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.     

Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8

The kinesin-8 family of microtubule motors plays?a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and its stabilizing activities. The Kip3 tail promotes Kip3's accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3 tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.     

Analysis of kinesin motor function at budding yeast kinetochores

Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome-MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.     

Mitotic motors in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.     

The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.     

Mitotic spindle form and function

The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.     

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.     

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.     

Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase

In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. the yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not been reported. We investigated Kip3''s role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.Key words: Cin8, cluster, GFP-tubulin, kinesin-5, kinesin-8, kinetochore, Kip3, metaphase, microtubule, mitosis, spindle     

Cell cycle control of kinesin-mediated transport of Bik1 (CLIP-170) regulates microtubule stability and dynein activation

CLIPs are microtubule plus end-associated proteins that mediate interactions required for cell polarity and cell division. Here we demonstrate that budding yeast Bik1, unlike its human ortholog CLIP-170, is targeted to the microtubule plus end by a kinesin-dependent transport mechanism. Bik1 forms a complex with the kinesin Kip2. Fluorescently labeled Bik1 and Kip2 comigrate along individual microtubules. Bik1 exists in distinct intracellular pools: a stable pool at the spindle pole body that is depleted during cell cycle progression, a soluble pool from which Bik1 can be recruited during microtubule initiation, and a dynamic plus end pool maintained by Kip2. Kip2 stabilizes microtubules by targeting Bik1 to the plus end and Kip2 levels are controlled during the cell cycle. As with Bik1, the targeting of dynein to the microtubule plus end requires Kip2. These findings reveal a central role for Kip2-dependent transport in the cell cycle control of microtubule dynamics and dynein-dependent motility.     

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this limit, which may explain why it has the smallest known mitotic spindle that still manifests the classic congression architecture.     

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Molecular motors translocate along cytoskeletal filaments, as in the case of kinesin motors on microtubules. Although conventional kinesin-1 tracks a single microtubule protofilament, other kinesins, akin to dyneins, switch protofilaments. However, the molecular trajectory—whether protofilament switching occurs in a directed or stochastic manner—is unclear. Here, we used high-resolution optical tweezers to track the path of single budding yeast kinesin-8, Kip3, motor proteins. Under applied sideward loads, we found that individual motors stepped sideward in both directions, with and against loads, with a broad distribution in measured step sizes. Interestingly, the force response depended on the direction. Based on a statistical analysis and simulations accounting for the geometry, we propose a diffusive sideward stepping motion of Kip3 on the microtubule lattice, asymmetrically biased by force. This finding is consistent with previous multimotor gliding assays and sheds light on the molecular switching mechanism. For kinesin-8, the diffusive switching mechanism may enable the motor to bypass obstacles and reach the microtubule end for length regulation. For other motors, such a mechanism may have implications for torque generation around the filament axis.     

Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase

In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. The yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not beenreported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.     

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.     

Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State

During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.     

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-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5⁺ and klp6⁺ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.     

Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells

We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae. The methods examine Cin8p rapidly purified from crude yeast cell extracts. We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p. This form was biotinated in yeast cells and provided Cin8p activity in vivo. Avidin-coated glass surfaces were used to specifically bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min in the presence of ATP. Force production by Cin8p was directed toward the plus ends of microtubules. A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by >10-fold, but reduced gliding velocity by only 35%. The cin8-3 mutant form, affecting the alpha2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected. cin8-F467A cells, but not cin8-3 cells, were greatly impaired in bipolar spindle forming ability. We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation.     

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 nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our?results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to?maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.