Lambie, Eric et al. (2021) International Worm Meeting "Examination of P5B ATPase function in vivo"

Lambie, Eric, Baumbach, Alexander, Zhou, Zhiyao

[International Worm Meeting, 2021]

P5B ATPases are evolutionarily conserved transporter proteins whose principal function is to pump polyamines from extracellular or vesicular spaces into the cytosol. The C. elegans genome encodes three paralogous P5B ATPase transporter proteins, CATP-5, CATP-6 and CATP-7. Each of these proteins has a distinct expression pattern and is likely to have a specific physiological function. For example, CATP-5 localizes to the intestinal brush border and is required for uptake of polyamines from the intestinal lumen. CATP-7 localizes to the basolateral membrane of the excretory cell. Since catp-7(0) mutants arrest when grown on polyamine-supplemented plates, CATP-7 probably pumps polyamines from the pseudocoelomic fluid into the excretory cell cytosol. CATP-6 localizes to endosomal vesicles and may be required to purge these of residual polyamines derived from the extracellular space. In order to better understand when and where the P5B ATPases are active, we are developing tagged versions that exhibit altered fluorescence properties in response to conformational changes. We are also performing screens to search for genes required for the transport of polyamines in the opposite direction, from the cytosol into the extracellular/vesicular space.

Eric Lambie et al. (2003) International Worm Meeting "Identification of genes that interact with gon-2"

Rachel West, Eric Lambie, Diane Church, Murline Gelin, Wenhua Wu

[International Worm Meeting, 2003]

gon-2 encodes a TRP-family cation channel whose function is required for the post-embryonic divisions of the gonadal precursors (1). In order to better understand how gon-2 is regulated and how it regulates the cell cycle, we have screened for extragenic suppressors of the hypomorphic allele, gon-2(q388). In this screen, we identified four loci, gem-1, 2, 3 and 4 (gon-2 extragenic modifier). We have determined that gem-4 encodes a member of the copine family. Copines were originally identified based on their ability to bind to phosphatidylserine-containing membranes in a Ca2+-dependent manner (2). The suppressor alleles of gem-4 all appear to be loss-of-function mutations. This suggests that gem-4(+) antagonizes gon-2(+). gem-4 mutations do not rescue gonad development when gon-2 activity is eliminated by gon-2[RNAi] in a gon-2(q388) background. Therefore, gem-4 acts either upstream of gon-2 or in a parallel, non-essential, negative regulatory pathway. As an independent method for identifying antagonists of gon-2 activity, we have begun screening the Fraser et al. (3) RNAi feeding library for clones that can suppress gon-2(q388). Unexpectedly, many of clones that suppress gon-2(q388) encode components of the protein synthesis machinery. The significance of this is not clear; however, we are currently testing whether these clones are able to suppress gon-2(0) and whether cycloheximide treatment can also suppress gon-2(q388). A progress report on the characterization of the various suppressor loci will be presented at the meeting.1. West et al., 2001. Gene 266:103; 2. Creutz et al., 1998. JBC 273:1393; 3. Faser et al., 2000. Nature 408:325.

Eric J Lambie et al. (2001) International C. elegans Meeting "gon-2 and interacting genes."

Diane L Church, Marc A Ginsburg, Eric J Lambie, Ian A White, Rachel J West

[International C. elegans Meeting, 2001]

gon-2 encodes a predicted cation channel belonging to the TRP superfamily. Loss-of-function mutations in gon-2 severely impair the mitotic divisions of the gonadal precursors. Our working hypothesis is that gon-2 functions within Z1 and Z4 to allow the influx of cations (probably Ca 2+ ), which then stimulate G1 progression. In order to identify genes that act in conjunction with gon-2 , we have screened for extragenic revertants of the temperature sensitive allele, gon-2(q388) . To date, we have identified four distinct gem loci ( g on-2 e xtragenic m odifier) that can mutate to suppress gon-2(q388) . gem-1 X and gem-4 IV are represented by multiple alleles, whereas gem-2 II and gem-3 III are represented by a single allele each. We are currently performing SNP mapping and transformation rescue assays to determine the molecular identities of the gem loci. As another means of identifying genes that interact with gon-2 , we screened for mutations that cause similar gonadogenesis defects. gon-11(dx5) is a recessive, temperature sensitive allele that produces a phenotype similar to that of gon-2(q388) . dx5 has relatively low penetrance, so it is only marginally useful. Therefore, we used non-complementation screening to search for more robust alleles of gon-11 . This resulted in the identification of gon-11(dx106) , which causes fully penetrant late embryonic lethality. We are currently using dx106 for SNP mapping and transformation rescue assays. This work is supported by NIH RO1 GM49785

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

Memar, Nadin et al. (2021) International Worm Meeting "The loss of psf-2 GINS leads to the inappropriate survival of cells programmed to die during C. elegans development"

Schnabel, Ralf, Conradt, Barbara, Memar, Nadin, Sherrard, Ryan, Lambie, Eric

[International Worm Meeting, 2021]

The genetic pathway required for programmed cell death during C. elegans development is highly conserved. The key activator of this pathway is the pro-apoptotic BH3-only gene egl-1. Earlier studies showed that egl-1 activity is controlled at the level of gene expression. Specifically, egl-1 expression is controlled both at the transcriptional level through lineage-specific transcription factors and at the post-transcriptional level through microRNAs. In this study, we present evidence that egl-1 expression is controlled at an additional level. The gene psf-2 encodes a subunit of the GINS complex, which is essential for DNA replication. We identified a temperature-sensitive loss-of-function mutation of psf-2, t3443ts, and found that it does not only cause a cell cycle defect but a general block in cell death i.e. a Ced phenotype. Furthermore, we provide evidence that the Ced phenotype exhibited by psf-2(t3443ts) mutants is independent of these animals' cell cycle defects. Based on these observations we propose that psf-2 GINS is required for programmed cell death during C. elegans development. To determine whether psf-2(t3443ts) affects the expression of egl-1, we quantified the number of egl-1 transcripts in cell death lineages i.e. lineages in which a cell death occurs. We found that psf-2(t3443ts) abolishes a spike in the number of egl-1 transcripts normally observed in cells programmed to die. This suggests that the loss of psf-2 GINS blocks programmed cell death by abolishing the transcriptional up-regulation of egl-1in cells programmed to die. Based on our findings, we propose that the GINS complex is necessary for changes in chromatin state at the egl-1 locus that permit the transcriptional up-regulation of the egl-1 gene in cells programmed to die.

Memar, Nadin et al. (2022) MicroPubl Biol

Memar, Nadin, Lambie, Eric J, Sethi, Aditya, Luehr, Sebastian, Conradt, Barbara

[MicroPubl Biol, 2022]

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated.

Ragnauth CD et al. (2000) European Worm Meeting "Molecular and functional studies on novel putative calcium channel protein in C. elegans"

Ragnauth CD, Baylis HA, Kwan CSM

[European Worm Meeting, 2000]

Ionized calcium is unique as a second messenger in that it cannot be metabolized and hence cells must regulate intracellular levels though an array of storage compartments and specialized binding and gating proteins. The molecular nature of many of the Ca2+ channels identified by physiological approaches remains unknown (1). The sequencing of the C. elegansgenome has added a new resource into the investigation of the molecular basis of Ca2+ signalling. Analysis of the C. elegans genome sequence has identified three families of proteins distantly related to the TRP family of cation channels. TRP channels are thought to gate Ca2+influx in response to Ca2+ store depletion and/or other signalling molecules (2). Of these families, the MGCs (Melastatin and GON-2 like channels) are widely distributed and expressed in nature with several homologues in man, Drosophila and mouse. In C. elegans, there are 3 members of the MGC family: gon-2, which is required for gonadogenesis (3 and E Lambie, pers comm.) and two further channels, C05C12.3 and F54D1.5. Like TRP channels, MGCs show similarity to the pore regions of voltage gated Ca2+ channels. At the amino acid level they are approximately '-24% identical to TRP channels whilst identity to each other and human homologues is 36-40%. Our working hypothesis is that they are Receptor Activated Ca2+ Channels (RACCs) and/or Store Operated Channels (SOCs). We are currently characterising C05C12.3 and F54D1.5. Using 5 RACE we determined the 5 ends of the mRNAs. We cloned the full length cDNAs of C05C12.3 and F54D1.5. This information was used to design GFP fusion constructs to determine expression patterns. Confocal microscopy shows that both proteins are strongly expressed in the intestine and F54D1.5 is also expressed in head neurons. In situ hybridization of C05C12.3 confirms this result. The expression pattern for gon-2 has not yet been determined (E Lambie, pers comm.). Currently we are using RNAi to specifically decrease the expression of the MGC genes in order to further characterise the function of these proteins. References: 1) Parekh A. B. and Penner, B. (1997) Physiol. Rev. :902-930 2) Barritt, G. J. (1999) Biochem. J. 337:153-169 3) Sun, A.Y. and Lambie, E. J. (1997) Genetics g:1077-1089

Ginsburg MA et al. (1994) Worm Breeder's Gazette "dx5 and Various Aspects of Gonadogenesis."

Ginsburg MA, Lambie EJ

[Worm Breeder's Gazette, 1994]

dx5 and various aspects of gonadogenesis. Marc Ginsburg and Eric Lambie. Department of Biological Sciences, Dartmouth College, Hanover NH 03755. One lucky day, we found a W-induced mutation (dx5) that maps to IR and has strikingly obvious gonadogenesis defects. First, the dx5/dx5 progeny of a dx5/+ hermaphrodite often have proximal germline proliferation in one (and sometimes both) of the gonad arms. Despite such an abnormality, all of these animals are fertile. This phenotype is reminiscent of that seen in lin-12(0) mutants, in which the anchor cell(s) induce proximal germline proliferation. However, dx5 animals do not exhibit either the two-anchor-cell phenotype or vulva morphogenesis defects characteristic of lin-12(0) mutants. To determine whether the anchor cell is responsible for inducing proximal proliferation in dx5 animals, we tested the effect of lin-12(n302), which prevents specification of the anchor cell fate. None of 20 Unc progeny of dx5 unc-75(e950)/ + + I; lin-12(n302) III hermaphrodites had proximal proliferation. 19/49 Unc progeny of dx5 unc-75(e950)/+ + controls had proximal proliferation. This strongly suggests that the anchor cell is the culprit; however, we'll have to do some laser ablation experiments to be certain. The second glaringly obvious effect of dx5 is seen among the progeny of dx5/dx5 homozygotes. These animals have very small gonads and are usually vulvaless (presumably due to lack of an anchor cell). More detailed inspection will be necessary to determine exactly which cell divisions have gone awry. We are optimistic that we can make good progress towards determining the normal function of the gene defined by dx5. For one thing, the dx5 mutation is temperature sensitive, which should permit the isolation of additional alleles by clonal noncomplementation screening. Another good thing is that dx5 maps very close to lin-11 (probably to the left); this region is fairly well-covered on the physical map by cosmids and YACs.

Hollopeter, Gunther et al. (2015) International Worm Meeting "The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex."

Hollopeter, Gunther, Jorgensen, Erik, Lambie, Eric, Slaughter, Brian, Zhang, Ying, Florens, Laurence, Vu, Thien, Lange, Jeffrey, Gu, Mingyu, Ailion, Michael, Unruh, Jay

[International Worm Meeting, 2015]

The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for C. elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

QF Boese et al. (1999) International C. elegans Meeting "The mut-2 mutator of C. elegans"

J Collins, QF Boese

[International C. elegans Meeting, 1999]

The mut-2 mutator plays multiple regulatory roles in the C. elegans germline. In addition to regulating activity of at least four distinct transposon families it is implicated in chromosome segregation, gametogenesis, germline gene silencing and DNA double-strand break repair. Elucidating the function of mut-2 will enhance the utility of transposons for functional genomic studies in this organism and shed light on mechanisms that maintain structural and functional integrity of eukaryotic genomes. Using the Him phenotype conferred by mut-2(r459), we mapped the gene between dpy-14 and sem-4 on LGI. However, efforts to identify a molecular clone of the gene were hampered because the available phenotypes were unsuitable for standard transformation rescue approaches. This roadblock led us to examine the temperature-sensitive (ts) behavior of mut-2: the original mut-2 isolates TR674 and TR679 are inviable at 25 o C. Analysis of ancestors and recombinants derived from these isolates demonstrated that the ts phenotype cosegregates with mut-2 and is unrelated to the Bergerac ancestry of these strains. Closer inspection (with assistance from Eric Lambie) revealed that sterility results from a defect in gametogenesis. Gonads of mut-2 animals raised at 25 o C appear normal but have a dearth of sperm which exhibit variable morphology. Oocytes show no gross abnormalities but may be affected as well. DAPI staining reveals no obvious chromosomal defects. We are testing if wild-type sperm can rescue sterility and determining the temperature-critical stage of sperm (and oocyte?) development. The ts sterile phenotype is rescued by sDp2, a large free duplication that covers the dpy-14 sem-4 interval. This suggests it may be a suitable phenotype for identifying a molecular clone of mut-2. Two smaller free duplications that bisect this interval, hDp62 and hDp65, were tested; hDp65 rescues, hDp62 does not. This positions mut-2 between +1.57 and +1.66, a region covered by three cosmids, containing ~20 ORFs. Work is in progress to clone mut-2 by rescuing sterility. Recent evidence suggests mut-2 strains may be resistant to RNAi (1). We are exploring the use of this phenotype in our cloning efforts as well. 1) Tabara, H and Mello, C. 1999. (this meeting)