Kinesin walks hand-over-hand

Kinesin is a processive motor that takes 8.3-nm center-of-mass steps along microtubules for each adenosine triphosphate hydrolyzed. Whether kinesin moves by a "hand-over-hand" or an "inchworm" model has been controversial. We have labeled a single head of the kinesin dimer with a Cy3 fluorophore and localized the position of the dye to within 2 nm before and after a step. We observed that single kinesin heads take steps of 17.3 +/- 3.3 nm. A kinetic analysis of the dwell times between steps shows that the 17-nm steps alternate with 0-nm steps. These results strongly support a hand-over-hand mechanism, and not an inchworm mechanism. In addition, our results suggest that kinesin is bound by both heads to the microtubule while it waits for adenosine triphosphate in between steps.     

Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos toward the microtubule minus end. How the two catalytic head domains interact and move relative to each other during processive movement is unclear. Here, we tracked the relative positions of both heads with nanometer precision and directly observed the heads moving independently along the microtubule. The heads remained widely separated, and their stepping behavior varied as a function of interhead separation. One active head was sufficient for processive movement, and an active head could drag an inactive partner head forward. Thus, dynein moves processively without interhead coordination, a mechanism fundamentally distinct from the hand-over-hand stepping of kinesin and myosin.     

Kinesin moves by an asymmetric hand-over-hand mechanism

Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.     

Nucleotide-dependent single- to double-headed binding of kinesin

The motility of kinesin motors is explained by a "hand-over-hand" model in which two heads of kinesin alternately repeat single-headed and double-headed binding with a microtubule. To investigate the binding mode of kinesin at the key nucleotide states during adenosine 5'-triphosphate (ATP) hydrolysis, we measured the mechanical properties of a single kinesin-microtubule complex by applying an external load with optical tweezers. Both the unbinding force and the elastic modulus in solutions containing AMP-PNP (an ATP analog) were twice the value of those in nucleotide-free solution or in the presence of both AMP-PNP and adenosine 5'-diphosphate. Thus, kinesin binds through two heads in the former and one head in the latter two states, which supports a major prediction of the hand-over-hand model.     

A processive single-headed motor: kinesin superfamily protein KIF1A

A single kinesin molecule can move "processively" along a microtubule for more than 1 micrometer before detaching from it. The prevailing explanation for this processive movement is the "walking model," which envisions that each of two motor domains (heads) of the kinesin molecule binds coordinately to the microtubule. This implies that each kinesin molecule must have two heads to "walk" and that a single-headed kinesin could not move processively. Here, a motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was shown to move processively along the microtubule for more than 1 micrometer. The movement along the microtubules was stochastic and fitted a biased Brownian-movement model.     

Myosin V walks by lever action and Brownian motion

Myosin V is a molecular motor that moves cargo along actin filaments. Its two heads, each attached to a long and relatively stiff neck, move alternately forward in a "hand-over-hand" fashion. To observe under a microscope how the necks move, we attached a micrometer-sized rod to one of the necks. The leading neck swings unidirectionally forward, whereas the trailing neck, once lifted, undergoes extensive Brownian rotation in all directions before landing on a site ahead of the leading head. The neck-neck joint is essentially free, and the neck motion supports a mechanism where the active swing of the leading neck biases the random motion of the lifted head to let it eventually land on a forward site.     

KIF1A alternately uses two loops to bind microtubules

The motor protein kinesin moves along microtubules, driven by adenosine triphosphate (ATP) hydrolysis. However, it remains unclear how kinesin converts the chemical energy into mechanical movement. We report crystal structures of monomeric kinesin KIF1A with three transition-state analogs: adenylyl imidodiphosphate (AMP-PNP), adenosine diphosphate (ADP)-vanadate, and ADP-AlFx (aluminofluoride complexes). These structures, together with known structures of the ADP-bound state and the adenylyl-(beta,gamma-methylene) diphosphate (AMP-PCP)-bound state, show that kinesin uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during the ATP hydrolysis cycle; loop L11 is extended in the AMP-PNP structure, whereas loop L12 is extended in the ADP structure. ADP-vanadate displays an intermediate structure in which a conformational change in two switch regions causes both loops to be raised from the microtubule, thus actively detaching kinesin.     

Evidence that the head of kinesin is sufficient for force generation and motility in vitro

Kinesin is a mechanochemical protein that converts the chemical energy in adenosine triphosphate into mechanical force for movement of cellular components along microtubules. The regions of the kinesin molecule responsible for generating movement were determined by studying the heavy chain of Drosophila kinesin, and its truncated forms, expressed in Escherichia coli. The results demonstrate that (i) kinesin heavy chain alone, without the light chains and other eukaryotic factors, is able to induce microtubule movement in vitro, and (ii) a fragment likely to contain only the kinesin head is also capable of inducing microtubule motility. Thus, the amino-terminal 450 amino acids of kinesin contain all the basic elements needed to convert chemical energy into mechanical force.     

Differential regulation of dynein and kinesin motor proteins by tau

Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.     

A kinesin-like motor inhibits microtubule dynamic instability

The motility of molecular motors and the dynamic instability of microtubules are key dynamic processes for mitotic spindle assembly and function. We report here that one of the mitotic kinesins that localizes to chromosomes, Xklp1 from Xenopus laevis, could inhibit microtubule growth and shrinkage. This effect appeared to be mediated by a structural change in the microtubule lattice. We also found that Xklp1 could act as a fast, nonprocessive, plus end-directed molecular motor. The integration of the two properties, motility and inhibition of microtubule dynamics, in one molecule emphasizes the versatile properties of kinesin family members.     

A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein

The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of bicoid mRNA and the posterior localization of oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive bicoid and oskar localization have been elusive, oocyte microtubules are known to be essential. Here we report that the plus end-directed microtubule motor kinesin I is required for the posterior localization of oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I.     

Directional switching of the kinesin Cin8 through motor coupling

Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end-directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end-directed motors, whereas they switched to plus-end-directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.     

SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus

The cascade of events that leads to vaccinia-induced actin polymerization requires Src-dependent tyrosine phosphorylation of the viral membrane protein A36R. We found that a localized outside-in signaling cascade induced by the viral membrane protein B5R is required to potently activate Src and induce A36R phosphorylation at the plasma membrane. In addition, Src-mediated phosphorylation of A36R regulated the ability of virus particles to recruit and release conventional kinesin. Thus, Src activity regulates the transition between cytoplasmic microtubule transport and actin-based motility at the plasma membrane.     

Conversion of Unc104/KIF1A kinesin into a processive motor after dimerization

Unc104/KIF1A belongs to a class of monomeric kinesin motors that have been thought to possess an unusual motility mechanism. Unlike the unidirectional motion driven by the coordinated actions of the two heads in conventional kinesins, single-headed KIF1A was reported to undergo biased diffusional motion along microtubules. Here, we show that Unc104/KIF1A can dimerize and move unidirectionally and processively with rapid velocities characteristic of transport in living cells. These results suggest that Unc104/KIF1A operates in vivo by a mechanism similar to conventional kinesin and that regulation of motor dimerization may be used to control transport by this class of kinesins.     

A Quantum State-Resolved Insertion Reaction: O((1)D) + H(2)(J = 0) --> OH((2) product operator product operator product operator, v, N) + H((2)S)

The O((1)D) + H(2) --> OH + H reaction, which proceeds mainly as an insertion reaction at a collisional energy of 1.3 kilocalories per mole, has been investigated with the high-resolution H atom Rydberg "tagging" time-of-flight technique and the quasiclassical trajectory (QCT) method. Quantum state-resolved differential cross sections were measured for this prototype reaction. Different rotationally-vibrationally excited OH products have markedly different angular distributions, whereas the total reaction products are roughly forward and backward symmetric. Theoretical results obtained from QCT calculations indicate that this reaction is dominated by the insertion mechanism, with a small contribution from the collinear abstraction mechanism through quantum tunneling.     

Inhibition of Plant Microtubule Polymerization in vitro by the Phosphoric Amide Herbicide Amiprophos-Methyl

The phosphoric amide herbicide amiprophos-methyl (APM) produced a concentration-dependent inhibition of taxol-induced rose microtubule polymerization in vitro. Parallel studies on taxol-induced assembly of bovine brain microtubules showed no effect of APM at a concentration ten times that required to give complete inhibition of rose microtubule assembly. The data indicate that (i) APM is a specific and potent antimicrotubule drug and (ii) APM directly poisons microtubule dynamics in plant cells, rather than indirectly depolymerizing microtubules through a previously proposed mechanism involving deregulation of intracellular calcium levels.     

A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase

Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule.     

Stathmin-tubulin interaction gradients in motile and mitotic cells

The spatial organization of the microtubule cytoskeleton is thought to be directed by steady-state activity gradients of diffusible regulatory molecules. We visualized such intracellular gradients by monitoring the interaction between tubulin and a regulator of microtubule dynamics, stathmin, using a fluorescence resonance energy transfer (FRET) biosensor. These gradients were observed both during interphase in motile membrane protrusions and during mitosis around chromosomes, which suggests that a similar mechanism may contribute to the creation of polarized microtubule structures. These interaction patterns are likely to reflect phosphorylation of stathmin in these areas.     

种子静滑动摩擦系数智能测定装置设计与试验

针对传统种子静滑动摩擦特性测定装置结构复杂、操作繁琐和读取误差较大问题,采用正切机构与Arduino单片机组合技术,设计了一种通过遥控控制能够实现角度自动测量与显示的种子摩擦特性智能测定系统。基于对称正切机构以及摩擦角测量理论,设计了种子静滑动摩擦系数智能测定装置、步进电机的控制系统与摩擦特性测量程序,通过调试系统修正系数提高了测量数据的精度。以玉米、小麦种子进行试验,与已有传统种子静滑动摩擦特性测定对比,结果表明,该装置所测数据与传统的试验数据一致,显著降低了试验工作量,可广泛用于不同种类种子静滑动摩擦特性测定。     

Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.