Podbilewicz B (1994) Worm Breeder's Gazette "What Snakes and Worms Have In Common: Disintegrins"

Podbilewicz B

[Worm Breeder's Gazette, 1994]

WHAT SNAKES AND WORMS HAVE IN COMMON: DISINTEGRINS Benjamin Podbilewicz, MRC Laboratory of Molecular Biology, Cambridge, UK

Gattegno T et al. (1997) Worm Breeder's Gazette "Characterization of duf-1: C. elegans first Hypofusion Mutant."

Gattegno T, Podbilewicz B

[Worm Breeder's Gazette, 1997]

We are interested in mechanisms of cell adhesion and cell fusion. Cell fusion in humans occurs during fertilization and during the development of multinucleate cells that are contained in bones, muscles and placenta. In C. elegans there are cell fusions during the formation of the vulva, pharynx, uterus, excretory gland and in the hypodermis. There are 46 multinucleate cells (1) of which hyp7 is the largest (133 nuclei).Hpy 7 is part of the hypodermal envelope that surround the adult worm. In a screen for mutations that are affected in the process of cell fusion many mutants were found and analyzed. The mutations were sorted into two main groups: hyperfusion and disordered hypodermis (1). No mutants with fewer fusions in the hypodermis were identified (hypofusion). duf-1(zu316cs) is a dorsal unfused zygotic lethal mutation that was isolated in a screen for mutants affected in the process of elongation (2). The phenotype was analyzed by immunofluoresence using the MH27 antibody. The MH27 protein is expressed in the adherens junctions at the apical domain of the hypodermis and it enable us to follow the fusions between hypodermal cells. After outcrossing we found that zu316cs is a cold sensitive mutation, at 15!C embryonic elongation arrests between 1.5 to 2 fold with fewer fusions in the dorsal hypodermis (hyp6 and hyp7) compared to the wild type and the tail is twisted (3). At 25!C some embryos arrest at 1.5 to 2 fold with fewer dorsal fusions, most embryos arrest at 3 fold and many hatch with normally fused hyp7 and variable posterior defects. duf-1 was mapped to the X chromosome, 10 centiM away from lon-2 (e678). Viral induced cell fusion and invasion by enveloped viruses are cold-sensitive processes. Many cold-sensitive mutations in different fusogenic viral proteins have been analyzed. Our characterization of duf-1(zu316) is consistent with the idea that membrane fusion is a cold-sensitive process. The future plans are to further characterize the phenotypes at the permissive and restrictive temperatures, find the cold sensitive period and to continue the mapping. 1. Podbilewicz B.(1997) Worm Meeting Abstract, p.300 2. Costa M. and Priess J.(1995) Worm Meeting Abstract, p.165 3. Gattegno T. and Podbilewicz B.(1997) Worm Meeting Abstract, p.546 We thank Mike Costa and Jim Priess for sending zu316 to us.

Hall DH (1996) Worm Breeder's Gazette "ImmunoEM Study of MH27 Reveals Sticky Junctions"

Hall DH

[Worm Breeder's Gazette, 1996]

Immunofluorescence studies using MH27 antibody have shown a wide variety of tissues to be labelled in patterns which define apical cell borders in C. elegans. Disappearance of MH27 binding during development often marks cell fusions in hypodermis and in the male tail (Podbilewicz and White, 1994; Fitch and Emmons, 1995). We have used an immunoEM method to learn the nature of membrane structures marked by MH27. A post-embedding technique was used to apply MH27 Ab to thin sectioned worms, followed by a gold-linked secondary Ab (for methods, see Selkirk et al., 1991; Hall, 1995). Electron microscopy reveals that MH27 binds only to a few types of cell junctions. By immunoEM, MH27 can be seen to bind to zonula adherens junctions in intestine, at hypodermal/seam cell borders, and at pharyngeal muscle/support cell borders. In adult pharyngeal muscle, small longitudinal stripes of adherens "junction" are also retained on the apical surface where pairs of muscle cells had fused to become syncytial. Thus MH27 binding can sometimes mark the vestiges of an old cell border, long after cell fusion. Spermathecal cells are held closely together by two types of "septate" junction, which together cover almost all of their lateral borders. Extensive, dark-staining apical junctions, which look vaguely adherens-like, are not labelled by MH27. In more basal regions, MH27 binds heavily to another class of extensive sinuous junctions, which lack any dramatic staining characteristics. Neither class of junction has osmiophilic septa visible in ordinary sections. The apical junctions are rather closely apposed and show periodic striping of the thick cytoplasmic density bordering the junctions. The basal, sinuous junctions show an even, widened extracellular space between adjacent plasma membranes, with faint periodic striations crossing the extracellular space between the cells. There is no cytoplasmic density associated with the sinuous junctions. Lanthanum infiltration has been used to negatively stain extracellular septa in the spermathecal junctions. The apical junctions have long, wavy septa; they may comprise a novel class of septate junction, characterized by the thick densely-staining coat on the cytoplasmic face of each cell. The sinuous junctions have not been well infiltrated with lanthanum yet. In one instance we did observe short septa at regular intervals, spanning the extracellular space, similar to those expected in "smooth septate junctions" as described in other invertebrate species. These two classes of septate junction may resist the stresses which would otherwise tear apart the spermatheca during the passage of an oocyte. Our current technique does not permit resolution of the exact locus of MH27 antigen within the adherens junction or the smooth septate junction. It could be cytoplasmic, extracellular, or within the plasma membrane. In glancing sections of both types of junction, it is clear the antigen is found evenly along the entire face of the cell-cell appositions, possibly at a fixed distance in relation to the plasma membrane. High resolution studies would require a more direct labelling method, such as attaching a small gold tag directly to a Fab fragment of the MH27 antibody. This would bring the gold tag into closer proximity to the true antigenic site within a thin section. We thank Jim Waddle and Ross Francis (Washington U.) for generously supplying MH27 antibody. Fitch and Emmons (1995) Dev. Biol. 170: 564-582. Hall (1995) In C. elegans: Modern Biological Analysis of an Organism. H.F. Epstein and D.C. Shakes (eds.). Academic Press, New York, pp. 395-436. Podbilewicz and White (1994) Dev. Biol. 161: 408-424. Selkirk et al. (1991) J. Biol. Chem. 266: 11002-11008.

Podbilewicz B et al. (1997) Worm Breeder's Gazette "A PATHWAY FOR VULVAL MORPHOGENESIS IN C. elegans"

Podbilewicz B, Kishore RS

[Worm Breeder's Gazette, 1997]

Cell interactions, fusions and migrations are processes involved in the formation of organs. In C. elegans these have been widely reported to occur in the formation of the hypodermis, uterus, excretory gland cells, pharynx and vulva. We have been studying the formation of the vulva in C. elegans by characterizing the pattern of cell recognitions, migrations and fusions by using immunofluorescent techniques and confocal microscopic reconstructions. Vulval sections at different times during the developmental process were stained for MH27, an adherens junction protein (1). Events have been followed from the time the fates of the six vulval precursor cells (VPCs) have been specified (27 hours; times are in hours after hatching), the generation of the VPC daughters (30 hours), VPC grand-daughters (32 hours; late L3), which results in 22 epithelial cells of the vulval lineage (34 hours) to the construction of a tube that consists of seven toroidal rings stacked together. These rings are designated as: vula, vulb1, vulb2, vulc, vuld, vule and vulf (2). These studies have revealed a definite pattern and order of cell migrations and fusions which allow us to propose a pathway for vulval morphogenesis, with the following intermediates being identified till now In the first stage a vulval primordium consisting of a longitudinal row of 12 cells (after two rounds of division), shows no signs of differentiation (Fig.1A) followed by a stage where the six central cells are polarized towards the center of symmetry before the last round of division (Fig.1B). During the third and final round of divisions six cells divide transversely, two cells do not divide and four divide longitudinally (3), (Fig.1C). This intermediate shows a central ring precursor containing four cells (vulf), four more that form the next ring (vule) and a third ring (vuld) with two cells. The next two cells (!c!) on each side have divided and are sending processes towards the centre of symmetry of the vulva surrounding vuld. The last schematic intermediates (Figs. 1D-H) show the seven rings that have completed the migrations and are in different stages of cell fusions starting with an intermediate where all seven rings are unfused followed by several intermediates: Fig.1D-rings vula and vulc are partially fusedand contain two binucleate cells each, Fig.1E-vuld has fused, Fig.1F-vula and vulc have fused, Fig.1G-vulf has fused, Fig.1H-vule has fused. These events studied in time thus reveal a pathway for vulva formation in C. elegans. 1. Podbilewicz, B. and White, J.G. (1994). Dev.Biol. 161, 408-424. 2. White, J.G. Personal Communication. 3. Sulston, J.E. and Horvitz, H.R. (1977). Dev. Biol. 56, 110-156.

Kariya K-i et al. (1997) Worm Breeder's Gazette "C. elegans homologs of ralGDS, AF-6, Cdc25 and phospholipase Cb interact with Let-60"

Barstead RJ, Watari Y, Shibatohge M, Kariya K-i, Goshima M, Kataoka T, Liao Y

[Worm Breeder's Gazette, 1997]

Mammalian Ras proteins regulate multiple effector molecules. They include Raf family (Raf-1, B-Raf, A-Raf), RalGDS family (RalGDS, RGL, RGF) and AF-6. C. elegans genetic studies contributed a lot to understanding of Ras function, especially regulation of Raf-MEK-MAPK pathway. However, studies have been limited to Raf and its downstream. Our long-term goal is to understand all pathways regulated by C. elegans Let-60. Here we report progress of our search for new Let-60 effectors and genetic study of their functions. We first screened for Let-60-binding proteins by using the yeast two-hybrid system. lACT-RB2, a random-primed mixed-stage C. elegans cDNA library was constructed in lACT that express cDNA clones fused to GAL4 activator domain. After conversion to the plasmid form (pACT-RB2), it was co-transformed into the test yeast strain CG-1945 with a plasmid pAS2-1-Let-60V12, expressing an activated Let-60 mutant as a fusion with GAL4 DNA-binding domain. After screening of approximately 106 transformants, 88 colonies were identified as both His+ and LacZ+. Plasmid clones were recovered and confirmed to confer both His+ and LacZ+ phenotypes when co-transformed again with pAS2-1-Let-60V12 but not when co-transformed with pAS2-1 vector. Inserts of confirmed clones were characterized by DNA sequencing. The sequencing data were used to identify cosmid clones containing overlapping genomic sequences by the BLASTN search (thanks to the C. elegans genome sequence project). The cDNA sequences together with the cosmid sequences were also used to identify EST clones containing overlapping sequences (thanks to Dr. Y. Kohara at NIG, Japan). When corresponding cosmid was not found, the cDNA sequences or the EST sequences were used to identify homologous proteins from the protein database by the BLASTX search. The clones comprised 6 classes. They included those encoding C. elegans Raf (Ce-Raf, 35 clones), C.elegans homologs of RalGDS (Ce-RalGDS, 29 clones), of AF-6 (Ce-AF-6, 6 clones), of Cdc25 (Ce-Cdc25, 10 clones) and of phospholipase Cb (Ce-PLCb, 2 clones), and those encoding other proteins (6 clones). Since lACT-RB2 represents 107 independent clones, this screen is not saturated yet. Predicted structures of newly found Let-60 effectors are shown in Fig. 1. We found Ce-RalGDS (F28B4.2) initially by simply performing a BLASTP search with mouse RalGDSA. Two-hybrid clones contained regions encoding the middle Cdc25 homology domain, the C-terminal Ras-interacting domain and a termination codon. pACT-RB2 plasmid library was used as a template to obtain sequence for N-terminal portion by PCR with a SL1 splice leader primer and a cDNA-specific primer. Clones for Ce-AF-6 contained the initiation codon and regions encoding the N-terminal Ras-interacting domain and the middle GLGF/DHR motif (no cosmid). Sequence for the C-terminal portion and the termination codon was found in a EST clone. Clones representing Ce-Cdc25 (T14G10.2/K04D7) encoded a Cdc25 homology domain most similar to that of Cdc25Mm/RasGRF. But regions around this domain were divergent from Cdc25Mm/RasGRF. It is possible that Ce-Cdc25 is a downstream effector of Ras, not a homolog of Cdc25Mm/RasGRF, an upstream regulator of Ras. Clones for Ce-PLCb (F31B12.1) encoded the X, Y and C2 domains found in the human counterpart. The Ras-binding domain was mapped to the C-terminal 300 amino acids by deletion analysis. F31B12.1 contains a very long N-terminal extension not found in human PLCb. But this prediction seems to be correct since PCR with a primer containing the predicted initiation codon and a downstream primer using the pACT-RB2 plasmid library gave a band with a predicted size. By computer analysis, Ponting and Benjamin recently proposed the existence of a family of Ras-associating domains (RA domain) including those of RalGDS and AF-6 [1]. Their list of proteins containing the RA consensus included Ce-RalGDS, Ce-Cdc25 and Ce-PLCb. However, Ce-AF-6 was not detected in their search, since it was not contained in any sequenced cosmids. Also, the list did not include Cdc25Mm/RasGRF, supporting the uniqueness of Ce-Cdc25. Search is in progress for mutant worms carrying Tc1-insertions within these genes. A PCR-based method was used to screen 108 plates, each started with ten MT3126 worms (thanks to the CGC). Two alleles each for Ce-RalGDS and Ce-PLCb were detected (we also found one allele for the previously reported Ach-1 gene[2]). After sib-selection, single worms were identified for all of them (thanks to Dr. Y. Andachi and the Japan C. elegans laboratory course at NIG). We are in the process of screening for worms carrying deletion of these genes by Tc1-excision.