Tzur, Yonatan B. et al. (2009) International Worm Meeting "LAB-1 cooperates with cohesin to ensure accurate homolog segregation during meiosis I."

de Carvalho, Carlos E., van Bostelen, Ivo, Colaiacovo, Monica P., Tzur, Yonatan B.

[International Worm Meeting, 2009]

Achieving accurate chromosome segregation is a critical outcome for meiotic cell divisions. Errors in correctly separating homologs during meiosis I, and sister chromatids during meiosis II, may lead to aneuploidy, with dramatic consequences such as congenital defects, miscarriages and infertility. It has therefore been of critical importance to understand the regulation involved in coordinating loading, stability and dissociation of cohesins from DNA. We have recently reported the finding of LAB-1, a protein which plays a key role in late prophase I where it localizes to the long arms of the bivalents during diakinesis and restricts the spreading of the Aurora B Kinase, AIR-2, thereby preventing the premature loss of sister chromatid cohesion. Here we identify a distinct and earlier role for LAB-1 in promoting the establishment and maintenance of sister chromatid cohesion during early/mid-prophase I. Utilizing fluorescence in situ hybridization we find that lab-1(RNAi) reduces homologous pairing levels on both autosomes and the X chromosome compared to wild type. Levels of RAD-51 foci, a marker for DNA double-strand break repair, dramatically increase following lab-1 depletion, and the foci persist throughout the late stages of prophase I. Further evidence of an impaired progression in DSB repair stems from the increased germ cell apoptosis observed in lab-1(RNAi) in late pachytene. Synapsis is also impaired in the lab-1 depleted gonads, as exemplified by the mixture of synapsed and unsynapsed chromosomes observed in pachytene, and the aberrant polymerization of SYP-1, a synaptonemal complex central region component, detected along unsynapsed tracks. Interestingly, SYP-1 can also partially polymerize along chromosomes in mutants for the meiosis specific cohesin rec-8, but it fails to do so in lab-1(RNAi);rec-8(ok958) mutants. A role for LAB-1 in the establishment and maintenance of sister chromatid cohesion is also implied by the delayed deposition of LAB-1 following depletion of smc-3, another member of the cohesin complex. Whereas SMC-3 localization during early prophase is not impaired in either lab-1(RNAi) or rec-8 mutants, SMC-3 signal is drastically reduced or completely absent in lab-1(RNAi);rec-8 mutants. We therefore propose that LAB-1 cooperates with the cohesin complex to bind sister chromatids, thereby promoting proper pairing, synapsis and recombination in C. elegans meiosis.

Tzur, Yonatan B et al. (2011) International Worm Meeting "LAB-1 cooperates with cohesin to ensure accurate homolog segregation during meiosis I."

Colaiacovo, Monica P, van Bostelen, Ivo, Tzur, Yonatan B, de Carvalho, Carlos E

[International Worm Meeting, 2011]

Successful execution of the meiotic program depends on the timely establishment and removal of sister chromatid cohesion. Errors in correctly separating homologs during meiosis I, and sister chromatids during meiosis II, may lead to aneuploidy, with dramatic consequences such as congenital defects, miscarriages and infertility. It is therefore critically important to understand how the loading, maintenance and dissociation of cohesin complexes from DNA is regulated. LAB-1, a functional homolog of Shugoshin in C. elegans, is required to protect sister chromatids from the premature removal of REC-8, a meiosis-specific cohesin member, at metaphase I. Here we identify an earlier role for LAB-1 in promoting the establishment of sister chromatid cohesion during early prophase I. Utilizing fluorescence in situ hybridization (FISH) we find that lab-1(RNAi) reduces homologous pairing levels on both autosomes and the X chromosome compared to wild type. Moreover, nuclei with between 3 to 4 FISH signals were present from the pre-meiotic tip to the end of pachytene in lab-1 depleted gonads, suggesting loss of sister chromatid cohesion. As a result, both the progression of meiotic recombination and synapsis are impaired, as evidenced by the elevated levels of a marker for DNA double-strand break repair (RAD-51 foci) and by the mixture of synapsed and unsynapsed chromosomes observed in pachytene, respectively. Further support for a role in the establishment of sister chromatid cohesion stems from the observation that both LAB-1 and REC-8 are required for the chromosomal association of SMC-3, a structural maintenance of chromosomes protein and cohesin member. Similar to rec-8 mutants, depletion of lab-1 results in partial loss of sister chromatid cohesion when combined with mutations in two of the rec-8 paralogs, coh-3 and coh-4, and further increases the number of separated chromatids when all three paralogs are mutated. LAB-1 has a putative degenerate Protein Phosphatase 1 (PP1) binding motif, and accordingly, during early meiotic steps, depletion of lab-1 leads to reduction in the nuclear localization of the PP1 homolog GSP-2, and nuclear accumulation of the Aurora B Kinase homolog AIR-2. We propose that LAB-1 cooperates with the cohesin complex to link sister chromatids, and maintain cohesion by antagonizing AIR-2 via GSP-2 in early prophase, thereby facilitating the C. elegans meiotic program.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Baer, Charles F. et al. (2013) International Worm Meeting "Revisiting the effects of spontaneous mutations on the (micro)environmental variance in Caenorhabditis."

Farhadifar, Reza, Baer, Charles F., Andersen, Erik C., Needleman, Daniel

[International Worm Meeting, 2013]

An obvious feature of living organisms is that their development is robust to variation in the environmental circumstances in which the organism finds itself - within limits. That is, the phenotype is more or less "canalized" (canalized "robust"). The "more or less" is of considerable interest to evolutionary biologists, for a variety of reasons, i.e., under what environmental or genetic circumstances does development become more or less canalized? Environmental canalization can be quantified as the phenotypic variation among genetically identical individuals raised in a uniform environment - the "(micro)environmental variance" in the lingo of quantitative genetics, V(E). Here we provide quantitative estimates of the effects of spontaneous mutations on V(E) for a variety of phenotypic traits subject to different selective regimes. Three general quantitative trends emerge. First, in almost all cases, mutation accumulation tends to de-canalize the phenotype (i.e., V(E) increases), typically at a rate similar to the rate of change of the trait itself. Second, there is a strong positive association between the rate of increase of V(E) for a trait and the mutational variance, V(M) for the trait itself. Third, and most intriguingly, mutations affecting V(E) for a trait appear to be usually under stronger selection than mutations affecting the trait itself.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Almendariz-Palacios, Carla et al. (2021) International Worm Meeting "The impact of calorie restriction mimetics on cellular phenotypes triggered by an Alzheimer disease-related presenilin-1 protein splice variant in Caenorhabditis elegans"

Waddell, Brandon M, de Carvalho, Carlos E., Almendariz-Palacios, Carla, Eskiw, Christopher H., Wu, Cheng-Wei, Mousseau, Darrell D

[International Worm Meeting, 2021]

As we age, our brain undergoes a number of changes at the cellular and molecular levels. These changes, such as those associated with protein removal through autophagy, lead to the aggregation of misfolded proteins and eventually to the disruption of the nuclear membrane. Age-related disruption of these cellular events is strongly associated with diseases, such as Alzheimer's disease (AD), the most common form of dementia. Much of the brain malfunction during AD is related with the beta-amyloid peptide that is generated because of incorrect cleavage of the amyloid precursor protein (APP). This incorrect cleavage of APP has been associated with dysfunction of the presenilin-1 (PS-1) protein. Alterations in the PS-1 gene have been associated with nuclear membrane disruption and autophagy impairment. A screen of mRNA transcripts in our laboratory identified a splicing variant of PS-1 (PS-1 (SV)) that is highly expressed in AD brain samples. The role of PS-1(SV) during AD and aging is unknown. Since current AD pharmacological therapies are not effective, there is critical need to find treatment strategies. One promising alternative is caloric restriction (CR), the reduction in dietary caloric intake without inducing malnutrition, which has been shown to increase autophagy, reduce risk of AD and extend lifespan in various species. A new class of compounds called CR mimetics (CRms), which mimic the effects of CR without the need for dietary intervention, have been identified. The C. elegans model is widely used to study aging and neurodegeneration. The sel-12(or131) strain, which carries a loss-of-function sel-12, the worm homologue of PS-1, exhibits an egg-laying defect (which is often associated with aging), a reduction in lifespan and a loss of nuclear membrane integrity. Our preliminary results, based on transient expression of PS-1 in the sel-12(or131) background, confirms that PS-1(WT) can rescue the egg-laying defect, but that PS-1(SV) cannot, suggesting that the PS-1(SV) is non-functional, which might underlie its influence on age-related phenotypes. Because the defects displayed by the sel-12 strain can be, in part, attributed to the damaged autophagy, it represents an ideal model to test if the CRms, metformin, resveratrol and everolimus, which have already shown to rescue experimental autophagy impairment, can alleviate age-related phenotypes found in C. elegans PS-1(SV)::sel-12.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Chen, P. et al. (2009) International Worm Meeting "The Early-Onset Torsion Dystonia Gene Product, torsinA, is a Mediator of ER Stress and Protein Homeostasis."

Roberts, N., Porter, J.C., Burdette, A.J., Berkowitz, L.A, Ricketts, J.C., Caldwell, K.A., Caldwell, G.A., Chen, P., Fox, S.A.

[International Worm Meeting, 2009]

The capacity of cells to carry out their various functions is wholly dependent upon efficient protein synthesis, processing, trafficking, and degradation. In this context, dysfunction or deficit of proteins that monitor and ensure the proper maintenance of cellular homeostasis can have serious consequences in terms of disease onset, penetrance, or progression. A single codon deletion (GAG = DE) in the gene encoding human torsinA causes a non-degenerative neurological disorder termed early-onset torsion dystonia. TorsinA, an endoplasmic reticulum (ER) resident protein, belongs to the diverse AAA+ (ATPases Associated with a variety of cellular Activities) family. Here we use a well established ER stress model in the nematode C. elegans (hsp-4::GFP) to investigate the role of torsinA in maintaining protein homeostasis in the ER. Using GFP as an ER stress reporter, we found that transgenic nematodes expressing wild type (WT) human torsinA exhibited a striking reduction in ER stress caused by tunicamycin, while animals with either DE or a mixture of WT/DE torsinA failed to do so. Interestingly, DE and WT/DE torsinA worms exhibited higher ER stress than WT torsinA animals, even in the absence of tunicamycin treatment. In addition, mutations within torsinA ER signal sequence and N-terminal hydrophobic region abolished its capacity to maintain an ER stress threshold against cellular stressors, indicating the importance of its localization to the ER for this activity. Furthermore, although torsinA possesses very low ATPase activity, the ATPase domain appeared important for ER stress suppression, as two different mutations within the ATPase domain, K108A and E171Q, resulted in loss of stress suppression. A single nucleotide polymorphism (SNP), D216H, diminishes the penetrance of dystonia from 30-40% (WT/DE) to 3% (D216H/DE) in patients. In C. elegans, coexpression of DE torsinA with D216H torsinA significantly protected worms from ER stress caused by tunicamycin, thereby recapitulating the effect on penetrance observed with patients. In order to find genetic interacting factors of torsinA, a small scale of RNAi screen for altered ER stress levels in DE torsinA worms, was carried out. Positives from the screen included glutaredoxin, H3 histones, and 13 core components and regulators of Ras signaling. Taken together, our data indicate that torsinA serves to maintain protein homeostasis in the ER, shedding new light on the pathophysiology of torsion dystonia.

Fuentes, A. et al. (2015) International Worm Meeting "Diet affects neurodegenerative processes in C. elegans."

Fuentes, A., Calixto, A., Garcia, V., Palominos, MF.

[International Worm Meeting, 2015]

C. elegans has been used to understand the dietary impact on development and behavior (Macneil & Walhout., 2013), but very little is known about the role of diet on neurodegenerative processes. To study the effect of diet on neuronal degeneration, we use animals expressing the hyperactivated degenerins mec-4d expressed in the touch receptor neurons (TRNs) and deg-1 expressed in the PVC neurons. We measured the impact of different bacterial diets on neuronal integrity in the respective degeneration models. mec-4d animals with GFP marked TRNs were fed with E. coli OP50, B, HT115 and K12; Commamonas aquatica, Commamonas testosteronii and Bacillus megaterium and their TRN integrity assessed both morphologically (by visual inspection) and functionally (by the touch response) at 72 hours where most axons are degenerated (Calixto et al., 2012). The maximum protection was elicited by E. coli HT115 diet, with up to 50% wild type axons while the control strain E. coli OP50 and its precursor B only protects 3% at 72 hours post hatching. Like in the mec-4d animals, deg-1 animals PVC show a higher percent of functional response when animals are fed E. coli HT115. E. coli HT115 is capable of protecting neurons during large periods of time after adulthood since animals at 168 hours post hatching still showed 16.5 % of wild type axons. E. coli K12, Comammonas aquatica, testosteronii and Bacillus megaterium protected less (between 10 and 17% of wild type axons) than E. coli HT115 and more than E. coli OP50. E. coli HT115 is still capable of generating neuroprotection when diluted 1:100 in E. coli OP50, showing that E. coli OP50 does not produce neurodegeneration. Additionally, this shows that a very small amount of the protective food is necessary to elicit neuronal protection. E. coli HT115 does not produce dietary restriction and supports well animal growth, with 100% animals reaching adulthood at 72 hours vs 100% in E. coli OP50. Dead bacteria produce the same levels of neuronal protection than live bacteria, indicating that the interaction between bacteria and intestine is not required. Our data suggest that E. coli HT115 is capable of triggering pathways involved in neuroprotective processes early in animal development (first 12 hrs after hatching), highlighting the relevance of developmental time when the protective diet is ingested. We are currently performing transcriptomics analysis to identify the bacterial components from E. coli HT115 that mediate neuroprotection.

Prasad BC et al. (1998) East Coast Worm Meeting "The O/E transcription factor, UNC-3, is required for axonal guidance and ASI function"

Reed RR, Seydoux GC, Prasad BC

[East Coast Worm Meeting, 1998]

The O/E family of vertebrate rHLH transcription factors has been implicated in neurogenesis and axonal pathfinding. The mammalian O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis. A Xenopus O/E homologue, Xcoe2, functions downstream of neurogenin and upstream of neuroD in neurogenesis. The murine O/E members are also expressed in olfactory neurons throughout development and may regulate components of the odorant signal transduction cascade. A C. elegans O/E (CeO/E) homologue has been identified in our laboratory and shown to be encoded by the unc-3 locus, mutations which result in an uncoordinated phenotype. Previous electron microscopic reconstruction of unc-3 mutants revealed that the axons of the ventral cord motor neurons are severely defasiculated, the neuromuscular junctions occur at ectopic sites and the motor neurons receive input from inappropriate interneurons as a result of the defasiculation. Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed in a large subset of the ventral cord motor neurons and in a pair of chemosensory neurons (ASI amphid neurons). The CeO/E protein is expressed transiently, during times of axonogenesis in the ventral nerve cord and throughout development in the ASI neurons. Several observations suggest that the protein may have distinct roles in the two cell types. Specifically, the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI and the unc-3 mutants enter the dauer pathway under inappropriate conditions. There is also evidence for autoregulation of the gene in ASI neurons - the expression of CeO/E is greatly reduced in unc-3 mutants. CeO/E lacks a conserved second repeat helix found that is highly conserved in the mammalian protein. Experiments suggest that CeO/E protein is able to homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. We are currently identifying the binding site for CeO/E by analysis of sequences essential for regulation of expression in the ASI neurons as well as by specific binding site selection methods (Selex). We are using PCR and differential expression methods to isolate downstream targets of CeO/E that might participate in signaling in the ASI neurons and mediate the axonal pathfinding activities in the ventral nerve cord. The isolation of putative cofactors and downstream targets of CeO/E should provide insight into the function of this transcription factor in neuronal differentiation in C. elegans and vertebrates.