Severson, Aaron F. et al. (2013) International Worm Meeting "Interchangeable a-kleisin subunits specify meiotic cohesin function in C. elegans."

Meyer, Barbara J., Severson, Aaron F.

[International Worm Meeting, 2013]

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. To achieve this, homologous chromosomes become linked through crossover recombination, the two sister chromatids of each homolog attach to microtubules from the same spindle pole (co-orient) in meiosis I and from opposite spindle poles (bi-orient) in meiosis II, and sister chromatid cohesion (SCC) is released in two steps to allow separation of homologs before sisters. In yeast, reducing ploidy during meiosis requires that Scc1, the "kleisin" subunit of cohesin complexes that mediate mitotic SCC, is replaced by the meiosis-specific paralog Rec8. We have shown that REC-8 is not the sole meiotic kleisin in C. elegans, and we predicted that this was also true in plants and mammals. This has now been proven. In C. elegans, REC-8 and two functionally redundant kleisins called COH-3 and COH-4 (henceforth, COH-3/4) perform specialized roles, indicating that interchangeable kleisin subunits determine cohesin function during meiosis. For example, only REC-8 cohesin can co-orient sister chromatids and mediate SCC that persists after anaphase I. Kleisin identity also influences the mechanism by which cohesin loads onto chromosomes and establishes cohesion between sisters. The axial element protein HTP-3 is required for loading of REC-8, but not COH-3/4. Once loaded, COH-3/4 cohesin is triggered to become cohesive by SPO-11-dependent double strand DNA breaks, while REC-8 cohesin generates SCC independently of SPO-11. Finally, REC-8 and COH-3/4 become asymmetrically distributed on meiotic chromosomes late in prophase of meiosis I: COH-3/4 becomes enriched where SCC is released at anaphase I and REC-8 becomes enriched where sister chromatids co-orient and SCC persists until anaphase II. Because REC-8 alone can co-orient sisters and mediate SCC that persists following anaphase I, we are testing whether achieving this reciprocal pattern of cohesin localization facilitates the stepwise separation of homologs and sister chromatids.

Severson, Aaron F. et al. (2011) International Worm Meeting "Kleisin Subunit Identity Determines Cohesin Loading Mechanism, Distribution, and Function during C. elegans Meiosis."

Meyer, Barbara J., Severson, Aaron F.

[International Worm Meeting, 2011]

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. Three processes unique to meiosis are crucial for reducing ploidy. First, homologous chromosomes become linked through crossover recombination. Second, the two sister chromatids of each homolog attach to microtubules (MTs) from the same spindle pole (co-orient) in meiosis I and attach to MTs from opposite spindle poles (bi-orient) in meiosis II. Third, sister chromatid cohesion (SCC) is released in two steps to allow separation of homologs and then sisters during meiosis I and II. Work in yeast has shown that establishing the meiotic pattern of chromosome segregation requires that Scc1, the "kleisin" subunit of cohesin complexes that mediate mitotic SCC, is replaced by the meiosis-specific paralog Rec8. Subsequently, rec8 mutations have been used to define meiotic cohesin functions in many organisms. We have shown that REC-8 is not the sole meiotic kleisin in C. elegans, and based on our work and other published data we propose that involvement of multiple kleisins is common in meiosis. C. elegans REC-8 and a pair of functionally redundant kleisins called COH-3 and COH-4 (COH-3/4 hereafter) perform specialized roles during meiosis. For example, although both REC-8 and COH-3/4 are required for meiotic SCC, only REC-8 cohesin can co-orient sister chromatids and mediate SCC that persists after anaphase I. Thus, cohesin complexes that differ in their kleisin subunit can perform distinct tasks in a single nucleus, and cohesin function is determined by kleisin identity. The kleisin also influences the mechanism by which cohesin loads onto chromosomes. The axial element protein HTP-3 is required for loading of REC-8 cohesin onto meiotic chromosomes, as has been shown previously for the timeless paralog TIM-1. Remarkably, neither HTP-3 nor TIM-1 is required for loading of COH-3/4 cohesin. Finally, REC-8 and COH-3/4 become asymmetrically distributed on meiotic chromosomes late in prophase of meiosis I, adopting an organization consistent with their distinct roles in meiotic chromosome segregation: COH-3/4 becomes enriched at regions where SCC is released at anaphase I and REC-8 becomes enriched where sister chromatids co-orient and SCC persists until anaphase II. Because REC-8 alone can co-orient sister chromatids and mediate cohesion that persists following anaphase I, we are currently testing whether establishing this reciprocal pattern of cohesin localization is important for the stepwise separation of homologs and sister chromatids.

Aaron F Severson et al. (2001) International C. elegans Meeting "The Profilin PFN-1 is required for cytokinesis and cell polarity in the C. elegans embryo"

Bruce Bowerman, Aaron F Severson

[International C. elegans Meeting, 2001]

Proper regulation of the actin cytoskeleton is crucial for diverse developmental asymmetries in the first cell cycle of the C. elegans embryo. In wild type embryos, the oocyte pronucleus migrates to meet the stationary sperm pronucleus in the posterior of the embryo. At the same time, polarized cytoplasmic flows can be detected in the posterior half of the embryo, and P-granules become asymmetrically distributed along the A/P axis. Finally, the first mitotic spindle becomes displaced toward the posterior of the embryo, resulting the production of daughter blastomeres with different sizes in the ensuing cytokinesis. We have identified three C. elegans homologs of the actin binding protein profilin based on sequence homology. Inactivation of one of these profilin genes, called pfn-1 , by RNAi results in defects in all of the actin-dependent processes described above. In pfn-1(RNAi) embryos, both pronuclei migrate to meet in the center of the embryo, and the first mitotic spindle is centrally positioned. Cytoplasmic flows are severely reduced, and P-granules persist in the middle of the embryo. Finally, pfn-1 mutant embryos fail in the related processes of polar body extrusion during meiosis, pseudocleavage, and cleavage furrow ingression during cytokinesis. Thus, PFN-1 is required for the formation or function of the contractile ring, as well as for the establishment of embryonic asymmetries. Because the phenotypes described above resemble those resulting from disruption of the actin cytoskeleton following treatment with the actin-depolymerizing drug cytochalasin D, we examined the effect of pfn-1 interference on the actin cytoskeleton by staining pfn-1 embryos with antibodies that recognize actin and nonmuscle myosin II (NMY-2). While both proteins accumulate at high levels around the entire cortex of wild-type embryos, we find actin and myosin only in dispersed patches in pfn-1 embryos. Additionally, the localization of the Formin Homology protein CYK-1, which is required for cytokinesis, and PAR-2 and PAR-3, which are essential for normal embryonic polarity, are severely disrupted in pfn-1 embryos. Thus, PFN-1 appears to function at a high level in the hierarchy of proteins regulating the assembly of the actomyosin cytoskeleton.

Aaron F Severson et al. (2004) West Coast Worm Meeting "The C. elegans condensin II complex maintains chromosome resolution in late meiotic prophase and promotes chromosome segregation in meiosis I and II"

Aaron F Severson, Raymond C Chan, Barbara J Meyer

[West Coast Worm Meeting, 2004]

Accurate segregation of meiotic chromosomes requires the removal of linkages between homologous chromosomes and sister chromatids prior to anaphase. Moreover, proper attachment of meiotic chromosomes to their spindle microtubules requires that the chromosomes be correctly organized. We have shown that a C. elegans condensin complex is required for both the resolution of homologs and sister chromatids and the condensation of meiotic chromosomes into properly organized bivalents. The prototypical condensin complex is composed of two Structural Maintenance of Chromosomes (SMC) subunits, Smc2 and Smc4, and three non-SMC subunits, called CAP-D2, -G, and -H. The single C. elegans Smc2 homolog MIX-1 functions in two condensin-like complexes. MIX-1 associates with the Smc4 homolog DPY-27 to mediate dosage compensation, a process that equalizes the expression of X-linked genes between XX hermaphrodites and XO males. MIX-1 associates with a second SMC partner, Ce SMC-4, to mediate the condensation of mitotic chromosomes. Through biochemical analysis of MIX-1 protein complexes, we identified HCP-6, the C. elegans ortholog of the condensin subunit XCAP-D3, and demonstrated the exclusive association of HCP-6 with the mitotic condensin complex. Chromosome condensation is a conserved property of meiotic prophase I, but the role of the condensin complex in meiosis has not been well characterized in metazoans. We therefore examined the requirements for condensin in the C. elegans meiotic germline. Reducing HCP-6 function disrupted chromosome segregation in meiosis I and II. Condensin was also required for the compaction and reorganization of meiotic chromosomes that occur after exit from pachytene and prepare chromosomes for their segregation. Homologs in pachytene nuclei were resolved in hcp-6 mutants as in wild-type, but chromosomes inappropriately decondensed in diplotene and individualized bivalents were rarely observed in diakinesis oocytes of animals with severely compromised HCP-6 function. In contrast, chromosomes in wild-type animals rapidly compacted to form six resolved bivalents in diplotene and diakinesis. Finally, condensin was necessary for the removal of linkages between diakinesis chromatids. In animals with wild-type HCP-6 function, homologs and sister chromatids separated prematurely following RNAi-mediated depletion of the meiosis-specific cohesin subunit REC-8. A partial loss-of-function hcp-6 allele prevented this precocious separation. Taken together, our results suggest that de novo linkages between homologs and sister chromatids arise in diplotene/diakinesis nuclei of hcp-6 mutants. We propose that one function of meiotic condensin is to maintain the resolution of chromosomes as the chromosomal axis is reorganized around the chiasma following breakdown of the synaptonemal complex in diplotene. Consistent with the genetic requirement for HCP-6 in late meiotic prophase, HCP-6 was first detectable on diplotene/diakinesis chromosomes, where it colocalized with MIX-1. Surprisingly, we found different loading dependencies for HCP-6 and MIX-1 on meiotic and mitotic chromosomes: while MIX-1 and HCP-6 loaded independently on mitotic chromosomes, MIX-1 required HCP-6 for its association with meiotic chromosomes. Additionally, the centromeric histone variant CENP-A was required for the association of HCP-6 with mitotic chromosomes but not with meiotic chromosomes. Our RNAi conditions effectively depleted CENP-A: meiotic chromosome segregation and the accumulation of the kinetochore proteins HIM-10 and MCAK on diakinesis chromosomes were disrupted. We conclude that condensin functions late in meiotic prophase, and that different rules govern the loading of HCP-6 and MIX-1 on chromosomes during meiotic and mitotic divisions.

Schilling, T. et al. (2019) International Worm Meeting "Non-Mendelian Inheritance in C. elegans: a Violation of the Law of Independent Assortment."

Severson, A. F., Schilling, T.

[International Worm Meeting, 2019]

Mendel's laws of inheritance can predict typical genotypic frequencies in subsequent generations. The second law, the Law of Independent Assortment, states that alleles determining different traits are inherited independently of each other. However, in C. elegans males, it has been previously observed that extrachromosomal DNA arrays and chromosomes containing integrated multicopy DNA arrays segregate away from the single male X chromosome, appearing more frequently in male than hermaphrodite offspring. For example, we observed that 80% of progeny inheriting a patroclinous copy of chromosome III balancer qC1[nIs281], are males and 20% are hermaphrodites instead of the expected 50% of each sex as predicted by Mendel's laws. A future goal is to perform FISH to visualize this unusual dependent assortment of chromosomes III and X. We also performed several crosses with different extrachromosomal and integrated arrays (oxEx229, mIs10, ccIs4251, syIs44) and analyzed the inheritance patterns in the offspring to demonstrate that the effects of non-Mendelian inheritance are not specific to the qC1[nIs281] balancer. The largest deviations from expected Mendelian frequencies resulted from crosses in which the array contained X chromosome homology. Finally, we are performing a forward genetic screen to identify mutations that disrupt non-Mendelian inheritance of the qC1[nIs281] balancer, shifting its transmission back to the expected ratio of 50% males and 50% hermaphrodites. Mutated pha-1/qC1[nIs281] males were crossed with pha-1 hermaphrodites and males fathering progeny in equal male and hermaphrodite proportions were considered candidates for having mutations resulting in expected inheritance patterns. Identifying genes required for non-Mendelian inheritance will define mechanisms that drive evolutionary change.

Rehain K et al. (2015) Curr Biol "Neuron migration: anillin protects leading edge actin."

Maddox AS, Rehain K

[Curr Biol, 2015]

Establishment of a neuronal system requires proper regulation of the F-actin-rich leading edges of migrating neurons and neurite growth cones. A new study shows that RhoG signals through the multi-domain protein anillin to stabilize F-actin in these structures.

Bennett JL et al. (1988) Parasitol Today "Pharmacology of ivermectin."

Williams JF, Bennett JL, Dave V

[Parasitol Today, 1988]

Ivermectin is a semi-synthetic macrocyclic lactone (Fig. I) active in single low doses against many parasites - particularly nematodes and arthropods. It has been registered for animal health use since early 1985, and was earlier this year approved for human use by the French Directorate o f Pharmacy and Drugs. Of particular interest is ivermectin's potential as a micro filaricide for treatment o f onchocerciasis. Clinical trials leave little doubt about the potential o f ivermectin as a therapeutic tool for symptomatic relief from the effects o f infection with Onchocerca volvulus, and the drug is also recognized to have potential in reducing transmission o f the parasite. The manufacturers (Merck, Sharp and Dohme) recently arranged to provide the drug free o f charge to the WHO for mass trials against onchocerciasis in 12 African and Central American countries. In this article we focus on the pharmacological properties o f ivermectin, with a brief consideration of its absorption, fate, excretion and side-effects, and a discussion o f its micro filaricidal action.

Kipreos ET et al. (2000) Genome Biol "The F-box protein family."

Kipreos ET, Pagano M

[Genome Biol, 2000]

SUMMARY: The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction. F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes (named after their main components, Skp I, Cullin, and an F-box protein), in which they bind substrates for ubiquitin-mediated proteolysis. The F-box motif links the F-box protein to other components of the SCF complex by binding the core SCF component Skp I. F-box proteins have more recently been discovered to function in non-SCF protein complexes in a variety of cellular functions. There are 11 F-box proteins in budding yeast, 326 predicted in Caenorhabditis elegans, 22 in Drosophila, and at least 38 in humans. F-box proteins often include additional carboxy-terminal motifs capable of protein-protein interaction; the most common secondary motifs in yeast and human F-box proteins are WD repeats and leucine-rich repeats, both of which have been found to bind phosphorylated substrates to the SCF complex. The majority of F-box proteins have other associated motifs, and the functions of most of these proteins have not yet been defined.

Kwiatkowski AV et al. (2010) Proc Natl Acad Sci U S A "In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans."

Hardin J, Weis WI, Nelson WJ, Choi HJ, Lynch AM, Kwiatkowski AV, Benjamin JM, Pokutta S, Maiden SL

[Proc Natl Acad Sci U S A, 2010]

The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.

Jayamani E et al. (2017) Antimicrob Agents Chemother "Characterization of a Francisella tularensis-Caenorhabditis elegans pathosystem for the evaluation of therapeutic compounds."

Ausubel FM, Nau GJ, Kim W, Mylonakis E, Coleman JJ, Okoli I, Lee K, RajaMuthiah R, Tharmalingam N, Hernandez AM, Jayamani E

[Antimicrob Agents Chemother, 2017]

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole animal Caenorhabditis elegans-F. tularensis pathosystem for high throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 MAP kinase cascade is involved in the immune response to F. tularensis and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z'-factor consistently >0.5, which was then utilized to screen a library of FDA approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, 5 FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the NSAID diflunisal, at 4X MIC, blocked the replication of a F. tularensis live vaccine strain (LVS) in primary human macrophages and non-phagocytic cells. Diflunisal was non-toxic to human erythrocytes and HepG2 human liver cells at concentrations 32-g/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans - F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.