The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor''s ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.     

Stepping and stretching. How kinesin uses internal strain to walk processively

The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.     

A seesaw model for intermolecular gating in the kinesin motor protein

Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1?microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.     

A synthetic ancestral kinesin-13 depolymerizes microtubules faster than any natural depolymerizing kinesin

The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a ‘super depolymerizer’ and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells.     

JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.     

Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic hela cells

We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at approximately 30 microm/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at approximately 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of approximately 5 microm/min, and microtubules glide for an average of 3 microm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.     

The mechanical properties of kinesin-1: a holistic approach

During the last 25?years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is presented that uses this information to simulate the motion of a bead carried by a kinesin molecule along a microtubule, with and without a weak optical trap present. A video sequence made from individual steps of the simulation gives a three-dimensional visual insight into these types of experiment at the molecular level.     

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.     

Mechanism of processive movement of monomeric and dimeric kinesin molecules

Kinesin molecules are motor proteins capable of moving along microtubule by hydrolyzing ATP. They generally have several forms of construct. This review focuses on two of the most studied forms: monomers such as KIF1A (kinesin-3 family) and dimers such as conventional kinesin (kinesin-1 family), both of which can move processively towards the microtubule plus end. There now exist numerous models that try to explain how the kinesin molecules convert the chemical energy of ATP hydrolysis into the mechanical energy to "power" their processive movement along microtubule. Here, we attempt to present a comprehensive review of these models. We further propose a new hybrid model for the dimeric kinesin by combining the existing models and provide a framework for future studies in this subject.     

Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd

Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.     

An InCytes from MBC Selection: The Aspergillus nidulans Kinesin-3 UncA Motor Moves Vesicles along a Subpopulation of Microtubules

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncA^rigor decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncA^rigor but reacted with a specific antibody against tyrosinated α-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules.     

Kinesin-1 plays multiple roles during the vaccinia virus life cycle

The cytoplasmic distribution of cellular structures is known to depend on the balance between plus- and minus-end-directed motor complexes. Among the plus-end-directed kinesins, kinesin-1 and -2 have been implicated in the outward movement of many organelles. To test for a role of kinesin-1 previous studies mostly relied on the overexpression of dominant-negative kinesin-1 constructs. The latter are often cytotoxic, modify the microtubule network and indirect effects related to altered microtubule dynamics should be excluded. In the present study we present a novel kinesin-1 construct, encompassing the first 330 amino acids of kinesin heavy chain fused to GFP (kin330-GFP) that does not alter microtubules upon its overexpression. Kin330-GFP functionally inhibits kinesin-1 because it induces the peri-nuclear accumulation of mitochondria and intermediate filaments. Using this construct and previously established siRNA-mediated knock-down of kinesin-2 function, we assess the role of both motors in the subcellular distribution of distinct steps of the vaccinia virus (VV) life cycle. We show that kinesin-1, but not kinesin-2, contributes to the specific cytoplasmic distribution of three of the four steps of VV morphogenesis tested. These results are discussed with respect to the possible regulation of kinesin-1 during VV infection.     

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin’s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.     

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.     

Backstepping Mechanism of Kinesin-1

Kinesin-1 is an ATP-driven molecular motor that transports cellular cargo along microtubules. At low loads, kinesin-1 almost always steps forward, toward microtubule plus ends, but at higher loads, it can also step backward. Backsteps are usually 8 nm but can be larger. These larger backward events of 16 nm, 24 nm, or more are thought to be slips rather than steps because they are too fast to consist of multiple, tightly coupled 8-nm steps. Here, we propose that not only these larger backsteps, but all kinesin-1 backsteps, are slips. We show first that kinesin waits before forward steps for less time than before backsteps and detachments; second, we show that kinesin waits for the same amount of time before backsteps and detachments; and third, we show that by varying the microtubule type, we can change the ratio of backsteps to detachments without affecting forward stepping. Our findings indicate that backsteps and detachments originate from the same state and that this state arises later in the mechanochemical cycle than the state that gives rise to forward steps. To explain our data, we propose that, in each cycle of ATP turnover, forward kinesin steps can only occur before Pi release, whereas backslips and detachments can only occur after Pi release. In the scheme we propose, Pi release gates access to a weak binding K⋅ADP-K⋅ADP state that can slip back along the microtubule, re-engage, release ADP, and try again to take an ATP-driven forward step. We predict that this rescued detachment pathway is key to maintaining kinesin processivity under load.     

A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.     

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.     

Kin I kinesins: insights into the mechanism of depolymerization

Kin I kinesins are members of the diverse kinesin superfamily of molecular motors. Whereas most kinesins use ATP to move along microtubules, Kin I kinesins depolymerize microtubules rather than walk along them. Functionally, this distinct subfamily of kinesins is important in regulating cellular microtubule dynamics and plays a crucial role in spindle assembly and chromosome segregation. The molecular mechanism of Kin I-induced microtubule destabilization is as yet unclear. It is generally believed that Kin Is induce a structural change on the microtubule that leads to microtubule destabilization. Recently, much progress has been made towards understanding how Kin Is may cause this structural change, and how ATPase activity is employed in the catalytic cycle.