Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie

[International Worm Meeting, 2009]

C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.

Lee RYN et al. (1995) International C. elegans Meeting "EAT-12 IS A PIVOTAL CONTROL OF MUSCLE EXCITATION IN C. ELEGANS"

Avery L, Lee RYN

[International C. elegans Meeting, 1995]

C. elegans feeds by stereotypic contraction and relaxation of pharygeal muscles. Contraction is dictated by muscle electrical activity, which can be monitored by electropharyngeograms (Raizen and Avery, Neuron 12: 483). To understand feeding behavior, we have isolated and characterized mutants that affect the pharyngeal muscle action potential. eat-12 is an important regulator of muscle excitation, not just in the pharynx but probably in all muscles. Gain-of-function mutations in eat-12 (one was kindly provided by Michael Hengartner and Bob Horvitz) prolong pharyngeal muscle action potentials and thus cause hypercontraction. The mutants are short and egg-laying constitutive. In contrast, partial loss-of-function mutants of eat-12 show feeble muscle contraction. Mutants are long, loopy and egg-laying defective. Preliminary evidence indicates that in these mutants pharyngeal muscle action potentials have a reduced rate of depolarization. eat-12 probably affects muscle excitation directly since null mutants arrest at the two-fold stage (Pat phenotype). Normally, embryonic muscle contraction occurs apparently independently of the nervous system function at this stage. To further characterize eat-12 genetically, we isolated 20 new alleles either by reverting the dominant alleles or by non-complementation of the recessive alleles. We found that eat-12 is the same locus as the previously identified genes egl-19 (Trent et al., Genetics 104: 619) and pat-5 (Williams and Waterston, JCB 124: 475). We also isolated intragenic suppressors of egl-19(n582), one of which reverts n582 to nearly wild-type phenotype. We are collaborating with Leslie Lobel to clone eat-12. Preliminary evidence suggests that eat-12 encodes a worm homolog of the alpha1-subunit of the mammalian L-type voltage-gated calcium channel. Leslie Lobel cloned a partial cDNA of a calcium channel gene by PCR and mapped it onto YACs close to eat-12 (Lobel et al., WBG 13.1: 46). We localized it to a cosmid and showed that the cosmid can rescue several eat-12 mutants by germ line transformation. We are analyzing eat-12 alleles to see if the calcium channel gene is mutated. Together, these observations suggest that eat-12, as a voltage-gated calcium channel, is a crucial component in generating muscle action potentials. It hastens depolarization and regulates the duration of an action potential. Our extensive collection of eat-12 mutants may facilitate structure-function analysis of calcium channels.

Ayesha N Shajahan et al. (2001) International C. elegans Meeting "Regulation of endocytosis of albumin in the intestinal epithelial cells of C.elegans by TGFbeta-like signaling"

Ayesha N Shajahan, Shahid S Siddiqui

[International C. elegans Meeting, 2001]

In C. elegans , TGF b -like signaling pathways regulate a range of developmental processes such as dauer formation (Estevez et al., 1993), body size growth and male tail formation (Morita et al., 1999; Suzuki et al., 1999), and axon guidance (Colavita et al., 1998). Recently, Siddiqui et al. have shown that daf-4 (TGF b -like-RII) and daf-1 (TGF b -like-RI) (at 25 o C) mutants show poor uptake of FITC-BSA (fluorescein-5-isothiocyanate isomer- bovine serum albumin) in the intestinal cells when compared to wild-type animals. Our aim is to identify the genes in the TGF b -like pathways that affect endocytosis of albumin in the intestinal epithelial cells. We will examine the uptake of FITC-BSA in the mutants of the TGF b -like pathway in comparison to its uptake in wild type. daf-4::gfp (Patterson et al., 1997) and daf-1::gfp (Gunther et al., 2000) are expressed in the intestinal cells. Expression and regulation of other TGF b -like receptors, Smad proteins and the downstream trancription factors in the TGF b -like signaling pathway will be examined in the intestinal cells of wild type and various daf mutants, in the presence and absence of albumin, by immunofluorescence and immunoprecipitation assays. This approach will determine the role of TGF b -like signaling in endocytosis of albumin, and also uncover any possible cross talk between such pathways in the process.

Sawa H et al. (1995) International C. elegans Meeting "lin-17 ENCODES A PROTEIN SIMILAR TO THE PRODUCT OF THE Drosophila TISSUE-POLARITY GENE Frizzled"

Horvitz HR, Sawa H

[International C. elegans Meeting, 1995]

Asymmetric cell divisions are involved in many aspects of development. lin-17 mutations disrupt the asymmetry of a number of cell divisions in C. elegans, suggesting that lin-17 is required to establish these asymmetries. We genetically localized lin-17 0.2 map units right of the RFLP nP58, which was identified by Leslie Lobel. Using cosmids and phage clones in this region, we searched for RFLPs in lin-17 mutant strains. A phage clone isolated by Kerry Kornfeld can detect RFLPs in rh41 and sy277. We identified cDNAs that detect both RFLPs. Because genomic clones containing the whole cDNA sequences are not available, we ligated a fragment of the genomic phage clone to one of these cDNAs and to the 3' region from the unc-54 gene. This construct rescued the Lin-17 phenotype. We determined the sequence of this cDNA. The predicted product of a 1.7 kb ORF is 28% identical to the product of the tissue-polarity gene frizzled of Drosophila. As in Lin-17, the polarities of a number of cells are disrupted in frizzled animals, resulting in the disordered orientation of hair, bristles, ommatidia and tarsi. Because lin-17 and frizzled encode proteins each with seven putative transmembrane domains, lin-17 is likely to be involved in cell-cell interactions required for the asymmetry of certain cell divisions. The similarity of lin-17 to frizzled raises the possibility that other genes related to tissue-polarity genes are involved in asymmetric cell divisions. The genome sequencing project identified an another frizzled-like cDNA that is different from lin-17 cDNAs. Also, a cDNA related to the Drosophila tissue-polarity dishevelled gene has been identified by the cDNA project led by Yuji Kohara. We are currently analyzing these cDNAs.

Kokel MJ et al. (1994) Worm Breeder's Gazette "Molecular Analysis of clr-1, A Negative Regulator of the FGFR in C. elegans"

DeLong L, Kokel MJ, Stern MJ

[Worm Breeder's Gazette, 1994]

MOLECULAR ANALYSIS OF clr-1, A NEGATIVE REGULATOR OF THE FGFR IN C. elegans. Michelle Kokel, Leslie DeLong, and Michael J. Stern, Department of Genetics, Yale University, New Haven CT 06536, USA The egl-15 encoded fibroblast growth factor receptor (FGFR) tyrosine kinase mediates multiple biological processes in C. elegans. egl-15 activity is required for viability, proper sex myoblast migration, and the expression of the Clear phenotype of clr-1 mutants. Genetic analysis suggests that clr-1 acts as a negative regulator of some of the activities of egl-15. In order to understand this interaction at the molecular level, we cloned and sequenced clr-1 and found that it encodes a receptor-linked protein tyrosine phosphatase (PTP). This suggests that CLR-1 regulates signaling through the FGFR either by dephosphorylation of EGL-15 directly, or of another component of the egl-15 signaling pathway. Thus, the Clear phenotype seems to be due to hyperactivity of FGFR signaling, perhaps due to increased tyrosine phosphorylation. There are a number of models which may explain why clr-1 only appears to affect a subset of egl-15 phenotypes. One possibility is that PTP activity is specific. For example, CLR-1 may only act on specific tyrosine residues in EGL-15 or downstream signaling components involved in specific pathways. Another possibility is that egl- 15 has different functions in different tissues and that clr-1 is only expressed in a subset of these tissues. To distinguish between these two possibilities, we plan to use antibody staining to determine the expression patterns of clr-1 and c91-15. The antibodies should also be useful to study the interaction of these two molecules biochemically. Suppressors of clr-1 (Soc) have identified three genes that could encode activators or mediators of egl-15 activity. One of these genes, sem-5, encodes an SH2/SH3 containing adaptor protein required to transduce signals from a variety of receptor tyrosine kinases. The function of SEM-5 supports the hypothesis that clr-1 suppressors may mediate signaling through the FGFR. Thus, we have begun to clone the other two soc genes with the hope that their analysis will provide additional insights into FGFR mediated signaling pathways.

Stephen R Wicks et al. (2002) European Worm Meeting "dyf-8 encodes a type I trans-membrane receptor with a zona-pellucida domain and may be a worm homologue of endoglin."

Stephen R Wicks, Ronald H A Plasterk

[European Worm Meeting, 2002]

TGF-like signaling cascades have been implicated both in the control of body size and the regulation of dauer stage entry and exit in C. elegans. In the dauer pathway, it is thought that a soluble signaling molecule of the TGF/BMP family (DAF-7) activates a DAF-1/DAF-4 (TGF type I/TGF type II) complex (Gunther et al, 2000). This activation leads to SMAD signaling. In some systems, a TGF type III receptor molecule has also been implicated in SMAD signaling. This type III receptor molecule is thought to be incapable of direct signaling in the absence of a Type I/Type II complex. The type III-receptor binds the soluble ligand with a very high affinity and hands the ligand off to the Type I/Type II complex, thus increasing the sensitivity SMAD signaling. No known type III receptor partners have been isolated in C. elegans screens for body size or dauer formation defects. Endoglin is a type III TGF-receptor protein found in some epithelial tissues. In humans, endoglin mutations are the causative agent of a disease called Hereditary Hemorrhagic Telangiectasia type I (HHT I). Patients present with bleeding lesions around the soft mucous tissue that arise due to weakness in vascular endothelium. This, and other evidence suggests that the type III co-receptor is required for the stabilisation of endothelial juxtapositions. Endoglin, and the related TGF Type III co-receptor, Beta-glycan, each have a weak signature for a zona pellucida domain. One notable hypodermal (epithelial) juxtaposition in the worm is made by the sheath cell and the socket cell in worm sensory organs, the amphid being the most well-studied of these. The structure of the amphid organ can quickly be assayed by soaking worms in the lipophillic dye, DiI. The penetrating amphid sensory neurons quickly backfill in wild type animals, but in the absence of an intact amphid organ, these neurons fail to fill. Dyf-filling (dyf) defective screens have yielded several mutants (Starich et al, 1995). We recently cloned one of these, dyf-8, using our SNP mapping methods (Wicks et al 2001). Although quite divergent at the level of primary sequence, the molecule possesses the modularity of an endoglin homologue, with a signal sequence, a zona pellucida domain, a trans-membrane domain and a short intracellular tail with no kinase domains. A clustal alignment of the ZP domains, including all of the TGF receptor subtypes from worm mouse and human show that dyf-8 clusters weakly with the endoglin family. The mutant phenotype is full rescued by re-introduction of the cosmid containing the gene.

Morita K et al. (1998) Worm Breeder's Gazette "CeBRAM-2 that binds to DAF-1 is a negative regulator of daf-7 TGF-b pathway."

Ueno N, Shibuya H, Shimizu M, Morita K

[Worm Breeder's Gazette, 1998]

TGF-b family of growth factors regulate many aspects of cellular function. The signaling is common in diverse animal species from vertebrates to C. elegans. We have been interested in the signaling pathway of the TGF-b family. We reported about cloning of a full-length cDNA (0.9 kb) of CeBRAM-2, a daf-1 binding protein (abstract, p 250), at the international C. elegans meeting in 1997. Briefly, CeBRAM-2 has a 57% amino acid identity over the carboxy-terminal 60 amino acids of human BRAM2 (BMP receptor associated molecule) identified by yeast two-hybrid system. HA-tagged CeBRAM-2 transiently expressed in COS7 cells was found to bind typeI receptors, both ALK-3 (mammalian) and DAF-1 (C. elegans). CeBRAM-2;;GFP fusion protein was found to be expressed dominantly in amphid and pasmid neurons (e.g. ASI, ASK, ASJ, etc.). This expression pattern was similar to that of daf-1 (C. Gunther and D. Riddle, pers. comm.). Subsequently, the null mutant was created by Tc-1 deletion method. However, phenotype of the CeBRAM2 single mutant was not obvious. We report here the genetic interaction of the CeBRAM-2 null mutant with mutants in daf-7 TGF-b pathway by examining dauer formation of the double mutants. The result suggests that CeBRAM-2 is involved in the daf-7 TGF-b pathway in C elegans as a negative regulator. 20C 23C 25C genotype + CeBRAM2 + CeBRAM2 + CeBRAM2 daf-1 m40 14(137) 2(373) 66(185) 10(348) 100(317) 51(495) m402 61(144) 6(231) 94(220) 24(329) 100(481) 72(750) m213 92(318) 27(245) 95(440) 37(308) 100(614) 80(482) daf-7 e1372 60(210) 4(316) 98(295) 52(261) 100(493) 89(335) daf-11m47 8(154) 5(281) N.D. N.D. 56(1488) 16(2323) daf-14m77 7(247) 3(492) N.D. N.D. 47(1283) 38(1260) TABLE Percent dauer formation of CeBRAM2 and daf-c double mutants at various temperatures .The percentage of animals forming dauers is given, with the total number of animals counted given in parentheses. + indicate the daf-c single mutants, and CeBRAM indicates daf-c;CeBRAM-2 double mutants. These results show that CeBRAM-2 mutant has a week suppressor of DAF-c phenotype. CeBRAM-2 was not suppressed daf-14 SMAD, so CeBRAM-2 is hypostatic to SMAD, and epistatic to daf-7 TGF-b ligand and daf-1 type I receptor. Rescue experiment was done with a CeBRAM-2 genomic fragment, to daf-1 (m40);CeBRAM-2 double mutant. Approximately 80 % of transgenic progeny formed dauers at 25!C, suggesting that suppression of dauer formation in the double mutant was recovered. Together, we hypothesize that CeBRAM-2 functions as a negative regulator by binding TGF-b type I receptors like as FKBP12 (Y., Chen, J., Massague, EMBO J. 16 3866-3876, 1997).