 gene, it regulates vegetative lateral meristem development. The architecture of maize has been a major target of selection for early agriculturalists and modern farmers, because it influences harvesting, breeding strategies and mechanization. By sampling nucleotide diversity in the barren stalk1 region, we show that two haplotypes entered the maize gene pool from its wild progenitor, teosinte, and that only one was incorporated throughout modern inbreds, suggesting that barren stalk1 was selected for agronomic purposes.  相似文献   

17.
Mechanism of force generation by myosin heads in skeletal muscle   总被引:1,自引:0,他引:1  
Muscles generate force and shortening in a cyclical interaction between the myosin head domains projecting from the myosin filaments and the adjacent actin filaments. Although many features of the dynamic performance of muscle are determined by the rates of attachment and detachment of myosin and actin, the primary event in force generation is thought to be a conformational change or 'working stroke' in the actin-bound myosin head. According to this hypothesis, the working stroke is much faster than attachment or detachment, but can be observed directly in the rapid force transients that follow step displacement of the filaments. Although many studies of the mechanism of muscle contraction have been based on this hypothesis, the alternative view-that the fast force transients are caused by fast components of attachment and detachment--has not been excluded definitively. Here we show that measurements of the axial motions of the myosin heads at ?ngstr?m resolution by a new X-ray interference technique rule out the rapid attachment/detachment hypothesis, and provide compelling support for the working stroke model of force generation.  相似文献   

18.
Invariant scaling relations across tree-dominated communities   总被引:36,自引:0,他引:36  
Enquist BJ  Niklas KJ 《Nature》2001,410(6829):655-660
Organizing principles are needed to link organismal, community and ecosystem attributes across spatial and temporal scales. Here we extend allometric theory-how attributes of organisms change with variation in their size-and test its predictions against worldwide data sets for forest communities by quantifying the relationships among tree size-frequency distributions, standing biomass, species number and number of individuals per unit area. As predicted, except for the highest latitudes, the number of individuals scales as the -2 power of basal stem diameter or as the -3/4 power of above-ground biomass. Also as predicted, this scaling relationship varies little with species diversity, total standing biomass, latitude and geographic sampling area. A simulation model in which individuals allocate biomass to leaf, stem and reproduction, and compete for space and light obtains features identical to those of a community. In tandem with allometric theory, our results indicate that many macroecological features of communities may emerge from a few allometric principles operating at the level of the individual.  相似文献   

19.
Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain.   总被引:30,自引:0,他引:30  
I R Gibbons  B H Gibbons  G Mocz  D J Asai 《Nature》1991,352(6336):640-643
Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.  相似文献   

20.
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