Wilkinson HA et al. (1993) International C. elegans Meeting "Molecular characterization of the lin-12 gene."

Wilkinson HA, Greenwald IS

[International C. elegans Meeting, 1993]

Wilkinson HA et al. (1991) International C. elegans Meeting "1991 LIN-12 UPDATE."

Kao G, Greenwald IS, Grant BD, Yochem JJ, Wilkinson HA, Fitzgerald K

[International C. elegans Meeting, 1991]

On this poster, we will report on our progress in a number of studies of lin-12 and associated genes that will not be covered in other presentations from our laboratory. In particular, we are currently examining transformed lines containing various lin-12--1acZ fusion constructs and generating monoclonal and anti-peptide antisera in order to characterize the expression and distribution of l in- 12 mRNA and protein products. We have also begun to clone certain sel genes and to construct a full-length lin-12 cDNA for various manipulations. We will also report on the various EGFrelated genes that we have isolated using lin-12 and glpI probes.

Wilkinson HA et al. (1995) International C. elegans Meeting "CLONING AND CHARACTERIZATION OF THE IVERMECTIN RECEPTOR IN FILARIAL NEMATODES"

Cully DF, Paress PS, Schaeffer JM, Wilkinson HA

[International C. elegans Meeting, 1995]

Ivermectin is a broad spectrum nematocidal agent which is widely used for the treatment of various filarial nematode diseases including dog heartworm (Dirofilaria imitis) and onchocerchiasis (Onchocerca vulvulus) in humans. The treatment for O.vulvulus requires yearly dosing due to the apparent insensitivity of the adult worms to ivermectin. One of the objectives of this study is to clone and characterize the filarial ivermectin receptor. Two ivermectin-sensitive glutamate-gated chloride channels have been cloned from C. elegans. Our approach has been to use degenerate oligonucleotides and the polymerase chain reaction (PCR) technique on cDNA made from D. imitis poly-A+ RNA since we can predict the exact size of the PCR product. Using degenerate primers located in a conserved region in the extracellular domain with primers in the first transmembrane spanning domain we have identified a PCR clone from D. imitis that appears to encode a member of the family of ivermectin-sensitive glutamate-gated chloride channels. This clone is 66% identical to the alpha subunit of the C.elegans ivermectin-sensitive chloride channel and 60% identical to the beta subunit of this channel. We are currently using this PCR clone to identify a full-length cDNA clone from a D. imitis cDNA library and we are also performing a low stringency screen to obtain a cDNA clone from an O.vulvulus cDNA library. Using similar primers we have also identified a third member of this family in C. elegans and are further characterizing this clone.

Greenwald IS et al. (1989) International C. elegans Meeting "everything else you wanted to know about lin-12."

Seydoux GC, Sundaram MV, Greenwald IS, Wilkinson HA, Yochem JJ, Rubens D

[International C. elegans Meeting, 1989]

Killeen MT et al. (1997) International C. elegans Meeting "Structure/function analysis of the C.elegans pioneer axon guidance molecule, UNC-5"

Krizus A, Merz DC, Nygiem M, Culotti JG, Killeen MT, Scott IM

[International C. elegans Meeting, 1997]

UNC-5 is required for guidance of the growth cones of pioneer motor axons and distal tip cells that migrate in the dorsal direction in C. elegans. It contains two immunoglobulin and two thrombospondin type I domains and its cytoplasmic region is largely novel (Leung-Hagesteijn et al., Cell, 71: 289-299, 1992). We hypothesize that UNC-5 is part of a cascade that is triggered by UNC-6 (netrin) which is present as a gradient in C. elegans (Wadsworth et al. Neuron, 16: 35-46, 1996) and may repel axons which express UNC-5 and UNC-40 (another putative receptor; Chan et al., Cell, 87: 187-195, 1996) while attracting those which express UNC-40 alone. To understand the spatial and temporal localization of UNC-5 we have made a HA tagged UNC-5 protein. This rescues the uncoordinated phenotype and we visualize UNC-5::HA by immunohistochemistry using anti-HA. The rescued strains express UNC- 5::HA in the ventral nerve cord and in motor axons affected by unc-5 mutations. Expression of UNC-5::HA in the distal tip cells is entirely coincident with their migration in the dorsal direction in the animal, suggesting that UNC-5 may be an instructive molecule for this migration. We have made deletions of portions of UNC-5 to establish the functional roles of the various protein structural domains. Most existing unc-5 mutants have nonsense codons that result in prematurely terminated products, splicing defects resulting in out of frame fusions, or gross rearrangements. Two mutants have missense mutations, one of a conserved cysteine to tyrosine and another of a leucine to phenylalanine in one of the immunoglobulin domains. The phenotypes of these mutant strains are being further characterized. This study will lead to a better understanding of the roles of the structural domains of the UNC-5 protein and will permit a clearer understanding of how this instructive molecule works.

K Kariya et al. (1999) International C. elegans Meeting "PLC210, a conserved Ras/Rap1A-associating phospholipase C is required for fertility in C. elegans"

T Kataoka, Y Liao, C Song, K Kariya, M Shibatohge, X Gao

[International C. elegans Meeting, 1999]

We have isolated a 210 kDa C. elegans phospholipase C (Ce-PLC210) through a two-hybrid screening for LET-60/Ras-binding proteins (WBG 14 #5, 34, 1997, J. Biol. Chem. 273, 6218, 1998). We have recently isolated its human homolog (Hs-PLC210) as well. Both of them possessed the X, Y and C2 domains highly homologous to those of the phosphoinositide-specific PLC (PI-PLC) and hydrolyzed phosphatidylinositol 4,5-bisphosphate in vitro . However, PLC210 possessed two additional functional domains unseen in any known PI-PLCs. One was the C-terminal Ras-associating (RA) domain bearing a structural homology with those of RalGDS and AF-6. This domain associated in vitro with human Ha-Ras and Rap1A in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other was the N-terminal CDC25-like domain, which possessed a structural homology to guanine nucleotide exchange proteins for Ras such as S. cerevisiae CDC25 and D. melanogaster Sos. Deletion mutations of the PLC210 gene in C. elegans resulted in sterility similar to that of a mutation of the gene for LET-23/EGF receptor, and the phenotype was rescued by transgenic expression of the wild-type PLC210 gene. These results suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras or Rap1A conserved through evolution.

Myeongwoo Lee et al. (2001) International C. elegans Meeting "Integrin function is required for gonad morphogenesis and ovulation in Caenorhabditis elegans"

Erin Cram, Myeongwoo Lee, Jean E Schwarzbauer, Bing Shen

[International C. elegans Meeting, 2001]

Extension of the hermaphrodite gonad arms in the nematode C. elegans depends on migration of distal tip cells along the body wall extracellular matrix (ECM). The migratory path is defined by UNC-6/netrin, UNC-129/TGF- b , matrix metalloproteases, and intracellular signaling through CED-10/Rac. Integrin receptors, which link the ECM and intracellular signaling, play a significant role in cell migration during vertebrate development. To determine the contribution of integrins to gonad morphogenesis, we inhibited the activity of the endogenous b pat-3 integrin. Transgenic nematodes were generated that express an HA- b tail transgene consisting of the b pat-3 transmembrane and cytoplasmic domains connected to a heterologous extracellular domain. High levels of HA- b tail expression in muscle and gonad caused transdominant inhibition of endogenous integrins as shown by induction of a paralyzed, arrested-at-two fold (Pat) embryonic lethal phenotype similar to the pat-3 null phenotype. Lower levels of transgene expression caused a small percentage of animals to exhibit uncoordinated (Unc) movement, disorganized muscle filaments, reduced egg-laying, aberrant migration of gonadal distal tip cells along the dorsal ECM, and accumulation of oocytes in the ovary. Conserved tyrosine residues in the beta integrin cytoplasmic domain regulate fibroblast chemotaxis in vitro. Mutation of these tyrosines did not alter the effects of HA- b tail on gonad migration, but did increase embryonic lethality during early morphogenesis, suggesting a role for these residues in early development. In order to investigate a possible link between integrins and Rac signaling, we constructed double mutants with HA- b tail transgenic animals expressing low levels of the transgene (mwIs25) and nematodes expressing mutant alleles of either ced-5 (DOCK180) or unc-73 (Rac-GEF), both of which normally activate Rac. Ced-5 mutant animals are defective in cell corpse engulfment and migration of the distal tip cells. Weak unc-73 mutant alleles result in defects in axon guidance and pathfinding. These phenotypes suggest a possible interaction of cell adhesion receptors and Rac signaling. Ced-5 (n1812); mwIs25 hermaphrodites have a dramatic increase in ovulation defects including prevalent endomitotic nuclei, whereas unc-73 (e936); mwIs25 hermaphrodites show defective muscle filament organization, including defects in vulval muscle. These results demonstrate an important role for integrins in gonadogenesis and early morphogenesis and suggest that integrin signaling through Rac contributes to gonad development and fertility in C. elegans .

Hanako Yashiro et al. (2007) International Worm Meeting "b (PAT-3) Integrin Plays Multiple Roles during Anchor Cell Invasion in C. elegans."

Hanako Yashiro, David R Sherwood

[International Worm Meeting, 2007]

Cell invasion through basement membranes (BM) is required in various developmental processes and is misregulated in diseases such as cancer. We investigate the underlying mechanisms regulating cell-invasive behavior by studying anchor cell (AC) invasion in C. elegans. During development, the gonadal AC invades through the BM to contact the underlying vulval precursor cells, which send a diffusible cue that guides AC invasion. In a whole-genome RNAi screen, we found that RNAi against both the <font face=symbol>b</font> integrin ina-1 and <font face=symbol>b</font>pat-3 caused defects in AC invasion. We also found that a loss-of-function allele in ina-1(gm39) showed a moderately penetrant invasion defect. Furthermore, in wild-type transgenic animals, PAT-3::GFP localization is increased at the basolateral invasive surface of the AC, suggesting a direct role in promoting invasion To further investigate the role of integrin, we have driven a dominant-negative construct of pat-3 (pAC&#62;HA-<font face=symbol>b</font>tail, a gift from Dr. Jean Schwarzbauer) specifically in the AC. In integrated lines of pAC&#62;HA-<font face=symbol>b</font>tail, AC invasion is severely disrupted. Examination of laminin::GFP, a BM marker, showed that the AC fails to breach the BM in pAC&#62;HA-<font face=symbol>b</font>tail worms and instead detaches from the membrane at the time of invasion. Notably, the AC fails to send cellular protrusions toward the vulval cells after detachment, suggesting an inability to respond to the vulval cue. Recent work in our lab has shown that the Rac protein MIG-2, F-actin, and PtdIns(4,5)P₂ localize to the basolateral membrane of the AC where they promote the formation of filapodia in response to the vulval cue. We have found that GFP::MIG-2 and PH::mCherry (a PtdIns(4,5)P₂-specific promoter) polarity at the invasive AC membrane is retained in pAC&#62;HA-<font face=symbol>b</font>tail animals as long as there is contact between the AC and the BM. Once the AC is detached, however, the polarization is significantly reduced, suggesting that integrin-mediated attachment of the AC to the BM is required to maintain polarity along the invasive membrane. Finally, we studied the loss of integrin in the AC on BM dynamics at the site of invasion by examining the localization of hemicentin::GFP. Hemicentin is a conserved extracellular matrix protein that is deposited under the AC at the site of invasion, where it promotes the movement of the AC through the BM. Strikingly, in pAC&#62;HA-<font face=symbol>b</font>tail worms, hemicentin is still made in the AC but is not deposited. Together, these results indicate that integrin function in the AC is crucial for establishing a properly organized invasive cell membrane and regulates the composition of the BM at the site of invasion.

Kariya K-i et al. (1997) International C. elegans Meeting "C. ELEGANS HOMOLOGS OF RALGDS, AF-6, CDC25 AND PHOSPHOLIPASE Cb INTERACT WITH LET-60"

Watari Y, Liao Y, Hu CD, Shibatohge M, Kataoka T, Kariya K-i, Goshima M

[International C. elegans Meeting, 1997]

Mammalian Ras proteins regulate multiple effectors including Raf family, RalGDS family and AF-6. To understand all signaling pathways regulated by Let-60 in C. elegans , we screened for Let-60-binding proteins by the yeast two-hybrid system (thanks to Dr. R. Barstead for the lACT-RB2 library). The screen identified partial cDNA clones encoding not only C. elegans Raf but also C. elegans homologs of RalGDS (Ce-RalGDS), of AF-6 (Ce-AF-6), of Cdc25 (Ce-Cdc25) and of phospholipase Cb (Ce-PLCb). Flanking cDNA sequences were obtained by the spliced leader sequence PCR or from Dr. Y. Kohara's EST clones. Ce-RalGDS contained the middle Cdc25 homology domain and the C-terminal Ras-interacting domain (RID) homologous to those of RalGDS. Ce-AF-6 contained the N-terminal RID and the middle GLGF/DHR motif homologous to those of AF-6. Ce-Cdc25 contained a proline-rich region, possible PH domain, and a Cdc25 homology domain similar to that of Cdc25Mm/RasGRF. Ce-PLCb contained PH, X, Y and C2 domains found in mammalian counterpart. However, the N-terminal Cdc25 homology domain (similar to those of Sos proteins) and the C-terminal RID (300 amino acids) of Ce-PLCb were unique. These proteins bound to mammalian Ha-Ras in the two-hybrid system, but the bindings were abolished by various mutations within the effector domain of Ha-Ras. MBP-fusion proteins of Ce-RalGDS RID and Ce-PLCb RID bound Ha-Ras in vitro in a GTP-dependent manner. To examine functions of the newly identified Let-60 effector candidates, worms carrying Tc1-insertions within the genes were isolated (for Ce-RalGDS, Ce-PLCb and Ce-AF-6), and deletion derivatives (only heterozygotes) were obtained (for Ce-RalGDS and Ce-PLCb). Phenotypic analyses will be presented if homozygotes are viable.

Marc D Perry et al. (1999) International C. elegans Meeting "Assays for interactions between HER-1 and TRA-2"

Roxana N Sultan, Nimerta Rajwans, Marc D Perry

[International C. elegans Meeting, 1999]

We are interested in how her-1 interacts with downstream genes in the sex determination pathway to specify cellular sexual fates. Given that the relative levels of HER-1 and TRA-2 affect the sexual fate decision of a cell (Kuwabara and Kimble, 1995 M.B.C. 121: 9), and that dominant tra-2(eg) alleles are insensitive to negative regulation by her-1 (Kuwabara, 1996 Devel. 122: 7), we wish to discern whether or not the large transmembrane protein pTRA-2A is the direct target of HER-1's masculinizing activity. We have engineered Sf9 insect tissue culture cells to express a construct encoding HIS-tagged HER-1 (Perry and Rajwans, 1997 11th Int. Worm Meeting) and are using the secreted protein in binding assays with mammalian COS cells which have been engineered to express a TRA-2-HA fusion protein. We have generated antibodies against HER-1 and are using these in conjunction with anti-HA antibodies and fluorescent secondary antibodies to detect co-localization of the two proteins. In parallel, we are using Kunkel mutagenesis to generate expression constructs encoding the tra-2(eg) allele in order to test the effects of this mutation on HER-1 binding. To correlate tra-2 function in vivo with its ability to bind HER-1 in vivo we will delimit the portions of TRA-2 which are essential to the interaction, by using truncated TRA-2 variants in binding assays with HER-1.