Length dependence of displacement fluctuations and velocity in microtubule sliding movement driven by sea urchin sperm outer arm beta dynein in vitro

We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules.     

Conventional kinesin mediates microtubule-microtubule interactions in vivo

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.     

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.     

Highly processive motility is not a general feature of the kinesins

Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily. Received: 16 September 1997 / Accepted: 4 November 1997     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.     

Tau Protein Diffuses along the Microtubule Lattice

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.     

Processivity of the Motor Protein Kinesin Requires Two Heads

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.     

Insight into the molecular mechanism of the multitasking kinesin‐8 motor

Members of the kinesin‐8 motor class have the remarkable ability to both walk towards microtubule plus‐ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin‐8 multitasks, we studied the structure and function of the kinesin‐8 motor domain. We determined the first crystal structure of a kinesin‐8 and used cryo‐electron microscopy to calculate the structure of the microtubule‐bound motor. Microtubule‐bound kinesin‐8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin‐8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non‐hydrolysable ATP analogue. This shows that, by collaborating, kinesin‐8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin‐13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin‐8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.     

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Theoretical formalism for kinesin motility I. Bead movement powered by single one-headed kinesins

The directional movement on a microtubule of a plastic bead connected elastically to a single one-headed kinesin motor is studied theoretically. The kinesin motor can bind and unbind to periodic binding sites on the microtubule and undergo conformational changes while catalyzing the hydrolysis of ATP. An analytic formalism relating the dynamics of the bead and the ATP hydrolysis cycle of the motor is derived so that the calculation of the average velocity of the bead can be easily carried out. The formalism was applied to a simple three-state biochemical model to investigate how the velocity of the bead movement is affected by the external load, the diffusion coefficient of the bead, and the stiffness of the elastic element connecting the bead and the motor. The bead velocity was found to be critically dependent on the diffusion coefficient of the bead and the stiffness of the elastic element. A linear force-velocity relation was found for the model no matter whether the bead velocity was modulated by the diffusion coefficient of the bead or by the externally applied load. The formalism should be useful in modeling the mechanisms of chemimechanical coupling in kinesin motors based on in vitro motility data.     

An isoform of microtubule-associated protein 4 inhibits kinesin-driven microtubule gliding

Recently, we revealed that microtubule-associated protein (MAP) 4 isoforms, which differ in the number of repeat sequences, alter the microtubule surface properties, and we proposed a hypothesis stating that the change in the surface properties may regulate the movements of microtubule motors [Tokuraku et al. (2003) J Biol Chem 278: 29609-29618]. In this study, we examined whether MAP4 isoforms affect the kinesin motor activity. When the MAP4 isoforms were present in an in vitro gliding assay, the five-repeat isoform but not the three- and four-repeat isoforms inhibited the movement of the microtubules in a concentration-dependent manner. The observation of individual microtubules revealed that in the presence of the five-repeat isoform, the microtubules completely stopped their movements or recurrently paused and resumed their movements, with no deceleration in the moving phase. The result can be explained by assuming that kinesin stops its movement when it encounters a microtubular region whose properties are altered by the MAPs. A sedimentation assay demonstrated that the MAP4 isoforms did not compete with kinesin for binding to microtubules, indicating that kinesin can bind to the MAP-bound microtubules, although it cannot move on them.     

The role of microtubules in processive kinesin movement

Kinesins are microtubule-based motors that are important for various intracellular transport processes. To understand the mechanism of kinesin movement, X-ray crystallography has been used to study the atomic structures of kinesin. However, as crystal structures of kinesin alone accumulate, it is becoming clear that kinesin structures should also be investigated with the microtubule to understand the contribution of the microtubule track to the nucleotide-induced conformational changes of kinesin. Recently, several high-resolution structures of kinesin with microtubules were obtained using cryo-electron microscopy. Comparison with X-ray crystallographic structures revealed the importance of the microtubule in determining the conformation of kinesin. Together with recent biophysical data, we describe different structural models of processive kinesin movement and provide a framework for future experiments.     

Poleward microtubule flux mitotic spindles assembled in vitro

In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase.     

Effects of Obstacles on the Dynamics of Kinesins,Including Velocity and Run Length,Predicted by a Model of Two Dimensional Motion

Kinesins are molecular motors which walk along microtubules by moving their heads to different binding sites. The motion of kinesin is realized by a conformational change in the structure of the kinesin molecule and by a diffusion of one of its two heads. In this study, a novel model is developed to account for the 2D diffusion of kinesin heads to several neighboring binding sites (near the surface of microtubules). To determine the direction of the next step of a kinesin molecule, this model considers the extension in the neck linkers of kinesin and the dynamic behavior of the coiled-coil structure of the kinesin neck. Also, the mechanical interference between kinesins and obstacles anchored on the microtubules is characterized. The model predicts that both the kinesin velocity and run length (i.e., the walking distance before detaching from the microtubule) are reduced by static obstacles. The run length is decreased more significantly by static obstacles than the velocity. Moreover, our model is able to predict the motion of kinesin when other (several) motors also move along the same microtubule. Furthermore, it suggests that the effect of mechanical interaction/interference between motors is much weaker than the effect of static obstacles. Our newly developed model can be used to address unanswered questions regarding degraded transport caused by the presence of excessive tau proteins on microtubules.     

Protein friction exerted by motor enzymes through a weak-binding interaction.

Recently Vale et al. (1989, Cell 59, 915-925.) reported an observation of the one-dimensional Brownian movement of microtubules bound to flagellar dynein through a weak-binding interaction. In this study, we propose a theoretical model of this phenomenon. Our model consists of a rigid microtubule associated with a number of elastic dynein heads through a weak-binding interaction at equilibrium. The model implies that (1) the Brownian motion of the microtubule is not directly driven by the atomic collision of the solvent particles, but is driven by the thermally-generated structural fluctuations of the dynein heads which interact with the microtubule; (2) dynein heads through a weak-binding interaction exert a frictional drag force on the sliding motion of the microtubule and the drag force is proportional to the sliding velocity the same as in hydrodynamic viscous friction. This protein friction, with such viscous-like characteristics, may well play a role as a velocity-limiting factor in the normal ATP-induced sliding movement of motile proteins.     

Arabidopsis thaliana protein,ATK1, is a minus-end directed kinesin that exhibits non-processive movement

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.     

Directional loading of the kinesin motor molecule as it buckles a microtubule.

Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.     

The role of kinesin and other soluble factors in organelle movement along microtubules

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.     

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion.