A non-motor microtubule binding site is essential for the high processivity and mitotic function of kinesin-8 Kif18A

Background

Members of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.

Methodology/Findings

Here, we investigate how Kif18A localizes to the plus-ends of kt-MTs. We find that Kif18A lacking its C-terminus does not accumulate on the tips of kt-MTs and fails to fulfill its mitotic function. In vitro studies reveal that Kif18A possesses a non-motor MT binding site located within its C-proximal 121 residues. Using single molecule measurements we find that Kif18A is a highly processive motor and, furthermore, that the C-terminal tail is essential for the high processivity of Kif18A.

Conclusion/Significance

These results show that Kif18A like its yeast orthologue is a highly processive motor. The ability of Kif18A to walk on MTs for a long distance without dissociating depends on a non-motor MT binding site located at the C-terminus of Kif18A. This C-proximal tail of Kif18A is essential for its plus-end accumulation and mitotic function. These findings advance our understanding of how Kif18A accumulates at the tips of kt-MTs to fulfill its function in mitosis.     

Kif18B interacts with EB1 and controls astral microtubule length during mitosis

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.     

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.     

Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in?vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.     

Localized Aurora B activity spatially controls non-kinetochore microtubules during spindle assembly

Efficient spindle assembly involves the generation of spatial cues around chromosomes that locally stabilize microtubule (MT) plus-ends. In addition to the small GTPase Ran, there is evidence that Aurora B kinase might also generate a spatial cue around chromosomes but direct proof for this is still lacking. Here, we find that the Aurora B substrate MCAK localizes to MT plus-ends throughout the mitotic spindle, but its accumulation is strongly reduced on MT plus-ends near chromatin, suggesting that a signal emanating from chromosomes negatively regulates MCAK plus-end binding. Indeed, we show that Aurora B is the kinase responsible for producing this chromosome-derived signal. These results are the first to visualize spatially restricted Aurora B kinase activity around chromosomes on an endogenous substrate and explain how Aurora B could spatially control the dynamics of non-kinetochore MTs during spindle assembly.     

Efficient mitosis in human cells lacking poleward microtubule flux

Chromosome segregation relies on the dynamic properties of spindle microtubules (MTs). Poleward MT flux contributes to spindle dynamics through the disassembly of MT minus ends at spindle poles coupled to the continuous poleward transport of spindle MTs. Despite being conserved in metazoan cells, the function of flux remains controversial because flux rates differ widely in different cell types. In meiotic systems, the rate of flux nearly matches that of chromosome movement, but in mitotic systems, flux is significantly slower than chromosome movement. Here, we show that spindles in human mitotic cells depleted of the kinesin-13 proteins Kif2a and MCAK lack detectable flux and that such cells frequently fail to segregate all chromosomes appropriately at anaphase. Elimination of flux reduces poleward chromosome velocity approximately 20%, but does not hinder bipolar spindle assembly, chromosome alignment, or mitotic progression. Thus, mitosis proceeds efficiently in human cells lacking detectable poleward MT flux. These data demonstrate that in human cultured cells, kinetochores are sufficient to effectively power chromosome movement, leading us to speculate that flux is maintained in these cells to fulfill other functional roles such as error correction or kinetochore regulation.     

Crystal structure of the motor domain of the human kinetochore protein CENP-E

The human kinetochore is a highly complex macromolecular structure that connects chromosomes to spindle microtubules (MTs) in order to facilitate accurate chromosome segregation. Centromere-associated protein E (CENP-E), a member of the kinesin superfamily, is an essential component of the kinetochore, since it is required to stabilize the attachment of chromosomes to spindle MTs, to develop tension across aligned chromosomes, to stabilize spindle poles and to satisfy the mitotic checkpoint. Here we report the 2.5A resolution crystal structure of the motor domain and linker region of human CENP-E with MgADP bound in the active site. This structure displays subtle but important differences compared to the structures of human Eg5 and conventional kinesin. Our structure reveals that the CENP-E linker region is in a "docked" position identical to that in the human plus-end directed conventional kinesin. CENP-E has many advantages as a potential anti-mitotic drug target and this crystal structure of human CENP-E will provide a starting point for high throughput virtual screening of potential inhibitors.     

Kinesin-5 inhibitor resistance is driven by kinesin-12

The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10⁷ cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process.     

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.     

The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression

BACKGROUND: The accurate alignment of chromosomes at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic integrity of eukaryotes. Although it is well established that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well understood. RESULTS: By combining RNAi-depletion experiments with in vitro biochemical assays, we demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells. We show that in vitro Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A like its yeast ortholog Kip3p depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies revealed that in Kif18A-depleted cells, chromosomes move at reduced speed and completely fail to align at the spindle equator. CONCLUSIONS: These studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.     

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.     

FAM29A promotes microtubule amplification via recruitment of the NEDD1–γ-tubulin complex to the mitotic spindle

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a γ-tubulin–dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1–γ-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.     

Modulated microtubule dynamics enable Hklp2/Kif15 to assemble bipolar spindles

Activity of the sliding motor Eg5 and coordinated microtubule dynamics are both essential for mitotic spindle pole separation. It is still a matter of controversy if changes in microtubule dynamics can compensate inhibition of Eg5 activity and re-enable bipolarization. Using a consistent live cell-imaging approach, we show that perturbation of microtubule dynamics can compensate inhibition of Eg5 through a spindle formation process reminiscent of meiosis: In Eg5-inhibited mammalian somatic cells, alteration of microtubule dynamics through depletion of TOGp or low doses of nocodazole induces the formation of multiple acentrosomal spindle poles which pass through an intermediate multipolar state followed by bipolarization. Pole separation depends on Hklp2/Kif15, an otherwise dispensable plus end-directed spindle motor and results in spindles with two centrosomal poles. Once bipolar, spindles do not rely on altered microtubule dynamics to maintain their bipolarity anymore and are functional in chromosome segregation. We conclude that altered microtubule dynamics enable Hklp2/Kif15 to replace Eg5 in pole separation through a mechanism involving the formation of acentrosomal poles. Our observations suggest that combination chemotherapy regimens involving microtubule-targeting drugs and Eg5 inhibitors might be less effective than expected.     

Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail.     

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.     

TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.     

MCAK facilitates chromosome movement by promoting kinetochore microtubule turnover

Mitotic centromere-associated kinesin (MCAK)/Kif2C is the most potent microtubule (MT)-destabilizing enzyme identified thus far. However, MCAK's function at the centromere has remained mechanistically elusive because of interference from cytoplasmic MCAK's global regulation of MT dynamics. In this study, we present MCAK chimeras and mutants designed to target centromere-associated MCAK for mechanistic analysis. Live imaging reveals that depletion of centromere-associated MCAK considerably decreases the directional coordination between sister kinetochores. Sister centromere directional antagonism results in decreased movement speed and increased tension. Sister centromeres appear unable to detach from kinetochore MTs efficiently in response to directional switching cues during oscillatory movement. These effects are reversed by anchoring ectopic MCAK to the centromere. We propose that MCAK increases the turnover of kinetochore MTs at all centromeres to coordinate directional switching between sister centromeres and facilitate smooth translocation. This may contribute to error correction during chromosome segregation either directly via slow MT turnover or indirectly by mechanical release of MTs during facilitated movement.     

A unique kinesin-8 surface loop provides specificity for chromosome alignment

Microtubule length control is essential for the assembly and function of the mitotic spindle. Kinesin-like motor proteins that directly attenuate microtubule dynamics make key contributions to this control, but the specificity of these motors for different subpopulations of spindle microtubules is not understood. Kif18A (kinesin-8) localizes to the plus ends of the relatively slowly growing kinetochore fibers (K-fibers) and attenuates their dynamics, whereas Kif4A (kinesin-4) localizes to mitotic chromatin and suppresses the growth of highly dynamic, nonkinetochore microtubules. Although Kif18A and Kif4A similarly suppress microtubule growth in vitro, it remains unclear whether microtubule-attenuating motors control the lengths of K-fibers and nonkinetochore microtubules through a common mechanism. To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A. Each of these chimeric kinesins localizes to K-fibers; however, K-fiber length control requires an activity specific to kinesin-8s. Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain. These data support a model in which microtubule-attenuating kinesins are molecularly “tuned” to control the dynamics of specific subsets of spindle microtubules.