Mary E Kosinski et al. (2003) International Worm Meeting "Release of the major sperm protein from spermatids and spermatozoa in vivo"

Jonathan Holder, David Greenstein, Ginger Winfrey, Gary Olson, Mary E Kosinski

[International Worm Meeting, 2003]

Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. During meiotic maturation, C. elegans oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in a spatially restricted manner such that the most proximal (-1) oocyte matures, is ovulated, and fertilized. Sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using the major sperm protein (MSP) as a signaling molecule. The discovery of MSPs signaling role raised the intriguing question of how sperm release MSP to signal oocytes and sheath cells at a distance. The release of MSP from sperm likely occurs through a non-canonical mechanism since spermatozoa do not possess cellular components required in standard models of protein secretion, such as ribosomes, ER, or Golgi. Moreover, MSP does not have a N-terminal leader sequence, and in vitro release does not involve protein processing. Non-canonical or leaderless secretory pathways are widespread, and in general, poorly understood, despite figuring prominently in cell signaling, disease, and host defense. Using specific antibodies, we detect MSP both inside and outside of spermatids and spermatozoa in vitro. MSP release does not require motility or a pseudopod, and does not involve sperm lysis. We observe extracellular MSP up to approximately 90 m away from spermatozoa in the spermatheca, frequently reaching the -1 oocyte. Examination of extracellular MSP reveals it is localized in a graded distribution with the highest levels of MSP adjacent to spermatozoa and lower levels distally. MSP receptors on the -1 oocyte appear to bind and concentrate MSP resulting in a sharp boundary of staining, which is eliminated when trafficking of oocyte membrane proteins is blocked. Confocal microscopy shows extracellular MSP to be punctate. We observe large (~100-200 nM) MSP-staining puncta near spermatids or spermatozoa in the uterus. We speculate that these puncta are MSP-containing vesicles. Using immuno-EM we examined MSP localization in spermatozoa to pinpoint its localization in the cell. In addition to localization of MSP within the pseudopod, we also found MSP located in the cell body. Interestingly, we found a fraction of MSP associated with the plasma membrane of the spermatozoa. Based on these results we propose a model in which spermatids and spermatozoa shed vesicles containing MSP. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical tests of this model are underway.

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Sternberg, Paul, Stein, Lincoln, Kersey, Paul, Harris, Todd, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.

Huan Wang et al. (2003) International Worm Meeting "Y110A2AL.14 is a glycosyltransferase that is important for proteoglycan tetrasaccharide linker biosynthesis."

Mark Sullivan, Fred Hagen, Huan Wang

[International Worm Meeting, 2003]

Galactosyltransferases modify carbohydrate chains that are attached to glycoproteins, proteoglycans, and glycolipids. Seven putative C. elegans genes have sequence motifs indicative of -1,3-galactosyltransferase function. Through an RNAi screen, one of these genes, Y110A2AL.14, was found to be required for early embryonic development. Sequence similarities using the BLAST program suggest that Y110A2AL.14 is a C. elegans homolog to human Galactosyltransferase II (GalT2) in the proteoglycan biosynthesis pathway. This cDNA encodes a type II membrane protein of 330 amino acids, shows 57 % amino acid similarity to human GalT2 and contains a DXD sequence motif that is potentially critical for enzymatic activity. A recombinant GalT2 fusion protein was expressed in insect cells and was purified to homogeneity. In vitro enzyme assays are consistent with this protein having GalT2 function. Glycosyltransferase activity was specific for galactose, utilizing the sugar donor UDP-galactose, and the acceptor, a terminal -linked galactose residue. We conclude that C. elegans Y110A2AL.14 encodes the enzyme likely act to attach the third sugar in the tetrasaccharide linker (GlcA--1,3-Gal--1,3-Gal--1,4-Xyl--1-O-Serine) for glycosaminoglaycan attachment. Glycosaminoglycan chains like heparan sulfate, chondroitin sulfate and dermatin sulfate inpact many properties of cells and require this tetrasaccharide for attachment. Therefore, reduction of C. elegans GalT II levels using RNAi, indicate that embryonic development depends on the expression of glycosaminoglycans. The RNAi knock-down phenotype is stronger in the F2 generation, suggesting that GalT2 is a maternal protein stored in oocytes at the time of fertilization. High yields of purified recombinant GalT2 has allowed us to produce single-chain anti-GalT2 antibodies to localize GalT2 in developing oocytes, embryos and larva. Y110A2AL.14 has recently been identified as sqv-2 by Hwang, Olson, Brown, Esko and Horvitz (JBC, in press)

Olson, Sara K. et al. (2013) International Worm Meeting "A semester-long investigative lab provides an authentic research experience in the cell biology of C. elegans embryos."

Olson, Sara K., Morgens, David

[International Worm Meeting, 2013]

Undergraduate biology education should provide students with authentic research experiences that allow them to think and act as scientists. The recent push to replace "cookbook" labs with inquiry-based investigative projects provides students with a more authentic research experience. At Pomona College, upper and lower division courses contain investigative laboratory components that typically last 1-4 weeks. In the fall semester of 2012, we piloted an investigative lab in an Advanced Cell Biology course that lasted an entire semester and was based on the research interests of the Olson Lab - C. elegans eggshell formation. Each lab group was assigned a different protein that was identified in a previous RNAi screen for genes involved in eggshell formation. None of the proteins were previously characterized, though worm strains expressing mCherry fusions were created prior to the start of the semester. Students spent the first 8 weeks conducting cell biological experiments on their assigned protein, learning methods such as worm picking, co-IP, mass spectrometry, Western blotting, RNAi, IF, and live cell fluorescence microscopy. Students then wrote a paper in the style of a journal article that highlighted their main findings, suggested a model for how their protein was involved in eggshell formation, and proposed future experiments that took into account current literature and classmates' data. Students then spent the next 5-6 weeks conducting experiments to address their hypotheses and proposed future directions. The students showed characteristics typical of dedicated scientists, such as taking ownership of "their proteins", returning voluntarily at night and on weekends to complete experiments, and being critical/skeptical of experiments conducted by other groups that did not meet perceived standards of experimental rigor. The investigative lab was a win-win for everyone. Students developed practical skills and learned what being a scientist is really like, while our research lab benefitted through generation of preliminary data for new projects and the recruitment of 5 new students to the lab (out of 20 enrolled in the course).

Swenson SI et al. (1995) International C. elegans Meeting "THE STEROID RECEPTOR HOMOLOGUE GENE, nhr-1, IN C. ELEGANS AND C. BRIGGSAE"

Eagen RM, Davison PJ, Swenson SI, Honda BM, Purac A

[International C. elegans Meeting, 1995]

We are studying the nhr-1 (nuclear hormone receptor) gene, one of a number of steroid receptor homologues identified in C. elegans. Sequencing of the cDNA has shown interesting features for this putative "orphan" receptor, including an unusual sequence in the P box of the DNA binding domain, which might indicate a very different DNA binding specificity and function, and a very long 3' untranslated region. The nhr-1 gene has been mapped to the right arm of the X chromosome, very close to sup-10. We are now characterizing the genomic organization of nhr-1 in C. elegans. We have also initiated work with C. briggsae in order to identify conserved, functionally important regions. The presence of an apparent homologue in C. briggsae was of special interest to us, given the unusual properties of nhr-1 (different P box, the long 3'UT region), and the apparent viability of homozygous deficiencies in this region around sup-10 (C. Cummins and P. Anderson). The C. elegans nhr-1 gene appears to contain 12 exons spread over 9.5 kb, including several relatively large introns, 1.3 to 2.3 kb in size. Work to date with genomic clones from C. briggsae shows conserved exons which share approximately 81-84% homology at the nucleotide level with the nhr-1 gene in C. elegans. This conservation between species, in the absence of a lethal phenotype for the homozygous deficiency, suggests that the nhr-1 gene may nevertheless have an evolutionarily relevant function. We wish to thank Ann Sluder, Gary Ruvkun, Claudia Cummins and Dave Baillie et al. for their assistance, and Alan Coulson et al. for help with mapping clones. Supported by a Research Reorientation Associateship (NSERC Canada) to SS, and an NSERC Operating grant to BMH.

John Willis et al. (2001) International C. elegans Meeting "PHYSIOLOGICAL ROLE OF K-CL COTRANSPORTERS IN C. ELEGANS"

John Willis, Edward Kipreos

[International C. elegans Meeting, 2001]

In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. This plays several documented roles: regulation of cytoplasmic chloride concentration, reduction of cell size in development, defense against cell swelling under hypoosmotic and isoosmotic conditions. The last, which includes the defense against swelling during moderate warming due to hyperactivity of the Na-K pump, has received the least attention but may be the most physiologically relevant. The gene sequences for two mammalian isoforms of this cotransporter were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (KO2A2.3, "KO2", on chromosome 2, and R13A1.2, "R13", on chromosome 4). Both C. elegans isoforms are expressed,as demonstrated by their presence in EST's and cDNA libraries. K02 has been shown to have K-Cl cotransport activity when transfected into mammalian cells. We have carried out dsRNAi blockages against both of these genes, singly and in combination, and found that about a third of the injected parents produced progeny that exhibit reduced survival at elevated temperature (33degC and 37degC) and increased sensitivity (seen as immobilization) to isoosmotic challenge induced by exposure to 100 or 200 mM ammonium chloride in M9 medium. A deletion mutant of K02 obtained from Gary Moulder at U. Oklahoma has not shown a phenotype by these tests and is now being used as a target for RNAi knockout of the R13 gene. GFP's expressed from upstream sequences of the two genes indicate that both isoforms are consistently expressed in pharyngeal muscle. The extent to which either is expressed elsewhere is still being determined. These studies may lead to establishing the role of K-Cl cotransporters in the whole organism and lay the foundation for a genetic analysis of their control

Aaron Bender et al. (2006) Development & Evolution Meeting "lin-35/Rb and spr-1/CoREST function redundantly to promote proper vulval and gonad morphogenesis"

David Fay, Aaron Bender

[Development & Evolution Meeting, 2006]

Using a previously described genetic screen (Fay et al., 2002), an allele of spr-1(fd6) was found to interact genetically with the C. elegans pocket protein ortholog, lin-35/Rb. lin-35; spr-1 double mutants display defects in germline as well as somatic gonad development, whereas neither single mutant is strongly affected. spr-1 was initially identified through its ability to suppress the egg-laying defective (Egl) phenotype of sel-12 presenillin mutants (Eimer et al., 2002; Jarriault and Greenwald, 2002). It is thought that SPR-1 functions as part of a multimeric complex known as CoREST to negatively regulate Notch signaling. Interestingly, RNAi of genes encoding other members of the complex causes similar synthetic defects with lin-35, indicating some degree of redundancy between the CoREST and LIN-35 repressor complexes. lin-35; spr-1 double mutants possess small morphologically aberrant gonads and contain reduced numbers of germ cells and cellularized oocytes compared with wild-type and single mutant animals. Additionally, lin-35; spr-1 mutants show a collapse of the vulval lumen that is characteristic of the squashed vulva (Sqv) phenotype. This collapse of the lumen is not due to cell lineage defects or alterations in the fates of vulval cells. Furthermore, unlike previously studied Sqv mutants, lin-35; spr-1 mutants do not show a pronounced attenuation of chondroitin biosynthesis, suggesting a distinct underlying cause for the Sqv phenotype in these animals (S. Olson and J. Esco, personal comm.). We have also identified a triple-synthetic phenotype between lin-35, spr-1, and a putative target of CoREST, the presenillin hop-1. Loss of all three genes leads to a Proximal germline proliferation (Pro) phenotype, whereby cells in the proximal germline fail to undergo meiosis resulting in a germline tumor. We are currently investigating the underlying mechanistic basis for this phenotype, including roles for Delta-Notch signaling and the somatic gonad.

AF Dernburg et al. (1999) International C. elegans Meeting "Getting Intimate with the Right Partner"

AF Dernburg, AM Villeneuve

[International C. elegans Meeting, 1999]

A unique and ubiquitous feature of sexual reproduction is the pairing between homologous chromosomes that occurs during meiosis. This intimate association allows partner chromosomes to undergo genetic exchange, which is in turn essential for accurate chromosome segregation at the first meiotic division. Although this process has intrigued biologists for decades, many fundamental questions about its mechanism remain; e.g., how do chromosomes first encounter their homologs? Once physical contact is made, how is their homology recognized? Using FISH and high-resolution 3-D microscopy, we can visualize chromosome pairing at specific chromosomal loci within intact gonads. Since the entire temporal sequence of meiosis is present within each adult worm, this approach quickly revealed the stage at which meiotic pairing occurs. Combined with reverse genetics, these cytological methods have resolved another crucial question: Conflicting results from yeast and Drosophila had fueled a debate as to whether recombination is essential for meiotic chromosome pairing. By knocking out a gene, spo-11 , whose function is necessary to initiate recombination (in collaboration with Mike Dresser, Gary Moulder, and Bob Barstead) we abolished all meiotic exchange, but found that pairing and synapsis were unperturbed in this mutant. This study demonstrated pairing can occur in the absence of recombination in a metazoan organism, and that this paradigm is probably extremely widespread. We are now focusing on the role of "pairing centers" in meiotic chromosome pairing and synapsis. Cytological examination of worms lacking X -chromosome pairing centers has revealed that these distal regions are indeed essential for intimate association between the X s. We are testing whether synaptonemal complex components can associate with defective X chromosomes. Two additional approaches have enabled us to perturb normal homolog pairing: First, genetic screens for Him mutants have uncovered trans-acting factors involved in pairing (see MacQueen abstract). Another strategy is to study rearrangements, which require the pairing machinery to contend with chromosomes that do not share homology along their entire lengths. We are currently testing the long-standing assumption that balancer chromosomes suppress recombination by disrupting pairing between homologous sequences.

Monsalve, G.C. et al. (2009) International Worm Meeting "Timing the Molting Cycle."

Monsalve, G.C., Frand, A.R., Davie, J.

[International Worm Meeting, 2009]

Nematodes develop through periodic molts but molecular mechanisms that set the pace of the molting cycle are not well understood. Molting involves detachment of the old exoskeleton from the underlying hypodermis and synthesis of the new exoskeleton, an extracellular matrix composed largely of collagens. Epidermal stem cells divide in sync with the first three molts but terminally differentiate at the final molt. A period of behavioral quiescence (lethargus) accompanies remodeling of the exoskeleton. Animals subsequently use specialized movements to escape the old skeleton (ecdyse). Improper coordination of these cell biological and behavioral events can be fatal. We aim to identify genes and molecules that regulate the timing of the molting cycle. The best-characterized biological timers control circadian rhythms in the physiology and behavior of humans and other animals. In theory, worm counterparts of the core circadian clock proteins might also regulate the ultradian rhythm of the molting cycle. We are therefore investigating the role of clock gene homologs in the reiterative molecular and behavioral events characteristic of molting. As a complementary approach, we conducted a forward genetic screen for mutants that molt prematurely, using expression of the mlt-10p::gfp-pest reporter to indicate synthesis of a new cuticle. In a pilot screen of approximately 18,000 genomes, we indentified one candidate that expressed the mlt-10 reporter too early and then partly shed the L1 exoskeleton. This particular animal perished, possibly due to the exposure of an incomplete L2 skeleton to the environment. In ongoing work, a clonal screen will allow the recovery of lethal mutations from siblings of affected animals. To further investigate the physiologic regulation of the molting cycle, we have cloned and characterized new mutations that cause supernumerary molts after puberty. These alleles emerged from a genetic screen conducted by Alison Frand and Sascha Russel in the laboratory of Gary Ruvkun. By studying the regulation of the molting cycle, we expect to uncover novel but conserved mechanisms for the endocrine control of development and behavior.

Sze JY (2000) West Coast Worm Meeting "Transcriptional regulation of the tryptophan hydroxylase gene tph-1"

Sze JY

[West Coast Worm Meeting, 2000]

tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. When I was in Gary Ruvkun's lab, I collaborated with Martin Victor in Yang Shi's lab and isolated a tph-1 deletion mutation, tph-1(mg280). tph-1(mg280) shows no detectable serotonin based on anti-serotonin antibody staining and exhibits several behavioral and metabolic defects (Sze et al., 2000). It has been proposed that in mammals changes in the level of TPH mRNA correlate with long-term changes in the cellular serotonin level and that the regulation of TPH mRNA tends to be "inducer"- and region-specific (Semple-Rowland et al., 1996; Clark and Russo, 1997; Siuciak et al., 1998. Bethea, et al., 2000). Thus, the regulation of TPH expression may be a key step by which the nervous system adjusts its long-term synaptic serotonin levels. To identify genes regulating tph-1, I have started a genetic screen for mutations that alter tph-1::gfp expression in specific neurons. Thus far, mutations identified can be classified into three classes. (1) I have previously reported that mutations in the POU-homeodomain transcription factor UNC-86 abolish tph-1::gfp expression in NSM and HSN, but the expression in ADF is not affected. UNC-86 is expressed in HSN and NSM, but not in ADF. (2) I have identified 18 mutations where tph-1::gfp expression in ADF is reduced or eliminated. Laser ablation of ADF and ASI causes animals to form dauers (Bargmann and Horvitz, 1993; Schackwitz et al., 1996) and the tph-1(mg280) mutation downregulates daf-7::gfp expression and enhances daf-7(e1372) dauer formation at 15oC. To test the role of ADF-produced serotonin is important for a non-dauer growth, I am testing if these mutations enhance daf-7(e1372) dauer formation at 15oC. (3) I have identified one mutation that has tph-1::gfp expressed in an extra neuron. These mutants provide me as useful means to characterize the role of serotonin produced in specific neurons.