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.     

The microtubule-binding protein Cep170 promotes the targeting of the kinesin-13 depolymerase Kif2b to the mitotic spindle

Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. In humans, there are three closely related kinesin-13 isoforms (Kif2a, Kif2b, and Kif2c/MCAK), which are highly conserved in their primary sequences but display distinct localization and nonoverlapping functions. Here we demonstrate that the N-terminus is a primary determinant of kinesin-13 localization. However, we also find that differences in the C-terminus alter the properties of kinesin-13, in part by facilitating unique protein–protein interactions. We identify the spindle-localized proteins Cep170 and Cep170R (KIAA0284) as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides Kif2b with a second microtubule-binding site to target it to the spindle. Thus the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family.     

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.     

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.     

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.     

KLP10A and KLP59C: The Dynamic Duo of Microtubule Depolymerization

Kinesin-13s are important effectors of microtubule depolymerization in cells. In a recent series of studies, we examined the roles played by kinesin-13s throughout the cell cycle in Drosophila. Our findings have revealed remarkable coordination between two family members, KLP10A and KLP59C, in which alterations in the relative targeting of these proteins allows them to participate in markedly different tasks at distinct points in the cell cycle. During mitosis, KLP10A and KLP59C function in parallel by targeting to and depolymerizing the opposite ends of kinetochore-associated microtubules, thereby driving poleward chromatid motility by a Pacman-Flux mechanism. Alternatively, during interphase, both proteins target to the same end of the microtubule but act in series to divide the labor of microtubule depolymerization. KLP10A initiates depolymerization while KLP59C perpetuates depolymerization after its initiation. Below, we detail these findings and examine some of their implications.     

The Kinesin-8 Kip3 Depolymerizes Microtubules with a Collective Force-Dependent Mechanism

Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes, in particular during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors’ residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times, suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called “bump-off” and “switching” models. Understanding the mechanics of kinesin-8’s microtubule end activity will provide important insights into cell division with implications for cancer research.     

Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.     

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.     

Rho of Plant GTPase Signaling Regulates the Behavior of Arabidopsis Kinesin-13A to Establish Secondary Cell Wall Patterns

Plant cortical microtubule arrays determine the cell wall deposition pattern and proper cell shape and function. Although various microtubule-associated proteins regulate the cortical microtubule array, the mechanisms underlying marked rearrangement of cortical microtubules during xylem differentiation are not fully understood. Here, we show that local Rho of Plant (ROP) GTPase signaling targets an Arabidopsis thaliana kinesin-13 protein, Kinesin-13A, to cortical microtubules to establish distinct patterns of secondary cell wall formation in xylem cells. Kinesin-13A was preferentially localized with cortical microtubules in secondary cell wall pits, areas where cortical microtubules are depolymerized to prevent cell wall deposition. This localization of Kinesin-13A required the presence of the activated ROP GTPase, MICROTUBULE DEPLETION DOMAIN1 (MIDD1) protein, and cortical microtubules. Knockdown of Kinesin-13A resulted in the formation of smaller secondary wall pits, while overexpression of Kinesin-13A enlarged their surface area. Kinesin-13A alone could depolymerize microtubules in vitro; however, both MIDD1 and Kinesin-13A were required for the depolymerization of cortical microtubules in vivo. These results indicate that Kinesin-13A regulates the formation of secondary wall pits by promoting cortical microtubule depolymerization via the ROP-MIDD1 pathway.     

Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle

The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.     

Molecular insight into the regulation and function of MCAK

Chromosome stability is ensured by precisely fine-tuned dynamics of mitotic spindles, which are controlled by a network of various microtubule-associated and interacting proteins including the kinesin-13 family. The best characterized member of this family is the mitotic centromere-associated kinesin (MCAK). By efficiently depolymerizing microtubules, MCAK influences various key events during mitosis. MCAK itself is regulated by its interaction partners, its intrinsic conformation switch and the phosphorylation of mitotic kinases like Aurora A/B, cyclin-dependent kinase 1 and Polo-like kinase 1. Perturbing its regulation alters MCAK’s conformation, catalytic activity, subcellular localization and stability, leading further to mitotic defects in spindle formation and chromosome movement. Indeed, MCAK is aberrantly regulated in various cancer types, which is linked to increased invasiveness, metastasis and drug resistance. In the current review, we summarize recently published data concerning MCAK, correlate its conformation changes with its depolymerization activity and function, propose a model of its regulation by multiple mitotic kinases and highlight its potential involvement in oncogenesis and drug resistance.     

The Drosophila Kinesin-13, KLP59D,Impacts Pacman- and Flux-based Chromosome Movement

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole–associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D''s impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.     

Crowding of molecular motors determines microtubule depolymerization

The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed.     

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.     

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 polymerases and depolymerases

The variety of shapes and sizes of the microtubule cytoskeleton is as great as the number of different cell types. This large variety is a consequence of the dynamic properties of microtubules, which allow them to adopt distributions of arbitrary size and form. How is the distribution of microtubule lengths controlled? Recent work suggests that the length distribution is controlled, at least in part, by the activity of microtubule polymerases and depolymerases, which accelerate microtubule growth and shrinkage. Specifically, biochemical and single-molecule studies have shown how MCAK (kinesin-13) and Kip3p (kinesin-8) accelerate depolymerization and how XMAP215 may accelerate growth. Studies on the yeast Dam1 complex have shown how proteins can couple a cellular structure, the kinetochore, to the ends of polymerizing and depolymerizing microtubules.     

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.     

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.     

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.