Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159-1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001-1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.     

MEL-28 is downstream of the Ran cycle and is required for nuclear-envelope function and chromatin maintenance

Early embryonic development depends on the faithful execution of basic cell biological processes whose coordination remains largely unknown. With a global network analysis, we found MEL-28 to be associated with two types of complexes, one implicated in nuclear-envelope function and the other in chromatin organization. Here, we show that MEL-28, a protein that shuttles between the nucleus and the kinetochore during the cell cycle, is required for the structural and functional integrity of the nuclear envelope. In addition, mel-28(RNAi) embryos exhibit defects in chromosome condensation, pronuclear migration, kinetochore assembly, and spindle assembly. This combination of mel-28(RNAi) phenotypes resemble those caused by depleting members of the Ran cycle in C. elegans, a conserved cellular signaling pathway that is required for mitotic spindle assembly, nuclear-envelope reformation after mitosis, and nucleocytoplasmic exchange (reviewed in). Although MEL-28 localization to the nuclear periphery is not dependent on nuclear pore components, it is dependent on RAN-1 and other key components of the Ran cycle. Thus, MEL-28 is downstream of the Ran cycle and is required for both proper nuclear-envelope function and chromatin maintenance.     

The BTB protein MEL-26 promotes cytokinesis in C. elegans by a CUL-3-independent mechanism

BACKGROUND: The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS: Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS: Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.     

SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.     

Bod1, a novel kinetochore protein required for chromosome biorientation

We have combined the proteomic analysis of Xenopus laevis in vitro-assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule-kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.     

Analysis of kinetochore assembly and function in Caenorhabditis elegans embryos and human cells

All eukaryotes rely on multi-protein assemblies, called kinetochores, to direct the segregation of their chromosomes in mitosis. The list of known kinetochore components has been growing rapidly in the post-genomic era: in animal cells, there are presently more than 80 proteins that show either exclusive or partial localization at kinetochores during mitosis. The future challenge is to elucidate how these proteins contribute to kinetochore structure, spindle microtubule attachment, regulation of microtubule dynamics, and the detection, signaling, and correction of microtubule attachment errors. Cultured human tumor cells, especially HeLa cells, are widely used for the study of kinetochores. Recently, the experimental advantages offered by the nematode Caenorhabditis elegans have been exploited for functional analysis of kinetochore components in the first embryonic division. Here, we discuss basic methods, largely based on fluorescence imaging, to study kinetochore structure and function in these two metazoan model systems.     

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.     

Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains

The maternally expressed C. elegans gene spd-5 encodes a centrosomal protein with multiple coiled-coil domains. During mitosis in mutants with reduced levels of SPD-5, microtubules assemble but radiate from condensed chromosomes without forming a spindle, and mitosis fails. SPD-5 is required for the centrosomal localization of gamma-tubulin, XMAP-215, and Aurora A kinase family members, but SPD-5 accumulates at centrosomes in mutants lacking these proteins. Furthermore, SPD-5 interacts genetically with a dynein heavy chain. We propose that SPD-5, along with dynein, is required for centrosome maturation and mitotic spindle assembly.     

The Caenorhabditis elegans UNC-87 protein is essential for maintenance, but not assembly, of bodywall muscle

Mutations in the unc-87 gene of Caenorhabditis elegans cause disorganization of the myofilament lattice in adult bodywall muscle. In order to assess the organization of specific bodywall muscle components in the absence of the unc-87 gene product, we examined the bodywall muscles of mutant animals using phalloidin and monoclonal antibodies to various muscle proteins. These studies indicated that the bodywall muscle of unc-87 embryos is initially almost wild type in its organization, but at later stages, the muscle becomes severely disorganized. To address the possibility that this disorganization is due to deterioration of the muscle as a result of contraction, we introduced into the unc-87 mutant background a mutation that decreases myosin heavy chain activity but does not substantially affect muscle structure. The improved muscle structure and motility of the double mutants are consistent with the hypothesis that at least part of the disorganization phenotype of unc-87 mutants is a consequence of the wild-type levels of force generated during muscle contraction. These results imply that the role of the unc-87 gene product is not in specifying organization but rather in serving as a structural component maintaining lattice integrity during and after contraction.     

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.     

The N-terminal tail of C. elegans CENP-A interacts with KNL-2 and is essential for centromeric chromatin assembly

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A–specific chaperone/targeting factor of the Scm3/HJURP family.     

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.     

The MIS12 complex is a protein interaction hub for outer kinetochore assembly

Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of ∼22 nm. Through biochemical analysis, cross-linking–based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms.     

kin-18, a C. elegans protein kinase involved in feeding.

TAO1 and TAO2 are recently described protein kinases whose initial characterization has placed them at the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase (MEKK) level of stress-responsive MAPK pathways. Because their physiological roles have not been identified, we sought to study their C. elegans homolog to learn more about their functions. kin-18 encodes a previously uncharacterized protein in C. elegans whose catalytic domain shares over 60% identity with TAO1 and TAO2. We demonstrate that KIN-18 is a protein of 120 kDa whose promoter is active in the pharynx and intestine of C. elegans. To learn more about TAO/KIN-18 function, we studied how expression of constitutively active forms of TAO1 or KIN-18 would affect the physiology of intact worms. Strains of C. elegans expressing active forms of TAO1 or KIN-18 exhibit altered pharyngeal electrophysiology as measured by electropharyngeogram. These worms grow more slowly and lay fewer eggs, phenotypes that could result from reduced feeding. We have also identified a C. elegans gene that encodes a protein kinase similar to mammalian MAPK/ERK Kinase (MEK) 4 whose promoter is active in the pharynx. It is phosphorylated by TAO1 in vitro and physically interacts with TAO1.     

Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system.     

The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus

The centrosome and nucleus are intimately associated in most animal cells, yet the significance of this interaction is unknown. Mutations in the zyg-12 gene of Caenorhabditis elegans perturb the attachment of the centrosome to the nucleus, giving rise to aberrant spindles and ultimately, DNA segregation defects and lethality. These phenotypes indicate that the attachment is essential. ZYG-12 is a member of the Hook family of cytoskeletal linker proteins and localizes to both the nuclear envelope (via SUN-1) and centrosomes. ZYG-12 is able to bind the dynein subunit DLI-1 in a two-hybrid assay and is required for dynein localization to the nuclear envelope. Loss of dynein function causes a low percentage of defective centrosome/nuclei interactions in both Drosophila and Caenorhabditis elegans. We propose that dynein and ZYG-12 move the centrosomes toward the nucleus, followed by a ZYG-12/SUN-1-dependent anchorage.     

mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.