Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity.

A late step in cytokinesis requires the central spindle, which forms during anaphase by the bundling of antiparallel nonkinetochore microtubules. Microtubule bundling and completion of cytokinesis require ZEN-4/CeMKLP-1, a kinesin-like protein, and CYK-4, which contains a RhoGAP domain. We show that CYK-4 and ZEN-4 exist in a complex in vivo that can be reconstituted in vitro. The N terminus of CYK-4 binds the central region of ZEN-4, including the neck linker. Genetic suppression data prove the functional significance of this interaction. An analogous complex, containing equimolar amounts of a CYK-4 ortholog and MKLP-1, was purified from mammalian cells. Biochemical studies indicate that this complex, named centralspindlin, is a heterotetramer. Centralspindlin, but not its individual components, strongly promotes microtubule bundling in vitro.     

Cooperative assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to form the centralspindlin complex

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.     

Structural analysis of the ZEN-4/CeMKLP1 motor domain and its interaction with microtubules

The centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. It is composed of two copies each of the kinesin-like protein ZEN-4, a Caenorhabditis elegans MKLP-1 (Kinesin-6 family), and the RhoGAP CYK-4. By using cryo-electron microscopy and helical 3D reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of ZEN-4 and microtubules. We have calculated helically averaged 3D maps of microtubules decorated with ZEN-4 motor domain in the presence of AMP-PNP, ADP, ADP-AlF(4)(-), and nucleotide-free conditions. We used statistical difference mapping to compare these maps among each other and to related maps obtained from microtubules decorated with a well-characterized Kinesin-1 motor domain from Neurospora crassa. Thereby, we found distinct structural features in microtubule-ZEN-4 complexes that may directly relate to the functional properties of ZEN-4 and centralspindlin. Furthermore, we investigated the location, structure, and function of a highly conserved extension of approximately 50 residues unique to the Kinesin-6 subfamily, located in the motor core loop6/beta4 region.     

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.     

An ECT2-centralspindlin complex regulates the localization and function of RhoA

In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the central spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the central spindle by binding to centralspindlin. Depletion of the centralspindlin component MKLP1 prevents central spindle localization of ECT2; however, RhoA, F-actin, and myosin still accumulate on the equatorial cell cortex. Depletion of the other centralspindlin component, CYK-4/MgcRacGAP, prevents cortical accumulation of RhoA, F-actin, and myosin. CYK-4 and ECT2 interact, and this interaction is cell cycle regulated via ECT2 phosphorylation. Thus, central spindle localization of ECT2 assists division plane positioning and the CYK-4 subunit of centralspindlin acts upstream of RhoA to promote furrow assembly.     

CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.     

ZEN-4/MKLP1 is required to polarize the foregut epithelium

BACKGROUND: Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. Here, we describe a novel function during tubulogenesis for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. RESULTS: Our analyses demonstrate that ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. CONCLUSION: We suggest that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ.     

The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly

The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora B(AIR-2) and the centralspindlin component MKLP1(ZEN-4) causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora B(AIR-2)-inhibited embryos, whereas inhibition of Rac does so in MKLP1(ZEN-4)-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.     

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 aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis

BACKGROUND: The Aurora/Ipl1p-related kinase AIR-2 is required for mitotic chromosome segregation and cytokinesis in early Caenorhabditis elegans embryos. Previous studies have relied on non-conditional mutations or RNA-mediated interference (RNAi) to inactivate AIR-2. It has therefore not been possible to determine whether AIR-2 functions directly in cytokinesis or if the cleavage defect results indirectly from the failure to segregate DNA. One intriguing hypothesis is that AIR-2 acts to localize the mitotic kinesin-like protein ZEN-4 (also known as CeMKLP1), which later functions in cytokinesis. RESULTS: Using conditional alleles, we established that AIR-2 is required at metaphase or early anaphase for normal segregation of chromosomes, localization of ZEN-4, and cytokinesis. ZEN-4 is first required late in cytokinesis, and also functions to maintain cell separation through much of the subsequent interphase. DNA segregation defects alone were not sufficient to disrupt cytokinesis in other mutants, suggesting that AIR-2 acts specifically during cytokinesis through ZEN-4. AIR-2 and ZEN-4 shared similar genetic interactions with the formin homology (FH) protein CYK-1, suggesting that AIR-2 and ZEN-4 function in a single pathway, in parallel to a contractile ring pathway that includes CYK-1. Using in vitro co-immunoprecipitation experiments, we found that AIR-2 and ZEN-4 interact directly. CONCLUSIONS: AIR-2 has two functions during mitosis: one in chromosome segregation, and a second, independent function in cytokinesis through ZEN-4. AIR-2 and ZEN-4 may act in parallel to a second pathway that includes CYK-1.     

Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy

Crystal structures of the molecular motor kinesin show conformational variability in a structural element called the neck linker. Conformational change in the neck linker, initiated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and 'docked'). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker conformation, which we confirmed by solving a new crystal structure. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility.     

Role of the kinesin neck linker and catalytic core in microtubule-based motility

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].     

CYK4 Promotes Antiparallel Microtubule Bundling by Optimizing MKLP1 Neck Conformation

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.     

Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone

In mammalian cultured cells the initiation of cytokinesis is regulated – both temporally and spatially – by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, polo-like kinase 1 (Plk-1), the centralspindlin complex, and the chromosome passenger complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis.     

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor''s properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.     

Dissecting the kinematics of the kinesin step

Kinesin walks processively on microtubules in an asymmetric hand-over-hand manner with each step spanning 16 nm. We used molecular simulations to determine the fraction of a single step due to conformational changes in the neck linker, and that due to diffusion of the tethered head. Stepping is determined largely by two energy scales, one favoring neck-linker docking and the other, ε(h)(MT-TH), between the trailing head (TH) and the microtubule. Neck-linker docking and an optimal value of ε(h)(MT-TH) are needed to minimize the probability that the TH takes side steps. There are three major stages in the kinematics of a step. In the first, the neck linker docks, resulting in ～(5-6) nm movements of the trailing head. The TH moves an additional (6-8) nm in stage II by anisotropic translational diffusion. In the third stage, spanning ～(3-4) nm, the step is complete with the TH binding to the αβ-tubulin binding site.     

Cytokinesis and Midzone Microtubule Organization in Caenorhabditis elegans Require the Kinesin-like Protein ZEN-4

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.     

The Caenorhabditis elegans Aurora B kinase AIR-2 phosphorylates and is required for the localization of a BimC kinesin to meiotic and mitotic spindles

BimC kinesins are required for mitotic spindle assembly in a variety of organisms. These proteins are localized to centrosomes, spindle microtubules, and the spindle midzone. We have previously shown that the Caenorhabditis elegans Aurora B kinase AIR-2 is required for the localization of the ZEN-4 kinesin protein to midzone microtubules. To determine whether the association of BimC kinesins with spindle microtubules is also dependent on AIR-2, we examined the expression pattern of BMK-1, a C. elegans BimC kinesin, in wild-type and AIR-2-deficient embryos. BMK-1 is highly expressed in the hermaphrodite gonad and is localized to meiotic spindle microtubules in the newly fertilized embryo. In mitotic embryos, BMK-1 is associated with spindle microtubules from prophase through anaphase and is concentrated at the spindle midzone during anaphase and telophase. In the absence of AIR-2, BMK-1 localization to meiotic and mitotic spindles is greatly reduced. This is not a consequence of loss of ZEN-4 localization because BMK-1 is appropriately localized in ZEN-4-deficient embryos. Furthermore, AIR-2 and BMK-1 directly interact with one another and the C-terminal tail domain of BMK-1 is specifically phosphorylated by AIR-2 in vitro. Together with our previous data, these results suggest that at least one function of the Aurora B kinases is to recruit spindle-associated motor proteins to their sites of action.     

ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.     

Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.