McMahon L et al. (2001) J Cell Sci "Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1."

Legouis R, Labouesse M, McMahon L, Vonesch JL

[J Cell Sci, 2001]

Specialised subapical junctions play a critical role in maintaining epithelial cell polarity and tissue integrity, and provide a platform for intracellular signalling. Here we analyse the roles of C. elegans genes let-413 and dlg-1, a homologue of Drosophila lethal discs large, in the assembly of the C. elegans apical junction (CeAJ), and provide the first characterisation of this structure, We have identified dlg-1 as an essential gene in an RNA interference screen against C. elegans homologues of genes encoding proteins involved in tight or septate junction formation. We show that DLG-1 colocalises with the junctional protein JAM-1 at CeAJs in a unit distinct from HMP-1/alpha -catenin, and apical to the laterally localised LET-413, Loss of dlg-1 activity leads to JAM-1 mislocalisation and the disappearance of the electron-dense component of the CeAJs, but only mild adhesion and polarity defects. In contrast, loss of let-413 activity leads to the formation of basally extended discontinuous CeAJs and strong adhesion and polarity defects. Interestingly, in LET-413-deficient embryos, CeAJ markers are localised along the lateral membrane in a manner resembling that observed in wildtype embryos at the onset of epithelial differentiation. We conclude that the primary function of LET-413 is to correctly position CeAJ components at a discrete subapical position. Furthermore, we propose that DLG-1 is required to aggregate JAM-1 and other proteins forming the electron-dense CeAJ structure. Our data suggest that epithelial adhesion is maintained by several redundant systems in C. elegans.

Madhumitha Nandakumar et al. (2005) International Worm Meeting "PUFAs play an important role in mediating the C.elegans immune response"

Madhumitha Nandakumar, Man-Wah Tan

[International Worm Meeting, 2005]

Lipids are known to play multiple roles in the immune system, ranging from functioning as immune precursors to acting as signaling molecules [1]. Recent studies have highlighted the importance of maintaining a balanced lipid composition in the body and speculated on the potential use of lipid supplements in improving the immune system [2]. Additionally, in the lab, a microarray analysis looking at the alteration of gene expression during infection showed an enrichment of lipid metabolic genes, with a particular down-regulation of genes involved in PUFA synthesis. This specific manipulation of lipid genes in response to infection points to potential roles for specific fatty acids in the innate immune response. We are interested in using the C.elegans model system to study the role of Poly Unsaturated Fatty Acids (PUFAs) in the immunity. We have shown that altering the composition of fatty acids, by interfering with PUFA synthesis, leads to an alteration of the immune response. Blocking the PUFA synthesis pathway at specific points leads to distinct alterations in fat composition [3], and each of these mutants show different resistance and susceptibility phenotypes against the pathogen Pseudomonas aeruginosa. Mutants such as elo-1, which has increased levels of 18:0 and 18:3n6 fatty acids, show increased resistance to infection, while mutants such as fat-3, which lacks many 18 and 20 carbon fatty acids, show increased susceptibility to infection. Gas chromatography analysis is being used to identify the individual fatty acids that could influence the immune response, and the results of this analysis will also be presented.1)Wallis JG, Watts JL, Browse J. Polyunsaturated Fatty Acid Synthesis: what will they think of next? Trends Biochem Sci, 2002. 27(9):467-473.2)Tricon S, Burdge GC, Kew S, Banerjee T, Russell JJ, Grimble RF, Williams CM, Calder PC, Yaqoob P. Effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid on immune cell function in healthy humans. Am J Clin Nutr. 2004. 80 (6): 1626-1633.3)Watts JL, Browse J. Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans. PNAS, 2002. 99: 5854-5859.

MOLIN, L. et al. (2019) International Worm Meeting "Screen for new actors of muscle aging."

Bessereau, JL., ROY, C., SOLARI, F., MOLIN, L.

[International Worm Meeting, 2019]

We previously demonstrated that muscle aging in C. elegans involves a stereotyped sequence of events characterized by a dramatic decrease in the expression of genes encoding proteins required for muscle contraction, followed by a change in mitochondria morphology, and an increase in autophagosome number (Mergoud et al., 2018). To further characterize the mechanisms underlying muscle aging we used CRISPR/CAS9 to generate a transcriptional reporter that mirrors the down-regulation of tnt-2 (troponine T) expression with age. We used CRISPR-CAS9 to generate a knock-in strain, in which tagRFP-T fluorescence in body wall muscles (BWM) and protein level decrease with age. Using this reporter strain we performed a semi-clonal screen after EMS mutagenesis in order to identify genes whose mutation maintains tagRFP-T fluorescence with age. Details of the screening strategy and some preliminary results will be presented. Mergoud dit Lamarche A., Molin L., Pierson L., Mariol MC., Bessereau JL., Gieseler K., Solari F. (2018). UNC-120/SRF independently controls muscle aging and lifespan in Caenorhabditis elegans. Aging Cell. https://doi.org/10.1111/acel.12713

Draper BW et al. (1991) International C. elegans Meeting "MATERNAL GENES WHICH AFFECT THE DEVELOPMENT OF BODY WALL MUSCLE IN C. ELEGANS"

Priess JR, Mello CC, Krause MW, Weintraub H, Draper BW

[International C. elegans Meeting, 1991]

A homolog of the mouse skeletal muscle-specific master regulatory gene MyoD-l (called CeMyoD) has recently been identified in C. elegans, and its pattern of embryonic expression make it a likely candidate for being a regulator of nematode myogenesis (see abstract by Krause). We have screened for maternal-effect lethal mutations which alter the number of body wall muscles to identify genes that may be involved in the early regulation of CeMyoD expression. Thus far we have identified five mutations which define three loci. All of these alleles cause excess muscle to be produced and we call them mex-l (Jl), mex-2 (II) and mex-3 (I) for muscle excess. mex-l and 2 mutant embryos can have as many as twice the normal number of CeMyoD positive cells. The temporal pattern of expression of CeMyoD appears altered in the mex mutant embryos such that expression begins early and in an abnormal number of cells. A wildtype AB blastomere does not produce any muscle or pharyngeal cells if its sister blastomere Pl is ablated with a laser microbeam. In contrast, a mex-l mutant AB blastomere produces both body wall muscle and pharyngeal cell when Pl is destroyed.

Tsukamoto T et al. (2016) J Biol Chem "X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION."

Shimono K, Honda N, Hasegawa T, Takahashi M, Hashimoto N, Sudo Y, Hayashi S, Mizutani K, Miyauchi S, Yamamoto M, Takagi S, Yamashita K, Tsukamoto T, Murata T

[J Biol Chem, 2016]

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

Scott, Alyssa et al. (2015) International Worm Meeting "Investigating the role of higher-order chromatin structure during gamete formation."

Turcotte, Carolyn, Checchi, Paula, Rigothi, Julia, Leonard, Emily, Scott, Alyssa, Romer, Megan

[International Worm Meeting, 2015]

Meiosis ensures the faithful segregation of genetic material to sperm and egg and is necessary for the propagation of all sexually reproducing species. Paradoxically, a requirement for successful meiosis is the deliberate induction of genome-wide double-strand breaks (DSBs), which in other contexts are considered highly toxic. As a result, organisms are equipped with an array of well-conserved DSB repair mechanisms as well as checkpoint pathways. While it is mechanistically understood how repair pathways and checkpoints respond to meiotic DSBs, the interplay between this complex network of pathways is poorly defined. Our hypothesis is that modifiers of higher order chromatin structure prevent the transmission of errors to offspring by regulating meiotic DSB repair. To test this, we have generated a library of transgenic C. elegans strains which allow us to induce targeted, site-specific DSBs1 as well as strains that possess LacO-"tagged" chromosomes that are easily identified in both meiotic nuclei and fixed embryos by adding purified LacI::GFP2. These technologies enable us to precisely define spatial and temporal events during meiotic recombination, determine the genetic consequences of faulty DSB repair in offspring, and identify the presence of aneuploid embryos. Using these tools, we discovered a role for the conserved structural maintenance of chromosomes (SMC) 5/6 complex in preventing chromosome missegregation. Strikingly, we found that in the absence of SMC-5/6, these defects are significantly more pronounced in oogenesis versus spermatogenesis. Further, we also discovered a requirement for the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex in maintaining the cellular integrity of germ cells during meiosis. We are currently using a combination of genetic, molecular and immunocytological approaches to address how these complexes ensure the production of gametes with the correct chromosome complement and an absence of DNA lesions.References:(1) Boulin T and Bessereau JL. (2007) Mos1-mediated insertional mutagenesis in Caenorhabditis elegans. Nat Protoc. 2(5):1276-87.(2) Severson AF and Meyer BJ. (2014) Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes. Elife. 3:e03467.

Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.

Sandrine Jacob et al. (2007) International Worm Meeting "dog-1: a useful tool for genetics."

Delphine Bernard, Bernard Lakowski, Sylvie Aubert, Virginie Rubio, Sandrine Jacob

[International Worm Meeting, 2007]

Certain, rare, mutations in the human gene BRIP1/BACH1/FANCJ cause Fanconi anemia, while others have been linked to familial breast cancer. Reduction of function of the C. elegans ortholog, dog-1, has been reported to produce a high frequency of small deletions upstream of long poly-guanine (poly G) stretches in the genome (1, 2). Based on the nature of the mutations identified, it has been hypothesized that dog-1may be required to resolve three dimensional structures such as Guanine quadruplexes (G4) on lagging strand DNA during DNA replication. As most dog-1deletions have been identified in individual worms by PCR, it is not clear if the full spectrum of mutations induced by dog-1has been characterized. To test the specificity of dog-1 for poly G stretches we screened for heritable presenilin suppressor (spr) gene mutations in a dog-1(gk10) I; sel-12(ty11) X background. Of the seven genes that mutate at high frequency to suppress sel-12, only one, spr-3, has a poly G stretch in, or near, the gene. As expected, 12/14 strong spr mutations recovered were small deletions affecting the poly G stretch of spr-3, indicating that dog-1 is highly specific for poly G stretches. The mutation frequency at the spr-3 poly G is 4 X 10 -4, very similar to the EMS frequency for this gene. To determine more completely the range of dog-1target sequences, we screened for spontaneous mutations with a strong visible phenotype. From analyzing many of these mutants we have found that dog-1mutations are almost always associated with a putative G4 sequence, however not just poly G but also other G rich sequences can become unstable. These results are a strong confirmation of the original model. The largest class of mutations is small deletions starting at G4 sequences but we have also recovered larger deletions, deletions with insertions and reciprocal translocations at lower frequency. The strong association of dog-1mutations with G4 sequences helped us to rapidly identify the sites of mutations in many alleles of previously characterized genes and helped us to positionally clone mutations in four genes, dpy-1, ngn-1, nhr-67 and pptf-1 that had not previously been cloned or recovered in forward genetic screens. Furthermore, dog-1 induced mutations often have different properties than alleles generated by the knock out consortiums. We are now testing whether we can use comparative genome hybridization (CGH) approaches to systematically detect dog-1 induced mutations in order to characterize a large collection of dog-1 induced mutations. 1 Cheung I et al. (2002) Nat. Genet. 31, 405-409. 2 Youds JL, et al. (2006) Genetics 173, 697-708.

Ahnn JH et al. (1994) Worm Breeder's Gazette "Update on Deficiency Screens."

Ahnn JH, Fire A

[Worm Breeder's Gazette, 1994]

Update on deficiency screens Joohong Ahnn and Andrew Fire Carnegie Institution of Washington, Department of Embryology, Baltimore, MD 21210 We have been screening available chromosomal deficiencies in order to identify genetic loci whose zygotic expression is required for early events in embryogenesis including formation of body wall muscle cellsl. A surprising result of deficiency screens (oursl and others2 3) has been the extensive tissue differentiation that occurs in many of the deficiency homozygote embryos. In our initial analysis we found only one region (on the left tip of LGV) which was required for accumulation of body-wall myosin. This gazette article reports updates on our deficiency analysis. Analysis of additional characterized deficiencies: Embryos homozygous for nDf3 (II)4 and nDf4 (Jl)4 arrest elongation before comma stage (although a small fraction develop to the 1-112 fold stage). These embryos form pharyngeal structures, have gut granules and stain with antibodies to unc-54 and myo-3 produsts. Deficiency sDf23 (IV)s homozygous embryos arrest before comma stage, have gut granules but no pharyngeal structures; these embryos do not show any movement but stain with both anti-myosin antibodies. nDf42 (V)6 homozygous embryos show an early arrest with no movement, no gut granules, and no pharyngeal structures. Homozygous embryos from nDfl 7 (m)4 and nDfl 9 (X)7 both arrest at 2-fold stage, show twitching movement, have gut granules, form pharynx and stain with myosin antibodies (n.b. mapping data on ACEDB suggests nDfl 7 and nDfl 9 could be compound deficiencies). Producing a bank of Dfs of VL: In order to isolate small deficiencies in the region of LG VL which appears to be essential for myogenesis, gamma-ray induced mutagenesis has been conducted. Mutagenized N2 males were crossed into unc-34 dpy-l l animals, and rare Unc Non- dpy F1 progeny were cloned. Seven deficiencies have been isolated among 17,000 haploid chromosomes screened so far. Further mapping of these deficiencies by physical and genetic means should allow us to narrow down the myogenic locus (or loci) in the region. A very early zygotic requirement ? In a more general screen for early arrest mutants, several very early lethals linked on LG m have been isolated. These segregate a high fraction of dead embryos, suggesting a chromosomal break or rearrangement. About 1/4 of the embryos from these strains show a characteristic very early terminal phenotypes. These embryos have 200-2S0 nuclei, with no overt tissue differentiation. The embryos fail to stain with myosin antibodies and do not have gut granules. Embryos from two of these strains were observed with 4-D microscopy to look for defects during early embryogenesis. The early cleavage pattern of early-arrest embryos look normal, but these embryos fail to complete last 1-2 cell divisions. In some cases, nuclear divisions appear to complete without subsequent cytokinesis. It is interesting to compare these terminal phenotypes with those of emb-29 (V). emb-29 produces embryos with comparable numbers of nuclei, but these embryos exhibit extensive differentiation8. This suggests that the putative chromosome m defect is not simply due to blockage or failure of cell divisions. WBG 12: 2, 106 Genetics (137:483-498) 2. F. Storfer-Glazer & W. Wood Genetics (137: 499-508) '93 C. elegans Meeting (Abt. 497) 4. I. Greenwald & R. Horvitz unpublished 6. M. Hengartner & R. Horvitz unpublished 7. V. Ambros & R. Horvitz unpublished 8. R. Hecht et. al J. of Cell Science (87:305- 314)

Mili Jeon et al. (2001) International C. elegans Meeting "THE TIMELESS-LIKE GENE TIM-1 IS CSG-5 AND IS REQUIRED FOR CHROMOSOME SEGREGATION"

Mili Jeon, Ann Rougvie

[International C. elegans Meeting, 2001]

The heterochronic gene lin-42 encodes a protein with high similarity to the PAS domain of the Drosophila circadian rhythm protein PERIOD (dPER). Because the PAS domain of dPER interacts with TIMELESS (dTIM), a second circadian rhythm protein, we investigated the role of TIM-1, a TIMELESS-like protein in C. elegans . Unlike the dtim null mutation, which shows no developmental defects (1), RNAi of tim-1 results in embryonic lethality. These embryos arrest development as a mass of cells with unevenly-sized nuclei. In early embryos, we observed multinucleate cells by nomarski optics. When stained with DAPI, these embryos showed DNA bridges between forming nuclei, lagging chromosomes, nuclei with abnormal DNA content and fragmented chromosomes. These observations are consistent with a role for TIM-1 in chromosome segregation. Animals that escape the early lethal dsRNA dose show no heterochronic defects and develop into sterile adults. In oocytes of DAPI-stained animals, we observed 11-12 fluorescent "spots", presumably univalents, in contrast to the 6 bivalents observed in wild-type animals. We are using FISH analysis to test whether these spots represent unpaired homologs. Affinity purified antibodies raised against TIM-1 showed nuclear accumulation and enrichment in the region of the kinetochore spindles during metaphase. Preliminary analysis revealed that TIM-1 is abundant in all cells during early embryogenesis and is present in the nuclei of seam cells, the intestine and the developing germ line during larval stages. To identify mutations in tim-1 , embryonic lethal mutations that mapped near the tim-1 locus on chromosome III were collected from the CGC. We analyzed candidate alleles by DAPI-staining and nomarski optics, looking for phenotypes similar to the tim-1 (RNAi) phenotype. We narrowed down the candidates to csg-5 (t1545) ( c hromosome s e g regation defective), isolated by Gonczy et al . in a maternal-effect embryonic lethal screen for genes involved in cell division (4). We sequenced t1545 and found a single base transition, resulting in a Gly924Asp mutation in a region moderately conserved among timeless proteins. A long-range PCR product containing the tim-1 coding region plus 3.3 kb 5'- and 1.3 kb 3'-flanking sequence fully rescued the embryonic lethality of t1545. These results indicate that tim-1 is csg-5. Recently, the identification of a second Drosophila timeless gene (dTIM2) was reported (2,3). dTIM2 is more closely related to TIM-1 and the single mammalian TIM protein than it is to dTIM. Interestingly, timeless knock-out mice die during early embryogenesis around a period of rapid cell proliferation (3), suggesting that mouse TIM and TIM-1 may play similar roles in the cell cycle and have functions distinct from the functions of the Drosophila clock protein dTIM. 1. Sehgal A, Price JL, Man B, Young MW. Science . (1994) 263 :1603-6. 2. Benna C, Scannapieco P, Piccin A, Sandrelli F, Zordan M, Rosato E, Kyriacou CP, Valle G, Costa R. Curr Biol . (2000) 10 :512-3. 3. Gotter AL, Manganaro T, Weaver DR, Kolakowski LF Jr, Possidente B, Sriram S, MacLaughlin DT, Reppert SM. Nat Neurosci . (2000) 3 :755-6. 4. Gonczy P, Schnabel H, Kaletta T, Amores AD, Hyman T, Schnabel R. J Cell Biol . (1999) 144 :927-46.