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.     

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)- induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.     

Loading direction regulates the affinity of ADP for kinesin

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.     

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.     

Jump-starting kinesin

When it is not actively transporting cargo, conventional Kinesin-1 is present in the cytoplasm in a folded conformation that cannot interact effectively with microtubules (MTs). Two important and largely unexplored aspects of kinesin regulation are how it is converted to an active species when bound to cargo and the related issue of how kinesin discriminates among its many potential cargo molecules. Blasius et al. (see p. 11 of this issue) report that either binding of the cargo linker c-Jun N-terminal kinase-interacting protein 1 (JIP1) to the light chains (LCs) or binding of fasciculation and elongation protein zeta1 (FEZ1) to the heavy chains (HCs) is insufficient for activation but that activation occurs when both are present simultaneously. A related paper by Cai et al. (see p. 51 of this issue) provides structural insight into the conformation of the folded state in the cell obtained by fluorescence resonance energy transfer analysis.     

Strain through the neck linker ensures processive runs: a DNA‐kinesin hybrid nanomachine study

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA‐kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA‐kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C‐terminal or Mid position) and sub‐nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13‐amino‐acid chain positioned at the C‐terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter‐head coordination through the neck linker facilitates long‐distance walking.     

Kinesin walks the line: single motors observed by atomic force microscopy

Motor proteins of the kinesin family move actively along microtubules to transport cargo within cells. How exactly a single motor proceeds on the 13 narrow lanes or protofilaments of a microtubule has not been visualized directly, and there persists controversy on the relative position of the two kinesin heads in different nucleotide states. We have succeeded in imaging Kinesin-1 dimers immobilized on microtubules with single-head resolution by atomic force microscopy. Moreover, we could catch glimpses of single Kinesin-1 dimers in their motion along microtubules with nanometer resolution. We find in our experiments that frequently both heads of one dimer are microtubule-bound at submicromolar ATP concentrations. Furthermore, we could unambiguously resolve that both heads bind to the same protofilament, instead of straddling two, and remain on this track during processive movement.     

Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release

Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.     

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

Conventional kinesin and Eg5 are essential nanoscale motor proteins. Single-molecule and presteady-state kinetic experiments indicate that both motors use similar strategies to generate movement along microtubules, despite having distinctly different in vivo functions. Single molecules of kinesin, a long-distance cargo transporter, are highly processive, binding the microtubule and taking 100 or more sequential steps at velocities of up to 700 nm/s before dissociating, whereas Eg5, a motor active in mitotic spindle assembly, is also processive, but takes fewer steps at a slower rate. By dissecting the structural, biochemical and mechanical features of these proteins, we hope to learn how kinesin and Eg5 are optimized for their specific biological tasks, while gaining insight into how biochemical energy is converted into mechanical work.     

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin.ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd).ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd.ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.     

A model of myosin V processivity

Cytoplasmic transport is mediated by a group of molecular motors that typically work in isolation, under conditions where they must move their cargos long distances without dissociating from their tracks. This processive behavior requires specific adaptations of motor enzymology to meet these unique physiologic demands. One of these involves the ability of the two heads of a processive motor to communicate their structural states to each other. In this study, we examine a processive motor from the myosin superfamily myosin V. We have measured the kinetics of nucleotide release, of phosphate release, and of the weak-to-strong transition, as this motor interacts with actin, and we have used these studies to develop a model of how myosin V functions as a transport motor. Surprisingly, both heads release phosphate rapidly upon the initial encounter with an actin filament, suggesting that there is little or no intramolecular strain associated with this step. However, ADP release can be affected by both forward and rearward strain, and under steady-state conditions it is essentially prevented in the lead head until the rear head detaches. Many of these features are remarkably like those underlying the processive movement of kinesin on microtubules, supporting our hypothesis that different molecular motors satisfy the requirement for processive movement in similar ways, regardless of their particular family of origin.     

Two distinct modes of processive kinesin movement in mixtures of ATP and AMP-PNP

An enzyme is frequently conceived of as having a single functional mechanism. This is particularly true for motor enzymes, where the necessity for tight coupling of mechanical and chemical cycles imposes rigid constraints on the reaction pathway. In mixtures of substrate (ATP) and an inhibitor (adenosine 5'-(beta,gamma-imido)triphosphate or AMP-PNP), single kinesin molecules move on microtubules in two distinct types of multiple-turnover "runs" that differ in their susceptibility to inhibition. Longer (less susceptible) runs are consistent with movement driven by the alternating-sites mechanism previously proposed for uninhibited kinesin. In contrast, kinesin molecules in shorter runs step with AMP-PNP continuously bound to one of the two active sites of the enzyme. Thus, in this mixture of substrate and inhibitor, kinesin can function as a motor enzyme using either of two distinct mechanisms. In one of these, the enzyme can accomplish high-duty-ratio processive movement without alternating-sites ATP hydrolysis.     

Configuration of the two kinesin motor domains during ATP hydrolysis

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.     

ESR reveals the mobility of the neck linker in dimeric kinesin

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site

Conventional kinesin and class V and VI myosins coordinate the mechanochemical cycles of their motor domains for processive movement of cargo along microtubules or actin filaments. It is widely accepted that this coordination is achieved by allosteric communication or mechanical strain between the motor domains, which controls the nucleotide state and interaction with microtubules or actin. However, questions remain about the interplay between the strain and the nucleotide state. We present an analysis of Saccharomyces cerevisiae Kar3/Vik1, a heterodimeric C-terminal Kinesin-14 containing catalytic Kar3 and the nonmotor protein Vik1. The X-ray crystal structure of Vik1 exhibits a similar fold to the kinesin and myosin catalytic head, but lacks an ATP binding site. Vik1 binds more tightly to microtubules than Kar3 and facilitates cooperative microtubule decoration by Kar3/Vik1 heterodimers, and yet allows motility. These results demand communication between Vik1 and Kar3 via a mechanism that coordinates their interactions with microtubules.     

Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head-Nkin neck chimera with conventional kinesin

Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.     

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.