MCAK associates with the tips of polymerizing microtubules

MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.     

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

MCAK is a Kinesin-13 that depolymerizes microtubules (MTs) and regulates MT dynamics. We used subtilisin-treated MTs (MTs lacking the C-termini of α- and β-tubulin) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization, which was not due to a reduction of end-specific binding. We also found that depolymerization of SMTs led to an increase in the stabilization of curved oligomeric tubulin products. Using alternative tubulin substrates with different geometries, we found that MCAK depolymerized parallel and anti-parallel tubulin sheets. However, MCAK did not depolymerize tubulin rings regardless of the presence or absence of the tubulin C-termini. We propose that localization of MCAK to the ends of MTs is independent of tubulin C-termini, that MCAK stabilizes a curved conformation at the end of the MT, and that efficient release of this complex is dependent on the presence of the C-termini of tubulin.αβ     

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.     

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.     

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.     

A new model for binding of kinesin 13 to curved microtubule protofilaments

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.     

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.     

A Second Tubulin Binding Site on the Kinesin-13 Motor Head Domain Is Important during Mitosis

Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.     

Microtubule Depolymerization by the Kinesin-8 Motor Kip3p: A Mathematical Model

Proteins from the kinesin-8 family promote microtubule (MT) depolymerization, a process thought to be important for the control of microtubule length in living cells. In addition to this MT shortening activity, kinesin 8s are motors that show plus-end directed motility on MTs. Here we describe a simple model that incorporates directional motion and destabilization of the MT plus-end by kinesin 8. Our model quantitatively reproduces the key features of length-versus-time traces for stabilized MTs in the presence of purified kinesin 8, including length-dependent depolymerization. Comparison of model predictions with experiments suggests that kinesin 8 depolymerizes processively, i.e., one motor can remove multiple tubulin dimers from a stabilized MT. Fluctuations in MT length as a function of time are related to depolymerization processivity. We have also determined the parameter regime in which the rate of MT depolymerization is length dependent: length-dependent depolymerization occurs only when MTs are sufficiently short; this crossover is sensitive to the bulk motor concentration.     

These motors were made for walking

Kinesins are a diverse group of adenosine triphosphate (ATP)‐dependent motor proteins that transport cargos along microtubules (MTs) and change the organization of MT networks. Shared among all kinesins is a ~40 kDa motor domain that has evolved an impressive assortment of motility and MT remodeling mechanisms as a result of subtle tweaks and edits within its sequence. Several elegant studies of different kinesin isoforms have exposed the purpose of structural changes in the motor domain as it engages and leaves the MT. However, few studies have compared the sequences and MT contacts of these kinesins systematically. Along with clever strategies to trap kinesin–tubulin complexes for X‐ray crystallography, new advancements in cryo‐electron microscopy have produced a burst of high‐resolution structures that show kinesin–MT interfaces more precisely than ever. This review considers the MT interactions of kinesin subfamilies that exhibit significant differences in speed, processivity, and MT remodeling activity. We show how their sequence variations relate to their tubulin footprint and, in turn, how this explains the molecular activities of previously characterized mutants. As more high‐resolution structures become available, this type of assessment will quicken the pace toward establishing each kinesin's design–function relationship.     

Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis

XMAP215/TOGp family members and KinI kinesins are conserved microtubule (MT)-regulatory proteins, and have been viewed as possessing prominent antagonistic stabilizing/destabilizing activities that must be balanced. Here, interdependencies between TOGp and the KinI kinesin MCAK were analyzed in human leukemia cells. A system was established that permits inducible overexpression in homogeneous cell populations that simultaneously synthesize interfering short hairpin RNAs. We present evidence that the functional interplay of TOGp and MCAK proteins is manifested as three distinct phenotypes during the cell cycle. The first involves a role for TOGp in protecting spindle MTs from MCAK activity at the centrosome, which appears essential to prevent the formation of disorganized multipolar spindles. The second phenotype involves TOGp-dependent counteraction of excessive MCAK activity during mitosis, which recapitulates the previously established plus-end specific counteractive activities in vitro. The third involves an unexpected destabilization of the interphase MTs by overexpressed TOGp, a phenotype that requires endogenous MCAK. We hypothesize that TOGp-dependent prevention of MCAK-mediated spindle disorganization, as evidenced by depletion experiments, reflects a primary physiological role for TOGp in human somatic cells.     

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 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.     

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.     

Aurora A,MCAK, and Kif18b promote Eg5-independent spindle formation

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve.     

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.     

Analysis of the weak interactions of ADP-Unc104 and ADP-kinesin with microtubules and their inhibition by MAP2c

Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed.     

TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.