Schiller EL et al. (1984) Am J Trop Med Hyg "Morphogenesis of larval Onchocerca volvulus in the Panamanian black fly, Simulium quadrivittatum."

Figueroa Marroquin H, Schiller EL, Petersen JL, Shirazian D

[Am J Trop Med Hyg, 1984]

Simulium quadrivittatum Loew (Diptera: Simuliidae), a man-biting black fly, was shown, for the first time, to be capable of supporting development of Onchocerca volvulus Leuckart (Nematoda: Filarioidea) from microfilariae to third-stage (infective) larvae. The black flies were collected in Chiriqui Province, Panama and transported alive to Guatemala, where they were allowed to feed on a human subject infected with O. volvulus. Samples of these flies were dissected over an 11-day period to assess morphogenesis of the parasite. Vigorously motile microfilariae were recovered from the mid-gut during the first 24 hours postfeeding; second-stage larvae were found in the thoracic musculature on day 4; and fully developed third-stage larvae were obtained from the cephalic capsule by day 10. This rate of larval development is similar to that observed in Guatemalan S. ochraceum. Onchocerciasis is not known to occur in Panama. The results of the present study direct attention to a potential public health hazard there and possibly elsewhere in Central America.

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

Johnson, Christina K. et al. (2021) MicroPubl Biol

Miller, David D., Johnson, Christina K., Bianchi, Laura

[MicroPubl Biol, 2021]

MEC-4 and UNC-8 are subunits of the DEG/ENaC family of voltage-independent Na+ channels in C. elegans (Driscoll and Chalfie 1991, Canessa, Horisberger et al. 1993, Waldmann, Champigny et al. 1996, Waldmann, Champigny et al. 1997, de Weille, Bassilana et al. 1998, Waldmann and Lazdunski 1998). While MEC-4 is expressed in body touch neurons where it mediates the transduction of gentle touch sensation (Driscoll and Chalfie 1991, O'Hagan, Chalfie et al. 2005), UNC-8 is primarily expressed in motoneurons where it is involved in synaptic remodeling during development (Tavernarakis, Shreffler et al. 1997, Miller-Fleming, Petersen et al. 2016). Both MEC-4 and UNC-8 can be hyperactivated by genetic mutations that hinder channel closing, called (d) mutations (Driscoll and Chalfie 1991, Shreffler, Magardino et al. 1995, Goodman, Ernstrom et al. 2002, Wang, Matthewman et al. 2013). C. elegans neurons and Xenopus oocytes expressing these hyperactive variants of MEC-4 and UNC-8 undergo cell death due to uncontrolled flux of ions into the cell. Cell death in Xenopus oocytes and in cultured C. elegans neurons can be prevented by incubation with the DEG/ENaC channel blocker amiloride (Goodman, Ernstrom et al. 2002, Suzuki, Kerr et al. 2003, Wang, Matthewman et al. 2013).

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.

Redo Riveiro, Alba et al. (2013) International Worm Meeting "egl-46 is a novel BAG cell fate modulator."

Pocock, Roger, Redo Riveiro, Alba

[International Worm Meeting, 2013]

Genetic studies in C. elegans attempt to identify molecular components required for neural circuits to function correctly. This study focuses on BAG neurons, which have the major role in oxygen (O2) and carbon dioxide (CO2) sensing. Development of BAG neurons is controlled by the conserved transcription factors ETS-5 (Brandt, Aziz-Zaman et al. 2012) and EGL-13 (Petersen et al. 2013-in press). However the effect of ets-5 and egl-13 loss of function mutants does not affect all BAG terminal fate markers. Therefore, other genes must act in parallel to these factors to specify BAG fate. In a classical forward genetic screen, we identified egl-46 as a BAG fate modulator. EGL-46 is a zinc finger transcription factor that regulates terminal cell divisions in the Q lineage where egl-46 mutants undergo extra rounds of terminal Q cell divisions (Wu, Duggan et al. 2001). The egl-46 mammalian ortholog Insm1 has a similar function in determining cell fates. Insm1 has been implicated in the development of pancreas (Farkas, Haffner et al. 2008), cortex and hindbrain (Jacob, Storm et al. 2009). Insm1 mutant mice present a thicker layer of proliferative progenitors and a thinner neuro-basal layer in the olfactory ephitelium (Rosenbaum, Duggan et al. 2011). Here we show that egl-46 mutants have defects in the expression of specific terminal markers in the BAG neurons. We have rescued these defects by transgenically expressing a fosmid containing the egl-46 locus. Currently, we are performing genetic interaction studies with BAG-fate regulators like ets-5 and egl-13.

Ken Norman et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Does the Rho family guanine nucleotide exchange factor VAV-1 function to regulate muscle excitability in C. elegans?"

Ken Norman, Villu Maricq

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Small G proteins of the Rho family are involved in many cellular functions, including cell cycle progression, cytoskeletal rearrangement, gene transcription, membrane transport and other signaling events. Due to their involvement in such a diverse array of cellular behaviors Rho proteins are highly regulated by several classes of proteins. One class that promotes Rho activity is the guanine nucleotide exchange factors (Rho GEFs). We have found that the Rho GEF VAV-1 is involved in regulating several rhythmic behaviors in C. elegans (Norman et al., 2005), including pharyngeal pumping, ovulation and defecation. In part, VAV-1 regulates these behaviors by modulating intracellular calcium levels via the signaling molecule IP3. vav-1 mutants have slow and asymmetric pharyngeal muscle contractions and abnormal calcium transients. These defects are rescued by specific expression of vav-1 in the pharyngeal muscle. Since mutations in the IP3 receptor, ITR-1, do not completely phenocopy vav-1 pharyngeal defects, vav-1 signaling may involve additional pathways. We have found that a gain of function mutation in the voltage gated potassium channel, UNC-103, is able to restore some normal pharyngeal activity in vav-1 mutants. The unc-103 gain of function mutation is thought to lead to hyperpolarized muscle cells resulting in less excitable muscle cells (Petersen et al., 2004). In another approach, we have found that vav-1 mutants are hypersensitive to the acetylcholine esterase inhibitor aldicarb. These data suggest that vav-1 mutants may have hyperexcitable muscle. To investigate this further, we generated egl-8; vav-1 double mutants. egl-8 encodes a phospholipase C beta; that promotes neurotransmitter release and when mutated results in aldicarb resistance. Introduction of the vav-1 mutation into an egl-8 mutant, restored normal aldicarb sensitivity. These results are consistent with VAV-1 regulating muscle excitability. We are now screening for additional genes that modify VAV-1 signaling.

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.