Karen Oegema et al. (2006) WormBook "Cell division."

Anthony A. Hyman, Karen Oegema

[WormBook, 2006]

The C. elegans embryo is a powerful model system for studying the mechanics of metazoan cell division. Its primary advantage is that the architecture of the syncytial gonad makes it possible to use RNAi to generate oocytes whose cytoplasm is reproducibly (typically > 95%) depleted of targeted essential gene products via a process that does not depend exclusively on intrinsic protein turnover. The depleted oocytes can then be analyzed as they attempt their first mitotic division following fertilization. Here we outline the characteristics that contribute to the usefulness of the C. elegans embryo for cell division studies. We provide a timeline for the first embryonic mitosis and highlight some of its key features. We also summarize some of the recent discoveries made using this system, particularly in the areas of nuclear envelope assembly/ dissassembly, centrosome dynamics, formation of the mitotic spindle, kinetochore assembly, chromosome segregation, and cytokinesis.

Karen Oegema et al. (2003) International Worm Meeting "KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans"

Karen Oegema, Anna Shevchenko, Sonja Rybina, Anthony Hyman, Thomas Muller-Reichert, Arshad Desai

[International Worm Meeting, 2003]

Kinetochores assemble on mitotic chromosomes to connect them to spindle microtubules and mediate their segregation. Here we use RNA interference-based genomics in C. elegans to identify a novel protein, KNL-1, that when depleted results in a kinetochore null phenotype characterized by total failure of chromosome segregation and the inability to form a stable mitotic spindle. To date, depletion of only two other kinetochore components, HCP-3 and HCP-4, thought to form the interface of the kinetochore with the underlying DNA, give this phenotype. Pair-wise RNAi-depletion and immunofluorescence experiments place KNL-1 downstream of HCP-3 and HCP-4 in kinetochore assembly. Consistent with this, KNL-1 is not required for chromosome condensation, kinetochore specification, or sister kinetochore resolution. Instead, KNL-1 directs assembly of the outer domains of the kinetochore that interact with spindle microtubules. KNL-1 is required to target five conserved outer kinetochore proteins. In extracts, KNL-1 is present in a complex with HIM-10 and the C. elegans homolog Ndc80/HEC, two widely conserved eukaryotic kinetochore proteins. Depletion of either CeNDC-80, HIM-10 or both results in less severe chromosome segregation defects, consistent with a more important role for KNL-1 in assembling the microtubule-binding interface of the kinetochore.

Karen Oegema et al. (2001) International C. elegans Meeting "Analysis of chromosome segregation and kinetochore assembly in the one-cell stage C. elegans, embryo"

Arshad Desai, Sonja Rybina, Anthony Hyman, Karen Oegema, Matthew Kirkham

[International C. elegans Meeting, 2001]

We have developed live and fixed assays to analyze chromosome movement and kinetochore function in one-cell stage C. elegans embryos. Unlike most eukaryotes, C. elegans has holocentric chromosomes, where the kinetochore is assembled along the length of the chromosome. Despite this dramatic structural difference, the C.elegans genome contains many genes whose homologues are implicated in the formation and function of localized kinetochores of other eukaryotes. Using strains expressing GFP-histone or GFP-tubulin we have examined chromosome segregation and spindle function in worms depleted of proteins involved in chromosome segregation by RNAi. Depletion of the C. elegans homologs of either CENP-A, (HCP-3) or CENP-C (HCP-4) results in an identical 'kinetochore-null' phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. Parallel analysis of embryos depleted of the C. elegans homolog of the chromosomal passenger protein INCENP (ICP-1) revealed mitotic chromosome segregation defects different from those observed in the absence of HCP-3 or HCP-4. Defects are observed before and during anaphase but the chromatin separates into two equivalently sized masses. Kinetochore components are recruited normally and mechanically stable spindles assemble that show defects later in anaphase and telophase. These detailed cytological phenotypes are also serving as high resolution 'fingerprints' for classifying new genes important for chromosome segregation that are being identified in comprehensive functional genomics screens. So far, we have one novel gene that has a 'kinetochore-null' phenotype from a RNAi based screen of the genes on chromosome III. We have also initiated an analysis of kinetochore assembly in this system. Using antibodies to kinetochore components and RNAi, we have developed a preliminary map of the dependency relationships during kinetochore assembly.

Zanin, Esther et al. (2011) International Worm Meeting "The RGA-3/4 RhoGAP and signalling by the mitotic asters restrict RhoA activity to cell equator during cytokinesis."

Zanin, Esther, Oegema, Karen

[International Worm Meeting, 2011]

Cytokinesis is accomplished by constriction of a cortical contractile ring. To ensure that each cell receives a single genomic complement, contractile ring assembly is directed by signals from the anaphase spindle. The spindle is thought to direct ring assembly by patterning activation of the small GTPase RhoA to generate a narrow equatorial zone of activated RhoA, which in turn directs assembly of the contractile ring. During cytokinesis, the RhoGEF ECT-2 activates RhoA. Active RhoA is anchored in the plasma membrane with a lipid moiety and freely diffuses within the membrane. Theoretical studies have suggested that in addition to localized activation, generation of a narrow equatorial zone of active RhoA would also require rapid constitutive inactivation of RhoA. Rapid RhoA flux would prevent active RhoA from distributing over the membrane by diffusion (Bement et al., BioEssays 28:983-993, 2006). The importance of RhoA flux and the identity of the critical inactivating RhoGAP are important current questions. In C. elegans two highly similar RhoGAPs, RGA-3 and RGA-4, have been shown to act preferentially on RhoA to control cortical contractility during polarization of the embryo (Schonegg et al., PNAS 104:14976-14981, 2007; Schmutz et al., Development 134: 3495-3505, 2007). Here, we test whether RGA-3/4 also promote Rho flux during cytokinesis to constrain RhoA activation. rga-3/4 mutant embryos have a hypercontractile cortex and 25% of rga-3/4 embryos form no cleavage furrow. Since hypercontractility complicates analysis, we analyzed the contribution of RGA-3/4 to the patterning of the contractile ring protein anillin in embryos in which we suppressed contractility by inhibiting none-muscle myosin II (nmy-2). In both wild type and nmy-2(RNAi) embryos, anillin is cleared from the poles of the cell and localizes in a ~10mm wide equatorial zone in anaphase. In nmy-2(RNAi);rga-3/4 embryos anillin is cleared from the polar regions, but localizes to a zone that is two times broader than in controls. Our results suggest that the RGA-3/4 RhoGAP opposes the ECT-2 RhoGEF to promote RhoA flux, which is critical to confine the equatorial zone of active RhoA. However, our data also indicate that the centrosomal asters inhibit the accumulation of cortical contractility at the poles via a mechanism independent of RGA-3/4. Thus, the RGA-3/4 RhoGAP and the centrosomal asters make independent contributions to control the spatial activation of RhoA during cytokinesis.

Worby, Carolyn et al. (2015) International Worm Meeting "Fam20C function in C. elegans."

Desai, Arshad, Dixon, Jack, Oegema, Karen, Worby, Carolyn, Gerson-Gurwitz, Adena

[International Worm Meeting, 2015]

A new family of atypical secreted kinases in human was recently identified, an archetypal member of which is Fam20C. Fam20C phosphorylates S-x-E motifs in caseins and several secreted proteins implicated in bone mineralization. In humans, mutations in Fam20C cause an osteosclerotic bone dysplasia known as Raine syndrome. In addition to biomineralization, phosphorylation by FAM20C is likely to be important for other functions, since many secreted hormones, proteases and other factors have been shown to be phosphorylated on S-x-E motifs.Intriguingly, the C. elegans genome encodes a FAM20C ortholog, which contains a signal-peptide for secretion and phosphorylates proteins within S-x-E motifs in vitro. Since mineralization does not occur in C. elegans, we are hoping to identify new roles for FAM20C in this system.In collaboration with the Oegema and Desai laboratories at UCSD, we are addressing the significance of FAM20C kinase activity in C. elegans. Preliminary work reveals that FAM20C is expressed and localized in the spermatheca and that its deletion leads to germline phenotypes and ~40% embryonic lethality. We are currently investigating potential substrates of FAM20C in C. elegans to determine the molecular basis for the observed phenotypes.

Lewellyn, Lindsay et al. (2009) International Worm Meeting "Aster separation is essential to couple furrow formation to anaphase onset and limit furrowing to a single site."

Lewellyn, Lindsay, Oegema, Karen, Desai, Arshad

[International Worm Meeting, 2009]

Cytokinesis is the final step of mitosis that physically divides a single cell into two daughter cells following chromosome segregation. Signaling from the centrosomal asters and the spindle midzone are coordinately required to promote furrow formation, but the relative contributions of these two signals is not known. Here, we explore the importance of aster separation in promoting furrow formation and ingression, as well as the relationship between aster and midzone-based signaling. In order to test the role of aster separation in cytokinesis, we use depletion of TPXL-1, which shortens the metaphase spindle, thereby delaying aster separation following anaphase onset. This effect that can be reversed by co-inhibition of kinetochore assembly. Using this controlled system, we show that delaying aster separation leads to a similar delay in furrow formation without affecting the subsequent rate of furrow ingression. In contrast to control embryos, when aster separation is delayed, two midzone based signaling complexes - Centralspindlin and the Chromosomal Passenger Complex - are essential for furrow formation. Remarkably, preventing aster separation, by simultaneously inhibiting TPXL-1 and cortical pulling forces on the asters, leads to the coincident formation of multiple furrows. Thus, aster separation is essential to: (1) couple furrow formation to anaphase onset and, (2) limit furrow formation to a unique site.

Moyle, Mark et al. (2013) International Worm Meeting "Mechanistic Insights Into The Recruitment of the Spindle Checkpoint Protein MDF-1 To Unattached Kinetochores."

Oegema, Karen, Desai, Arshad, Moyle, Mark

[International Worm Meeting, 2013]

Correct alignment and segregation of chromosomes during cell division is essential for proper development and growth. When chromosomes are improperly attached an intracellular signaling cascade, termed the spindle checkpoint, generates a "wait anaphase" signal which delays cell cycle progression until all chromosomes are properly aligned. This signal originates from unattached kinetochores, the macromolecular spindle microtubule-binding machines built on the centromeric regions of chromosomes. Central to the "wait anaphase" signal is the protein MDF-1 (Mad1 in human), but it is still unclear how MDF-1 is recruited to and activated at unattached kinetochores. To address this question, we performed a yeast two-hybrid screen with MDF-1 and a library of kinetochore constituents. We identified an interaction between MDF-1 and BUB-1, a conserved kinase implicated in spindle checkpoint signaling. We then performed a mutagenic yeast two-hybrid screen, which identified alleles of MDF-1 that no longer interact with BUB-1. Prior work had shown that BUB-1 is required for the kinetochore localization of MDF-1. Employing the MosSCI system, we generated MDF-1 mutants that do not interact with BUB-1 and found that the mutant forms of MDF-1 are compromised in their localization to unattached kinetochores. Consistent with this, the MDF-1 mutants that do not interact with BUB-1 were defective in generating a spindle checkpoint signal. As BUB-1 is a kinase, we generated kinase-dead mutants of BUB-1 and found that these mutants no longer interact with MDF-1 in the two-hybrid assay, suggesting that the kinase activity of BUB-1 is required for this interaction. Currently, we are investigating BUB-1 kinase-dead mutants in vivo. Finally, we performed a compensatory mutagenic yeast two-hybrid screen and found a mutation in BUB-1 that restores interaction with a MDF-1 mutant, strongly suggesting that MDF-1 and BUB-1 directly interact. Overall, our findings elucidate the recruitment of MDF-1 to unattached kinetochores via the kinase BUB-1, and show that the specific interaction underlying this recruitment is required for generation of the "wait anaphase" checkpoint signal.

Wang, Shaohe et al. (2013) International Worm Meeting "NOCA-1 isoforms regulate non-centrosomal microtubule array formation in different C. elegans tissues."

Desai, Arshad, Oegema, Karen, Wang, Shaohe

[International Worm Meeting, 2013]

In contrast to the radial microtubule arrays organized by centrosomes in dividing cells, many differentiated cells assemble non-centrosomal microtubule arrays adapted for specific cellular functions. In a genome-wide RNAi screen, we identified NOCA-1, a novel protein essential for organizing non-centrosomal microtubule arrays in C.elegans germline and epidermis. NOCA-1 has six described isoforms and we identified a seventh that is necessary and sufficient for its germline function. A combination of live imaging and immunofluorescence revealed that NOCA-1 is found in multiple tissues, including the germline, epidermis, intestine, muscles and neurons. In the germline, NOCA-1 inhibition results in a catastrophic phenotype similar to inhibition of the microtubule nucleating protein g-tubulin. NOCA-1 and g-tubulin both target to the plasma membrane in the germline, but independently of each other. Due to its essential germline function, embryos homozygous for a noca-1 deletion grow up to become sterile adults. We also find that deletion of noca-1 is synthetically larval lethal with a deletion of the gene encoding the C. elegans homolog of the microtubule minus end-binding protein Patronin (PTRN-1). We used MosDEL to generate a null allele of ptrn-1 that deletes the majority of its coding sequence including the start codon, and found the deletion worms are superficially wildtype. Compared to wildtype control or to worms harboring the single deletions, noca-1D;ptrn-1D worms grow much slower post-embryonically and most die during the first three days after L1. The synthetic interaction of NOCA-1 and PTRN-1 likely occurs in the epidermis of larval and adult worms, since the synthetic lethal phenotype can be rescued by a NOCA-1 isoform that is mainly expressed in the epidermis. Our results suggest that different isoforms of NOCA-1 function together with microtubule minus-end associated proteins to generate non-centrosomal microtubule arrays that are essential to support the architecture of the syncytial germline and to maintain the barrier function of the epidermis.

Olson, Sara et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Assembly of the protective eggshell barrier in C. elegans"

Muller-Reichert, Thomas, Oegema, Karen, Olson, Sara

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Metazoan oocytes have an extracellular coating that governs fertilization. Following fertilization, this covering is altered to prevent polyspermy and protect the developing embryo. In C. elegans, a vitelline layer covers oocytes prior to fertilization. Fertilization initiates conversion of the vitelline layer into a trilaminar eggshell consisting of an outer vitelline layer, a middle chitin-containing layer, and an inner layer proposed to serve as a permeability barrier. Here, we characterize CPG-1 and CPG-2, functionally redundant chondroitin proteoglycans that are the first described protein eggshell components. We show that CPG-1 and CPG-2 are delivered to the extracellular space after formation of the chitin layer by cortical granule exocytosis during meiosis I. Although they contain multiple chitin binding domains, CPG-1 and CPG-2 localize to the inner eggshell layer, whereas chitin is confined to the middle eggshell layer. We show that the inner eggshell layer is not the permeability barrier for small molecular weight solutes. Instead, this function resides in a previously undescribed layer that resides between the eggshell and the plasma membrane. Disruption of the permeability barrier leads to solute permeability and osmotic stress. Disruption of the inner CPG-1/2 eggshell layer causes these phenotypes, as well as adhesion of the embryonic plasma membrane to the eggshell and cytokinesis failure. Interfering with chitin layer assembly results in the inner layer phenotypes, plus polyspermy and catastrophic eggshell rupture. We conclude that the eggshell layers and permeability barrier are laid down in a step-wise fashion from outermost to innermost, with later assembly events requiring successful completion of previous ones.

Canman, Julie C. et al. (2009) International Worm Meeting "The cloning of three conditional fast-inactivating icp-1 (INCENP) alleles."

Bowerman, Bruce, Canman, Julie C., Oegema, Karen

[International Worm Meeting, 2009]

Cell division is a dynamic process controlled by a number of proteins, among which some are multifunctional and are required at multiple steps. Loss-of-function of these master regulators, by classical techniques of reverse genetics, leads to a complex pleiotropic phenotype due to the combination of defects at these various steps. The role of these master regulators in a specific aspect of cell division is thus challenging to address. One such important master regulatory complex is the Chromosome Passenger Complex (CPC), which participates throughout meiosis, mitosis, and during cytokinesis. Indeed, disrupting function of the CPC by traditional forward or reverse genetics results in massive chromosome segregation defects as well as a failure to divide. To examine the role of the CPC during cytokinesis specifically, we have taken a forward genetics approach in C. elegans to isolate conditional alleles which phenocopy loss-of-function of the CPC showing both massive chromosome segregation and cytokinesis defects. Using this approach we found three mutant lines that are all completely impaired in chromosome segregation and cytokinesis. These mutants all failed to complement with each other indicating they are alleles of the same gene. Using traditional visible marker mapping, we mapped these mutants to the left arm of chromosome I, which contains the C. elegans INCENP homolog, ICP-1INCENP, the core scaffolding protein of the CPC. We sequenced the icp-1 locus in these mutants and found that all three mutants contain single mis-sense mutations in icp-1 resulting in single amino acid changes. All three alleles are fast-inactivating, showing a fully penetrant loss-of-function phenotype within seconds of shifting to the restrictive temperature. Thus, they will be powerful tools for studying the role of the CPC in cytokinesis independent of chromosome segregation during meiosis and mitosis.