Grant BD et al. (1994) Worm Breeder's Gazette "sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein."

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1994]

sel-l, a negative regulator of lin-12 and glp-l activity, encodes a novel extracellular protein. Barth Grant and Iva Greenwald HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

Shah, P. et al. (2017) International Worm Meeting "Investigating redundancy among tubulin glutamylating enzymes."

Chakraborty, M, Lee, JD, Barth, ZK, Shah, RV, Chawla, DG, Shah, P., Peel, N

[International Worm Meeting, 2017]

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. Redundancy between the TTLL enzymes has been observed in other species and we are therefore constructing combinations of ttll mutations to explore potential redundancy between the enzymes. We have found that a ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. We are currently investigating whether further redundancies exist.

Chawla DG et al. (2016) Biol Open "Caenorhabditis elegans glutamylating enzymes function redundantly in male mating."

Shah RV, Chawla DG, Salmon TP, Peel N, Lee JD, Badecker KE, Naik A, Barth ZK, Brewster MM

[Biol Open, 2016]

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons.

Hyun-Ok Song et al. (2007) International Worm Meeting "Calcineurin, a Ca2+/calmodulin dependent Ser/Thr protein phosphatase, is important for endocytosis in C. elegans."

Joohong Ahnn, Hyun-Ok Song, Meenakshi Dwivedi

[International Worm Meeting, 2007]

Endocytosis is an essential cellular mechanism required for the internalization of fluid from the extracellular medium, nutrient uptake, and the recycling of membrane components. The significance of endocytosis has been shown in the down-regulation of signaling, synaptic vesicle recycling, and antigen presentation in immune cells. One dramatic example of receptor-mediated endocytosis is yolk uptake observed in C. elegans oocytes. Yolk proteins are synthesized in intestine and secreted into pseudocoelomic cavity. Finally oocytes take yolk protein up by receptor-mediated encodytosis. Many rme (receptor mediated endcytosis defective) mutants have been identified using vitellogenin::GFP (YP170::GFP) fusion system (Barth Grant et al., 1999). In addition, C. elegans use coelomocytes, scavenger cells, to endocytose fluid from pseudocoelom. The endocytosis using coelomocytes has been suggested to be mediated by clathrin. Coelomocyte uptake defective (cup) mutants have been identified based on the uptake of fluid-phase GFP markers from the pseudocoelom by coelomocytes (Hanna Fares and Iva Greenwald, 2001). Here, we report a possible function of calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, in C. elegans endocytosis using above analyses. We show that mutants of calcienurin A and B [tax-6(jh107gf), tax-6(p675lf), cnb-1(jh103null)] have partial defects in endocytosis.

Caffaro CE et al. (2008) Biochemistry "A single Caenorhabditis elegans Golgi apparatus-type transporter of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine."

Caffaro CE, Vestweber D, Samuelson J, Berninsone P, Hirschberg CB, Bakker H, Luhn K

[Biochemistry, 2008]

The genome of Caenorhabditis elegans encodes for 18 putative nucleotide sugar transporters even though its glycome only contains 7 different monosaccharides. To understand the biological significance of this phenomenon, we have begun a systematic substrate characterization of the above putative transporters and have determined that the gene ZK896.9 encodes a Golgi apparatus transporter for UDP-glucose, UDP-galactose, UDP- N-acetylglucosamine, and UDP- N-acetylgalactosamine. This is the first tetrasubstrate nucleotide sugar transporter characterized for any organism and is also the first nonplant transporter for UDP-glucose. Evidence for the above substrate specificity and substrate transport saturation kinetics was obtained by expression of ZK896.9 in Saccharomyces cerevisiae followed by Golgi enriched vesicle isolation and assays in vitro. Further evidence for UDP-glucose transport was obtained by expression of ZK 896.9 in Giardia lamblia, an organism recently characterized as having endogenous transport activity for only UDP- N-acetylglucosamine. Expression of ZK896.9 was also able to correct the phenotype of a mutant Chinese ovary cell line specifically defective in the transport of UDP-galactose into the Golgi apparatus and of a mutant of the yeast Kluyveromyces lactis specifically defective in the transport of UDP- N-acetylglucosamine into its Golgi apparatus. Because up to now all three other characterized nucleotide sugar transporters of C. elegans have been found to transport two or three substrates, the substrate specificity of ZK896.9 raises questions as to the evolutionary ancestry of this group of proteins in this nematode.

Law, Fiona et al. (2013) International Worm Meeting "Regulation of TBC-2, an endosomal Rab5 GAP."

Law, Fiona, Chotard, Laetitia, Rocheleau, Christian, Karbassi, Farhad

[International Worm Meeting, 2013]

Diseases such as cancer, metabolic disorders and neuronal degeneration can originate from endocytic trafficking defects. The proper trafficking of endosomes depends upon regulating proteins such as Rab GTPases that associate with their vesicular membrane. These proteins alternate between active GTP and inactive GDP bound forms through the actions of Guanine nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs). Different Rab GTPases function at discrete stages of the endocytic pathway to provide directionality to vesicular traffic and to recruit effectors for mediating downstream processes. During early to late endosome maturation, Rab5 is replaced by the Rab7 GTPase in a process called Rab conversion. Using C. elegans, our lab identified TBC-2 as a RAB-5 GAP that regulates RAB-5 to RAB-7 conversion. Loss of tbc-2 function or expression of constitutively active RAB-5 leads to the formation of large RAB-7 positive endosomes in intestinal cells. Our data suggest that TBC-2 is recruited to endosomes by RAB-7 where it inactivates RAB-5, thus facilitating the RAB-5/RAB-7 conversion. Recently, in collaboration with the lab of Barth Grant, we found that TBC-2 physically interacts with CED-10/Rac1 and that both proteins regulate endosome recycling. In ced-10 mutants, TBC-2 localization is strongly reduced, yet these mutants do not display the tbc-2 mutant phenotype. Therefore, is membrane localization important for TBC-2 function? To answer this question, I am conducting transgenic rescue experiments to identify which domains of TBC-2 are required for its localization and/or function. If there exists a TBC-2 domain that is required for both of these roles, I will test whether targeting the catalytic domain of TBC-2 alone to late endosomes is sufficient for rescue of TBC-2 function. Aside from CED-10/Rac1, TBC-2 has also been found to bind to two other C. elegans Rac GTPases: RAC-2 and MIG-2. Future experiments involve testing for genetic redundancy among these Rac proteins in early to late endosome trafficking. This work is funded by CIHR.

M Paupard et al. (1999) International C. elegans Meeting "ImmunoEM Localization of YP170::GFP Reveals Yolk Transport"

M Paupard, DH Hall, A Miller

[International C. elegans Meeting, 1999]

We have tested a variety of antibodies in post-embedding protocols to localize proteins in thin sections of C. elegans. Despite recent improvements, about 50% of tested antibodies fail to work satisfactorily. Those that fail apparently involve antigens that are denatured during alcohol dehydration prior to LR Gold embedment. Fixation of chilled whole worms within a microwave oven provides more uniform, rapid fixation than worms fixed by immersion or cut open in fixative by a blade (1,2). Just as for immunofluorescence, it is still necessary to compare fixative strengths to obtain optimal preservation while retaining antigenicity. Here we compare the merits of several monoclonal and polyclonal antibodies against GFP to localize yolk protein (YP170::GFP strain) in thin sections, using a gold-linked secondary Ab. Commercial anti-GFP antibodies were obtained from Clontech, Quantum and RDI. Until now, it has been difficult to prove which organelles contain yolk, or to trace the exact path by which yolk travels from intestine to gonad and embryos (3). The YP170::GFP label has also been used by light microscopy to monitor endocytosis of yolk in wild type and mutant tissues (4). By immunoEM, GFP label in YP170::GFP animals is concentrated in two classes of dark-staining organelles in the intestine. Labeled yolk particles are found in the pseudocoelom. Labeled granules and free label above background levels are seen in the cytoplasm of oocytes in the proximal gonad and egg chamber. We will present TEM evidence for yolk passage through sheath pores and endocytosis into oocytes separately (5). We thank Barth Grant (Columbia U.) for providing the YP170::GFP strain used in this study. 1. Li and Kimble, International C. elegans Meetings, 1995, 1997 2. Miller and Hall, Worm Breeder's Gazette 15: 15, 1998 3. Bossinger and Schierenberg, Int. J. Dev. Biol. 40: 431-9, 1996. 4. Grant, Zhang, Pedraza, Hall and Hirsh, this meeting. 5. Hall, Winfrey, Blauer, Hoffman, Furuta, Rose, Hobert and Greenstein, this meeting.

Mattingly, Brendan C. et al. (2009) International Worm Meeting "EXC-5 Maintains Subcellular Organelle Distribution."

Buechner, Matthew, Mattingly, Brendan C., Brown, Elinor M.

[International Worm Meeting, 2009]

The C. elegans excretory canal cell is a long "H"-shaped tubular structure that serves as the osmoregulatory organ for the animal. Null mutations in exc-5 cause the canals to swell into large fluid-filled cysts, sometimes expanding to as large as the entire body diameter of the worm. These cysts form as a result of a mechanical failure of the actin-based cytoskeleton lining the canal apical surface. Overexpression of exc-5 leaves the apical surface intact, but causes defects in the formation of the basal surface of the canal. This results in a convoluted tubule phenotype, similar to that of laminin alpha chain mosaics. EXC-5 represents the sole nematode homologue of a family of guanine nucleotide exchange factors called FGD, that is conserved throughout animal evolution. Humans carrying defective copies of either FGD1 or FGD4 develop the genetic disorders FacioGenital Dysplasia (Aarskog Syndrome) or Charcot-Marie-Tooth Disease type 4H, respectively. In the latter disease, the single-celled tubular glia of the peripheral nervous system are no longer able to maintain their apically-secreted myelin layer. In order to determine the subcellular processes altered by loss or overexpression of exc-5, we created lines of animals expressing subcellular markers (the generous gift of Barth Grant) within the canals. mCherry-labeled proteins include EEA-1 (early endosomes), CHC-1 (clathrin), CDC-42, RAB-5 (endocytic vesicles) EEA-1 (early endosomes), RAB-7 (late endosomes), RAB-11 and RME-1 (recycling endosomes), GRIP (Golgi), and GLO-1 (lysosomes). These lines show punctate location and organization of many of these compartments along the length of the canals. In addition, we have crossed these labeled strains to an exc-5 (rh232) null mutant, and to a line overexpressing exc-5. We have examined the overlapping expression of these markers with EXC-5 to determine the subcellular location of this protein. In addition, the effects of mild overexpression of EXC-5::GFP on canal morphology are enhanced when combined with expression of some of the markers. We conclude that EXC-5 activity on the actin cytoskeleton regulates passage of material through recycling endosomes to regulate replacement of material necessary for maintaining the apical tubular surface.

Haley, R. et al. (2019) International Worm Meeting "The small GTPase RAB-35 leads a novel third pathway for apoptotic cell clearance."

Wang, Y., Zhou, Z., Haley, R.

[International Worm Meeting, 2019]

The Rab family small GTPases are important regulators of membrane trafficking and play pivotal roles in many cellular events, including phagocytosis. Given that C. elegans RAB-2, RAB-5, and RAB-7 all actively participate in the degradation of apoptotic cells, we performed a candidate gene screen for additional rab genes playing a role in the clearance of apoptotic cells among 30 C. elegans genes that encode homologs of mammalian Rab GTPases. In this screen, we identified rab-35 as a new gene important for apoptotic cell clearance. Previously, rab-35 was discovered to function in the receptor recycling and yolk endocytosis by Barth Grant's lab. We have found that RAB-35 acts to regulate both the recognition and degradation of apoptotic cells by engulfing cells. Our extensive cellular and molecular characterizations of RAB-35 have revealed a few unique features of RAB-35 functions. First of all, RAB-35 leads a third pathway independent and in parallel to the well known ced-1/-6/-7/dyn-1 and the ced-2/-5/-10/-12 pathways to promote the clearance of apoptotic cells. This pathway acts to ensure the stability of the clearance activity under stress, as when rab-35 is inactivated, temperature changes further enhance the clearance defects. Secondly, unlike RAB-2, -5, and -7, which specifically regulate phagosome maturation, RAB-35 has an additional function in the recognition of apoptotic cells. Thirdly, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Last but not least, RAB-35 and TBC-10, its GTPase activating protein (GAP), mutually regulate each other to establish a precise temporal pattern of phagosomal localization and function. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells. In addition, RAB-35 defines a largely unexplored initial phase of phagosome maturation that precedes the canonical RAB-5+ stage.

Eric G et al. (1995) Worm Breeder's Gazette "MARINER-LIKE ELEMENTS IN THE ENTOMOPARASITIC NEMATODE HETERORHABDITIS BACTERIOPHORA (NEMATODA RHABDITIDA)"

Pierre A, Monique A, Christian L, Eric G

[Worm Breeder's Gazette, 1995]

The mariner transposable element is a small member of the transposable element class II. This element is thought to transpose through a DNA intermediate and shows short inverted terminal repeats (ITR) bordering the entire element. Originally described in Drosophila mauritiana, it has been identified in many other insect species representing ten insect orders. Mariner-like elements (MLE) have also recently been identified in the planarian Dugesia tigrina and in the nematode Caenorhabditis elegans. Using a polymerase chain reaction (PCR) strategy with degenerated oligonucleotides deduced from conserved sequences of the mariner central region, we have been able to detect the presence of MLE in the entomoparasitic nematode Heterorhabditis bacteriophora. These MLE appear to be either deleted forms or full-sized elements of 1279 bp with imperfect ITR of 30 bp. In a full-sized element, the transposase part codes for 358 amino acids including three stop codons and two frame shifts. Therefore, none of the sequences has an open reading frame encoding for a putative transposase. The H. bacteriophora sequences present a 5' transposase longer than those usually described in the Tc/mariner-like family (addition of 14 amino acids). Most of the MLE conserved mariner amino acids (Robertson H.M., 1995, J.Ins.Physiol., 41(2):99) are present and 90% of those missing result from a single nucleotidic mutation. Interestingly, the H. bacteriophora transposase shares more similarities with insect MLE transposase than with C. elegans one. Among the conserved H. bacteriophora amino acids, only 14% are typical C. elegans amino acids (compared with C. elegans PM and C. elegans ZK 370 MLE) while 28% are typical insect ones (compared with Drosophila mauritiana Mos 1 and Hyalophora cecropia MLE). When analysed with Genbank data system, the most important similarity has been found with a Carpelimus sp. mariner (Genbank accession number U04455). The H. bacteriophora MLE is 73% similar, at the nucleotidic level, to this coleopteran MLE, while it is only 52% similar to a C. elegans mariner. On another hand, the H. bacteriophora MLE ITR are more similar to C. elegans MLE ITR (up to 53%) than to insect ones (about 38%). The distribution of H. bacteriophora MLE has been studied by Southern blot analysis. All the tested H. bacteriophora isolates contain MLE, while they seem to be absent from the Steinernema genus, a very closed genus of entomopathogenic nematodes too. These results, together with the fact that H. bacteriophora is an entomoparasitic rhabditid, strongly suggest a case of trans-phyla horizontal transfer. studies are still carried out to further caracterize this new mariner element and to understand the phylogenetic relationships between the mariner transposable elements isolated so far.