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feh-1 and apl-1, the Caenorhabditis elegans orthologues of mammalian Fe65 and ß-amyloid precursor protein genes, are involved in the same pathway that controls nematode pharyngeal pumping

Nicola Zambrano1, Marida Bimonte1, Salvatore Arbucci2, Davide Gianni1, Tommaso Russo1 and Paolo Bazzicalupo2

1 Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Via S. Pansini, 5, I-80131, Napoli, Italy
2 International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Via G. Marconi, 10, I-80125, Napoli, Italy



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  		Fig. 1. The structural similarity between C. elegans FEH-1 and mammalian Fe65, Fe65-L1 and Fe65-L2 proteins. (A) The FEH-1 sequence is deduced from translation of the putative exon I of feh-1 (italic characters) and of the yk423e6 cDNA clone (bold characters). Methionines 1 and 142 are underlined on the FEH-1 sequence. The alignment of FEH-1 with mammalian Fe65s has been performed with ClustalW. The sequence identities are shown by black boxes; at the bottom of each line, the asterisks (*) indicate matching amino acids; the double dots (:) show strong amino acid similarities, whereas the dots (.) indicate weaker similarities. The positions of the WW, PTB1 and PTB2 domains are indicated. (B) Schematic representation of FEH-1 and mammalian Fe65s. The size of the spacers between the WW and the PTB1 domains, or the PTB1 and the PTB2 domain, is comparable among the four proteins, and the N- and the C-terminal regions of FEH-1 are similar in length to Fe65 and Fe65-L1.

 


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  		Fig. 2. FEH-1 and APL-1 maintain the binding properties of mammalian homolog proteins. (A) C. elegans (lanes 1-3) or mouse brain (lane 4) lysates were resolved by SDS-PAGE and probed with pre-immune serum (lane 1), pre-adsorbed (lane 2) or purified FEH-1 antibody (lane 3) or anti-Fe65 serum (lane 4). In lanes 5 and 6, immunoprecipitated FEH-1 treated (lane 6) with calf intestine alkaline phosphatase (CIP) or untreated (lane 5) was assayed by western blot analysis with FEH-1 antibody. (B) Glutathione-sepharose beads were saturated with wild-type GST (lane 1) or with fusions of GST with the APL-1 cytodomain (GST-APL-1, lane 2), the FEH-1 PTB1 (GST-PTB1, lane 3) or PTB2 (GST-PTB2, lane 4) domains and incubated with 500 µg each of C. elegans proteins. Bound proteins were resolved by SDS-PAGE and probed with FEH-1 antibodies. Lanes 1' to 4' show Ponceau S staining of the filter to indicate the amounts of each GST proteins used. (C) Glutathione-sepharose beads were saturated with wild-type GST (lane 2) or with fusions between GST and the FEH-1 PTB2 domain (GST-PTB2, lane 3); the FEH-1 PTB1 domain (GST-PTB1, lane 4) and were incubated with 2 mg of C. elegans proteins. Bound proteins were resolved by SDS-PAGE and probed with APL-1 antibodies. Lane 1 contains a C. elegans protein lysate sample. Lanes 2'-4' show Ponceau S staining of the filter.

 


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  		Fig. 3. gb561 is a feh-1-null allele. (A) shows the structures of the predicted Y54F10AM.2 coding region (adapted from Wormbase) and of the actual feh-1 gene. The numbered bar at the top shows approximate sizes, in base pairs (bp). The exons are represented as filled boxes (black boxes, translated region; grey boxes, untraslated regions); roman numerals above the exons indicate their relative number. The yk423e6 sequence starts at the 3' end of exon 1. The ATG codons in exons 1 and 2 are also shown. The black bars below the feh-1 structure indicate the double deletion of the mutagenised allele gb561, whose structure is shown at the bottom. (B) Western blot analysis of protein lysates from +/+ (lane 1), +/gb561 (lane 2) and gb561/gb561 (lane 3) worms. Worms from the three populations were transferred into SDS buffer for protein extraction and western blot analysis with FEH-1 antibody. (C) Western blot analysis of protein lysates from untreated, mixed-stage Bristol N2 worms (lane 1) or from the F1 of feh-1 dsRNA-injected worms (lane 2). Harvested worms were lysed in SDS buffer and subjected to western blot with the FEH-1 antibody.

 


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  		Fig. 4. Embryonic defects in feh-1(gb561/gb561) worms and feh-1 dsRNA-injected worms. (A) Embryos laid by feh-1 (+/gb561) worms. The white arrowheads indicate two arrested embryos. (B) Two embryos laid by feh-1 dsRNA-injected worms. The arrowhead indicates the arrested embryo. Bars, 10 µm.

 


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  		Fig. 5. Pharyngeal localisation of FEH-1 by whole-mount immunofluorescence analysis. Anterior is to the left. Bar, 20 µm. (A) Immunodetection analysis of FEH-1 expression in adult hermaphrodite. Whole worms were fixed and stained with FEH-1 purified antibodies, followed by Texas-Red conjugated anti-rabbit antibody. (B) Worms were stained with FEH-1 and MH27 antibodies, followed by FITC-conjugated anti-rabbit antibodies to detect FEH-1 (green) and the Texas-Red conjugated anti-mouse antibody to detect JAM-1, the epithelial antigen recognised by MH27 (red). Samples were analysed by confocal microscopy. The merged image, at the bottom, shows no overlapping localisation of the two proteins. (C) Worms were stained with FEH-1-purified antibodies, followed by FITC-conjugated anti-rabbit antibodies (green); pharyngeal muscle was stained with Texas-Red-conjugated phalloidin (red). Samples were analysed by confocal microscopy. The merged image (yellow) at the bottom shows that FEH-1 is present both in muscle cells and outside.

 


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  		Fig. 6. Reporter analysis reveals feh-1 expression in the nervous system and in the neuromuscular structures of the pharynx. (A-C) show the expression pattern of a feh-1::GFP reporter fusion in living worms. The GFP gene from vector pPD95.75 fused to the 5' end of feh-1 is strongly expressed in neurons of the ventral cord and in the head, at the level of the pharynx, in a larva (A) and in an adult hermaphrodites (B,C). Arrowheads in (C) indicate some strongly expressing neuronal bodies, whereas the arrows show the neurites. The left panels show Nomarski images of the worms. Anterior is to the left, ventral is to the top of the figures. Bars, 100 µm. (D-G) shows the expression pattern of a 5'-feh-1::NLS::lacZ reporter fusion in fixed worms. lacZ gene from vector pPD21.28 fused to the 5' end of feh-1 produces a nuclear-targeted fusion protein, which allows us to detect the nuclei of the cells expressing the reporter. (D,E) show the heads of two worms, in which the staining of extra-pharyngeal neurons (D), and of pharyngeal cells (E) is evident, and is indicated as follows: white arrowheads, m3 cells; black arrowheads, m4 cells; arrows, m5 cells. The worm in (F) shows a detail of the central region of the body: the nucleus of a ventral chord neuron (black arrowhead), and a faint staining of cells close to the vulva (white arrowheads) is also seen. In (G), two tail neuronal nuclei are intensely stained. Bars, 20 µm.

 


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  		Fig. 7. Decreased levels of FEH-1 or APL-1 protein result in increased pharyngeal pumping. (A) The rate of pharyngeal contractions were determined in (+/+) individuals (N2), in feh-1 (+/gb561) heterozygous worms (+/-), as well as in feh-1(gb561/gb561) individuals rescued by feh-1 transgene (R). Significance of the difference between wild-type and feh-1 (+/gb561) individuals was evaluated using the Student's t test (P<0.0001). Bars indicate standard deviation. (B) The rate of pharyngeal contractions was determined in wild-type Bristol N2 individuals fed to bacteria producing vector-derived ds RNA (vec) or vector/feh-1 dsRNA (feh) or vector/apl-1 dsRNA (apl). Significance of results obtained from feh-1 and apl-1 dsRNAs versus control measurementes was evaluated using the Student's t test (P<0.0001). Bars indicate standard deviation.

 

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