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.     

The Role of the Kinesin-13 Neck in Microtubule Depolymerization

To ensure genetic integrity, replicated chromosomes must be accurately distributed to daughter cells—a process that is accomplished on the microtubule spindle. Kinesin-13 motors play an essential role in this process by performing regulated microtubule depolymerization. We set out to dissect the depolymerization mechanism of these kinesins, and in particular, the role of their conserved neck sequence. We used a monomeric kinesin-13 MCAK, consisting of the neck and motor core, which has strong depolymerizing activity. In the presence of a non-hydrolysable ATP analogue, this construct induced formation of rings around microtubules. The rings are built from tubulin protofilaments that are bent by the kinesin-13 motor engaged at the ATP-binding step of its ATPase cycle. Our data suggest that the ring-microtubule interaction is mediated by the neck and support the idea of a role for the kinesin-13 neck in depolymerization efficiency, acting by optimising release of tubulin from microtubule ends.     

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.     

The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tension-remarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ～1?pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through a 'side-sliding, end-catching' mechanism.     

Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.     

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.     

A mechanism for microtubule depolymerization by KinI kinesins

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.     

New Insights into the Coupling between Microtubule Depolymerization and ATP Hydrolysis by Kinesin-13 Protein Kif2C

Kinesin-13 proteins depolymerize microtubules in an ATP hydrolysis-dependent manner. The coupling between these two activities remains unclear. Here, we first studied the role of the kinesin-13 subfamily-specific loop 2 and of the KVD motif at the tip of this loop. Shortening the loop, the lysine/glutamate interchange and the additional Val to Ser substitution all led to Kif2C mutants with decreased microtubule-stimulated ATPase and impaired depolymerization capability. We rationalized these results based on a structural model of the Kif2C-ATP-tubulin complex derived from the recently determined structures of kinesin-1 bound to tubulin. In this model, upon microtubule binding Kif2C undergoes a conformational change governed in part by the interaction of the KVD motif with the tubulin interdimer interface. Second, we mutated to an alanine the conserved glutamate residue of the switch 2 nucleotide binding motif. This mutation blocks motile kinesins in a post-conformational change state and inhibits ATP hydrolysis. This Kif2C mutant still depolymerized microtubules and yielded complexes of one Kif2C with two tubulin heterodimers. These results demonstrate that the structural change of Kif2C-ATP upon binding to microtubule ends is sufficient for tubulin release, whereas ATP hydrolysis is not required. Overall, our data suggest that the conformation reached by kinesin-13s upon tubulin binding is similar to that of tubulin-bound, ATP-bound, motile kinesins but that this conformation is adapted to microtubule depolymerization.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

The mechanism, function and regulation of depolymerizing kinesins during mitosis

Kinesins are motor proteins that use the hydrolysis of ATP to do mechanical work. Most of these motors translocate cargo along the surface of the microtubule (MT). However, a subfamily of these motors (Kin-I kinesins) can destabilize MTs directly from their ends. This distinct ability makes their activity crucial during mitosis, when reordering of the MT cytoskeleton is most evident. Recently, much work has been done to elucidate the structure and mechanism of depolymerizing kinesins, particularly those of the mammalian kinesin mitotic centromere-associated kinesin (MCAK). In addition, new regulatory factors have been discovered that shed light on the regulation and precise role of Kin-I kinesins during mitosis.     

Kinesin, 30 years later: Recent insights from structural studies

Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. They share a conserved motor domain (head) which harbors both the ATP‐ and microtubule‐binding activities. The kinesin that has been studied most moves toward the microtubule (+)‐end by alternately advancing its two heads along a single protofilament. This kinesin is the subject of this review. Its movement is associated to alternate conformations of a peptide, the neck linker, at the C‐terminal end of the motor domain. Recent progress in the understanding of its structural mechanism has been made possible by high‐resolution studies, by cryo electron microscopy and X‐ray crystallography, of complexes of the motor domain with its track protein, tubulin. These studies clarified the structural changes that occur as ATP binds to a nucleotide‐free microtubule‐bound kinesin, initiating each mechanical step. As ATP binds to a head, it triggers orientation changes in three rigid motor subdomains, leading the neck linker to dock onto the motor core, which directs the other head toward the microtubule (+)‐end. The relationship between neck linker docking and the orientations of the motor subdomains also accounts for kinesin's processivity, which is remarkable as this motor protein only falls off from a microtubule after taking about a hundred steps. As tools are now available to determine high‐resolution structures of motor domains complexed to their track protein, it should become possible to extend these studies to other kinesins and relate their sequence variations to their diverse properties.     

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.     

K-loop insertion restores microtubule depolymerizing activity of a "neckless" MCAK mutant

Unlike most kinesins, mitotic centromere-associated kinesin (MCAK) does not translocate along the surface of microtubules (MTs), but instead depolymerizes them. Among the motile kinesins, refinements that are unique for specific cellular functions, such as directionality and processivity, are under the control of a "neck" domain adjacent to the ATP-hydrolyzing motor domain. Despite its apparent lack of motility, MCAK also contains a neck domain. We found that deletions and alanine substitutions of highly conserved positively charged residues in the MCAK neck domain significantly reduced MT depolymerization activity. Furthermore, substitution of MCAK's neck domain with either the positively charged KIF1A K-loop or poly-lysine rescues the loss of MT-depolymerizing activity observed in the neckless MCAK mutant. We propose that the neck, analogously to the K-loop, interacts electrostatically with the tubulin COOH terminus to permit diffusional translocation of MCAK along the surface of MTs. This weak-binding interaction may also play an important role in processivity of MCAK-induced MT depolymerization.     

Arp2/3 phosphorylation kickstarts cells

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.     

ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy

Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.     

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.     

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.     

Full-length dimeric MCAK is a more efficient microtubule depolymerase than minimal domain monomeric MCAK

MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.MCAK cosedimentation assays to compare the activity of full-length MCAK, which is a dimer, with MD-MCAK, which is a monomer. Full-length MCAK exhibits higher ATPase activity, more efficient microtubule end binding, and reduced affinity for the tubulin heterodimer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Multi-talented MCAK: Microtubule depolymerizer with a strong grip

Microtubule-depolymerizing motor proteins regulate microtubule dynamics during chromosome segregation, but whether they can independently grip the ends of shrinking kinetochore microtubules has remained unresolved. MCAK, a member of the kinesin-13 motor protein family, is now shown to grip microtubules on its own and harness the forces of microtubule disassembly.