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.     

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.     

Binding of the CYK-4 Subunit of the Centralspindlin Complex Induces a Large Scale Conformational Change in the Kinesin Subunit

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.     

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.     

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.     

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.     

RhoA activation during polarization and cytokinesis of the early Caenorhabditis elegans embryo is differentially dependent on NOP-1 and CYK-4

The GTPase RhoA is a central regulator of cellular contractility in a wide variety of biological processes. During these events, RhoA is activated by guanine nucleotide exchange factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs at the proper time and place. During cytokinesis, RhoA is activated by the RhoGEF ECT-2. In human cells, ECT-2 activity requires its association with CYK-4, which is a component of the centralspindlin complex. In contrast, in early Caenorhabditis elegans embryos, not all ECT-2–dependent functions require CYK-4. In this study, we identify a novel protein, NOP-1, that functions in parallel with CYK-4 to promote RhoA activation. We use mutations in nop-1 and cyk-4 to dissect cytokinesis and cell polarization. NOP-1 makes a significant, albeit largely redundant, contribution to cytokinesis. In contrast, NOP-1 is required for the preponderance of RhoA activation during the establishment phase of polarization.     

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.     

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.     

The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement

In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.     

The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function.     

The conserved KMN network constitutes the core microtubule-binding site of the kinetochore

The microtubule-binding interface of the kinetochore is of central importance in chromosome segregation. Although kinetochore components that stabilize, translocate on, and affect the polymerization state of microtubules have been identified, none have proven essential for kinetochore-microtubule interactions. Here, we examined the conserved KNL-1/Mis12 complex/Ndc80 complex (KMN) network, which is essential for kinetochore-microtubule interactions in vivo. We identified two distinct microtubule-binding activities within the KMN network: one associated with the Ndc80/Nuf2 subunits of the Ndc80 complex, and a second in KNL-1. Formation of the complete KMN network, which additionally requires the Mis12 complex and the Spc24/Spc25 subunits of the Ndc80 complex, synergistically enhances microtubule-binding activity. Phosphorylation by Aurora B, which corrects improper kinetochore-microtubule connections in vivo, reduces the affinity of the Ndc80 complex for microtubules in vitro. Based on these findings, we propose that the conserved KMN network constitutes the core microtubule-binding site of the kinetochore.     

A network of nuclear envelope membrane proteins linking centromeres to microtubules

In the fission yeast S. pombe, nuclei are actively positioned at the cell center by microtubules. Here, we show that cytoplasmic microtubules are mechanically coupled to the nuclear heterochromatin through proteins embedded in the nuclear envelope. This includes an integral outer nuclear membrane protein of the KASH family (Kms2) and two integral inner nuclear membrane proteins, the SUN-domain protein Sad1 and the previously uncharacterized protein Ima1. Ima1 specifically binds to heterochromatic regions and promotes the tethering of centromeric DNA to the SUN-KASH complex. In the absence of Ima1, or in cells harboring mutations in the centromeric Ndc80 complex, inefficient coupling of centromeric heterochromatin to Sad1 leads to striking defects in the ability of the nucleus to tolerate microtubule-dependent forces, leading to changes in nuclear shape, loss of spindle pole body components from the nuclear envelope, and partial dissociation of SUN-KASH complexes. This work highlights a framework for communication between cytoplasmic microtubules and chromatin.     

Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain.

We describe a detailed genetic analysis of the DNA-binding regions in the HAP2/HAP3 CCAAT-binding heteromeric complex. The DNA-binding domain of HAP2 is shown to be a 21 residue region containing three critical histidines and three critical arginines. Mutation of an arginine at position 199 to leucine alters the DNA-binding specificity of the complex to favor CCAAC over CCAAT. Residues in HAP3 that are critical for DNA-binding comprise a short, seven amino acid region. Three different mutations in the HAP2 DNA-binding domain are suppressed by a mutation in the HAP3 DNA-binding domain. This HAP3 mutation also suppresses mutations in a different region of HAP2 which promotes subunit assembly of the complex. These findings suggest that short regions of HAP2 and HAP3 comprise a hybrid DNA-binding domain and that this domain can help hold the two subunits together in the CCAAT-binding complex.