Harvey, L-M. et al. (2019) International Worm Meeting "Identification and characterization of new actors of the RNAi pathway."

Harvey, L-M., Simard, M.J., Kakumani, P.K.

[International Worm Meeting, 2019]

The RNA interference pathway (RNAi) uses either ectopic (exo-RNAi) or endogenous (endo-RNAi) short non-coding RNA molecules known as short interfering RNA (siRNA) to regulate gene expression. To this day, knowledge on this regulation pathway was mainly obtained with studies made in plants and nematodes where it has been described as important for the genome protection and stability against pathogens. In contrast, our understanding of this regulatory pathway in higher eukaryotes is underwhelming. In mammals, a strong presence of these endogenous siRNA (endo-siRNA) has been detected in embryonic stem cells (ESCs), but in opposition to plants and nematodes, their function is yet to be fully understood. In an effort to address this question, we recently identified specific proteins associated to the short interfering RNA induced silencing complex (siRISC) in mouse ESCs using a 2'-O-methyl pulldown of an endo-siRNA followed by mass spectrometry. Beside Argonaute proteins, we identified 10 proteins associated with the mouse siRNA that are also encoded in the C. elegans genome, suggesting that they may play a conserved role in the endo-siRNA pathway. Therefore, we initiated the characterization of these new siRNA interactors in C. elegans, a biological system in which the tools and reagents needed to study components of the endo-siRNA pathway are well established in contrast to mammalian cells. Several molecular and genetic approaches are currently used to quickly understand the implication of these new factors in the RNAi pathway. During this meeting, we will present our recent progress on the characterization of some of the identified proteins. At term, this characterization will contribute to define the role of new conserved actors in the endo-siRNA pathway and lead to a better understanding of the pathway in mammals.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Lackner MR et al. (1995) International C. elegans Meeting "The role of mpk-l in vulval development"

Kim SK, Lackner MR

[International C. elegans Meeting, 1995]

The mpk-l gene encodes a MAP kinase protein that is approximately 75% identical to vertebrate MAP kinases. Previously, we have shown that mpk-l plays an important role in ras-mediated induction of vulval cell fates, as reduction-offunction mutations in mpk-l completely suppress excess vulval differentiation caused by a gain-of-function mutation in let-60 ras. We have now characterized a strong reduction-offunction allele of mpk-l that results in temperature-sensitive vulvaless phenotype (84% Vul at 25 degrees Celsius) and a completely penetrant sterile phenotype at all temperatures. Epistasis analyses suggest that mpk-l acts downstream of lin-15 and let-60 ras, but upstream of the putative transcription factors lin-l and lin-31. To determine whether mpk-l is absolutely essential for vulval induction, or whether some induction may occur in the absence of mpk-l activity, we are in the process of isolating a null allele of mpk-l. We used mutant activated forms of mpk-l and Drosophila MEK to address the question of whether activation of these two kinases is sufficient to promote vulval fates in the absence of upstream signaling. Coexpression of both activated mpk-l and activated D-MEK causes a strong Muv phenotype (~90% Muv), whereas coexpression of wild-type mpk-l and wild-type D-MEK results in a weak Muv phenotype (~10% Muv). The strong Muv phenotype is unaffected by ablation of the anchor cell or a reduction-offunction mutation in lin-45 raf, consistent with activation of mpk-l and MEK being at least partially independent of upstream signaling. Our future plans are to identify targets of mpk-l by isolating suppressors of this strong Muv phenotype.

Mango SE et al. (1991) International C. elegans Meeting "A NOVEL glp-1 ALLELE MIMICS THE EFFECT OF lin-12(d) ON VULVAL DEVELOPMENT."

Kimble JE, Maine EM, Mango SE

[International C. elegans Meeting, 1991]

The lin-12 and glp-l genes encode homologous transmembrane proteins ( l) that may act as receptors for cell interactions during development ( 2,3). The glp-l gene is required zygotically for germ line proliferation and maternally for embryogenesis (2, 4); lin-12, in contrast, is involved in various postembryonic cell interactions in the soma, including those between vulval precursor cells (VPCs) that specify vulval fates (5). The novel allele glp-l (q35) has a semidominant Multivulva phenotype (Muv) in addition to the typical recessive Glp phenotypes. We find that the Muv phenotype of glp-l (q35) is similar to that of lin-12(d) in three ways. First, VPCs that would normally fuse with the hypodermal syncytium (3 fate) follow a 2 -1ike vulval lineage instead. Second, the Muv phenotype is not dependent on the anchor cell. Third, the Muv phenotype results from increased or novel, rather than decreased, glp-l activity. Because glp-l (q35) remains Muv in animals lacking lin-12, this effect does not depend on lin-12 activity. We conclude that the glp-l (q35) product is functionally very similar to that of lin-12. The glp-l (q35) gene bears a nonsense mutation predicted to truncate the glp-l protein 122 amino acids from its carboxy terminus. The region which should be missing from the glp-l (q35) protein includes a PEST sequence thought to destabilize proteins. A simple explanation of these results is that the carboxy terminus bears a negative regulatory domain that normally keeps glp-l activity off in the VPCs. In glp-l ( q35) animals, inappropriate glp-l activity may drive these cells to proliferate, and thereby channel them toward a 2 -like fate.

Schedl TB et al. (1991) International C. elegans Meeting "GLD-1, A GENE NECESSARY FOR MULTIPLE STEPS IN GERMLINE DEVELOPMENT"

Kimble JE, Barton MK, Maine EM, Kahn S, Schedl TB, Francis GR, Jones AR

[International C. elegans Meeting, 1991]

Three crucial aspects of germline development are the switch from proliferation to meiosis, sex determination, and gametogenesis. An important question is how these processes are controlled and coordinated. Mutations in previously identified genes effect only one of these processes. We describe a novel gene gld-l (defective in germline development, formerly called fog-3), in which mutations effect each of these processes in the hermaphrodite germline. Twenty- five mutations were isolated and demonstrated to be gld-l alleles by complementation tests, mapping, and intragenic reversion. 8 mutants are defective in the switch from proliferation to meiosis: homozygotes have a tumorous germline phenotype (Tum), with major proliferative regions proximally and diqtally, and no gametogenesis. gld-l(Tum) mutations are probably null as they behave like a deficiency in complementation tests. 11 other mutants are defective only in germline sex determination: homozygotes for 10 alleles display masculinization of the germline (Mog), while one allele displays feminization of the germline (Fog). The semi-dominant Mog mutations were shown to be gain- of-function alleleg of gld-l by intragenic reversion to gld-l recessive phenotypes at high frequency (1/2300 haploid genomes). 6 mutants are defective in gametogenesis: homozygotes for two alleles produce small oocytes that can not be fertilized, while 4 other alleles display germline nuclei arrested in meiotic prophase with no obvious signs of gametogenesis. Somatic phenotypes have not been observed. The role of gld-l in germline proliferation has been analyzed by: l) laser ablation of somatic gonad cells or their ancestors which promote germline proliferation [distal tip cell (DTC) or anchor cell (AC)], and 2) interactions in double mutants with glp-l (which causes premature entry into meiosis). Ablations demonstrate that gld-l proximal proliferation is independent of the AC or DTCs, but distal proliferation remains dependent on a DTC. The gld-l(Tum); glp-l(null) double mutant undergoes two additional rounds of proliferation followed by entry into meiosis and differentiation of sperm. As gld-l(Tum) can increase germline proliferation in the absence of the glp-l product, gld-l is likely to act downstream or on a pathway parallel to glp-l. However, full expression of the gld-l tumorous phenotype is dependent on some glp-l activity.

Rogalski TM et al. (1991) International C. elegans Meeting "GENETIC APPROACHES TO C. ELEGANS RNA POLYMERASE III."

Golomb M, Watson N, Lancy ED, Rogalski TM, Riddle DL, Albert PS

[International C. elegans Meeting, 1991]

RNA polymerase III (Rpo m) transcribes genes encoding SS and transfer RNA's. This enzyme and its associated factors have been studied extensively in other systems, but Rpo m function has not been studied genetically in a metazoan. Developmental regulation of Rpo m activity in C. elegans has been suggested by the molecular genetic analysis of nonsense suppressors (Kondo et al., J. Mol. Biol. 215:7-19, 1990). The gene for the largest subunit of Rpo m, rpc-l, has been cloned (Bird and Riddle, Mol. Cell. Biol. 9:4119-4130, 1989) and positioned on the physical map between unc44 and unc-24 on chromosome IV. The availability of cloned genes and an in vitro transcription system (Honda et al., Nucl. Acid Res. 14B:869-881, 1986) provides potential tools for a detailed analysis of Rpo III structure and function using mutants, if appropriate mutants can be identified. Two approaches to genetic analysis of rpc-l have been employed. The first approach utilized resistance to the fungal toxin, ct-amanitin. A unique strain with an extremely amanitin-resistant Rpo II (Rogalski et al., Genetics 126:889-898, 1990) was used as a parent to select even higher levels of resistance. One such mutation was mapped on chromosome IV, and preliminary nuclear run-on assays indicate that Rpo III is abnormally resistant to amanitin, in appropriate recombinants derived from this mutant strain. The mutant Rpo III transcribes SS RNA in the presence of 200 g/ml amanitin, whereas wild-type Rpo m is inhibited by 25 g/ml. These results suggest that the resistance mutation affects Rpo III, and it may be within the rpc-l gene. A second approach to rpc-l genetics utilized a collection of EMSinduced lethal mutations generated by Denise Clark. We mapped fourteen of her lethal mutations to the interval between unc44 and unc-24, a region estimated to contain approximately 30 essential genes. These lethal mutations are now being assigned to complementation groups. Lethal derivatives of the putative amanitin-resistant rpc-l allele will also be sought, and selected lethal strains will be injected with the cloned rpc-l gene in an effort to identify the genetic correlate to rpc-l by DNA transformation. Genetic, phenotypic, and biochemical analysis of these mutants may provide a useful entree into structure- function relationships in this important enzyme.