First published online 15 November 2005
doi: 10.1242/jcs.02665
Journal of Cell Science 118, 5575-5587 (2005)
Published by The Company of Biologists 2005
The C. elegans homologs of nephrocystin-1 and nephrocystin-4 are cilia transition zone proteins involved in chemosensory perception
Marlene E. Winkelbauer1,
Jenny C. Schafer1,
Courtney J. Haycraft1,
Peter Swoboda2 and
Bradley K. Yoder1,*
1 Department of Cell Biology, University of Alabama at Birmingham Medical Center, Birmingham Alabama, 35294, USA
2 Karolinska Institute, Department of Biosciences, Södertörn University College, Section of Natural Sciences, S-14189 Huddinge, Sweden
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Fig. 1. Expression of nph-1 and nph-4 in C. elegans. (Top) Diagram showing the position of the neurons examined in A and B. (A) nph-1::CFP (L1 Stage) and (B) nph-4::DsRed2 (adult) are expressed in the ciliated sensory neurons of the worm including the amphid and labial neurons in the head (left panels) as well as the phasmid neurons in the tail (right panels). In this figure and all following figures, anterior is toward the left.
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Fig. 2. DAF-19 regulation of nph-1 and nph-4. RT-PCR analysis of (A) nph-1 and (B) nph-4 showed a marked decrease in expression of both genes in the daf-19(m86) mutant background compared to that seen in the daf-19(+) background. Synaptotagmin (snt-1) was used as a control for a neuronal gene whose expression is DAF-19 independent. (C) In vivo expression analysis of nph-1::CFP in daf-19(m86) mutant (left panels) and daf-19(+) (right panels) backgrounds. nph-1::CFP expression was greatly diminished in the daf-19(m86) background compared with daf-19(+). A similar reduction in expression is seen for che-13::DsRed2, a gene encoding an IFT protein that is known to be regulated by DAF-19. The merged image of nph-1::CFP and che-13::DsRed2 expression in the daf-19 (+) background shows that both of these genes are expressed in the same cells. (D) In vivo expression analysis in wild-type worms using the nph-4(mut)::DsRed transgene in which the X-box has been mutated. Mutation of the X-box results in the loss of nph-4 expression in the ciliated sensory neurons relative to the same nph-4 transgene with a wild-type X box (see Fig. 1B) or the IFT gene osm-5::CFP with wild-type X-box.
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Fig. 3. Colocalization of the NPH-1 and NPH-4 proteins to the transition zone at the base of cilia in C. elegans. (A) Transgenic lines were generated that express NPH-1::CFP and NPH-4::YFP under control of their endogenous promoters. NPH-1 and NPH-4 colocalized to the distal end of the dendrites of the ciliated sensory amphid and labial neurons in the head of the worm (left panels) and the sensory rays of the male tail (right panels). (B,C) The localization of NPH-1 (B)and NPH-4 (C)was further evaluated by generating transgenic lines that co-express the IFT protein, CHE-13. CHE-13 was detected at the transition zones (arrowheads) and in the cilium axoneme (arrows); however, both NPH-1 and NPH-4 were restricted to the transition zone. There was no detectable signal for NPH-1 or NPH-4 in the axoneme. For enlarged images of the cilium region see Fig. S1 in supplementary material.
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Fig. 4. C. elegans NPH-1 and NPH-4 protein domain structures. (A) The C. elegans NPH-1 protein consists of two coiled-coil domains, an E-rich domain, a QP-rich domain (glutamate-proline rich domain), cytochrome P450E, an SH3 domain, and the nephrocystin homology domain (NHD). The deleted region (amino acids 281-641) in the nph-1(ok500) mutant is indicated as well as the nephrocystin-4 mammalian interaction region (C-terminal 131 amino acids). The protein sequence is based on the mRNA sequence derived from NM_063897. (B) The NPH-4 protein in C. elegans does not exhibit any well-characterized domains. The deletion, which spans amino acids 86-264 in nph-4(tm925) mutants, and the region involved in the interaction with mammalian nephrocystin-1 (N-terminal 176 amino acids) are indicated. The protein sequence is based on the mRNA sequence from AY959881.
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Fig. 5. Cilium structure analysis of nph-1;nph-4 double mutants. (A) Cilium structure was analyzed by evaluating the ability of nph-1, nph-4 and double nph-1;nph-4 mutants to absorb DiI (dye-filling assay). There were no overt differences detected between any of the mutant strains when compared to that of wild-type controls. (B) Cilium structure was further analyzed in nph-1;nph-4 double mutants using the CHE-13::YFP fusion protein. Cilium structure was indistinguishable from that of wild-type controls and CHE-13::YFP was properly localized throughout the cilium. (C) To evaluate possible effects of the nph mutants on cilium structure, the length of cilia was determined in nph-1;nph-4 double mutants expressing CHE-13::YFP. There were no statistically significant differences detected in the cilia of wild type and double mutant lines. Identical results were obtained with nph-4 single mutants (data not shown).
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Fig. 6. Lifespan analysis of nph-1 and nph-4 single and double mutants in C. elegans. The lifespan of nph-1, nph-4 and nph-1;nph-4 double mutants was compared to N2 wild-type controls and the long lived osm-5 cilia mutants. The nph-1(ok500), nph-4(tm925) and nph-1(ok500); nph-4(tm925) double mutant strains had a significant expansion in lifespan compared to the N2 worms. Although the nph-1(ok500) and nph-1(ok500); nph-4(tm925) double mutants did not have as severe of an increase in lifespan as osm-5 mutant worms, single nph-4(tm925) mutants were indistinguishable from osm-5 mutant worms. Similar data were obtained on three independent lifespan experiments (average of 75 total worms in each genetic category) conducted for this analysis.
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Fig. 7. The nph-1 and nph-4 single and nph-1;nph-4 double mutants exhibit defects in chemotaxis toward volatile attractants. Comparison of chemotaxis indices for the wild-type N2 and the nph mutants in response to (A) benzaldehyde and (B) diacetyl indicate that both single mutants and the double mutant exhibit abnormal response to these attractants. To demonstrate that the phenotypes were the result of mutation of the nph genes, the chemotaxis defect in nph-1 and nph-4 mutants was rescued by expression of nph-1::CFP and nph-4::YFP, respectively. Both the nph-1 and nph-4 rescued lines exhibited a strong chemotactic response to (A) benzaldehyde or (B) diacetyl similar to that obtained for wild-type controls. Error bars represent the standard deviation and n refers to the number of independent experiments with an average of 100 worms on each plate being evaluated.
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Fig. 8. The NPH-4 protein is required for proper localization of NPH-1 to the transition zone. To explore the possibility that the NPH proteins function as part of the same complex, we analyzed the localization of the NPH proteins in the reciprocal mutant background. (A) Compared to wild-type controls, NPH-4::YFP localization was not altered by loss of nph-1. (B) In contrast, NPH-1::CFP was not detected at the transition zone in nph-4 mutants indicating that NPH-4 is required for NPH-1 localization. (C) The localization of NPH-1::CFP to the transition zone was restored by crossing the strain back to wild-type worms.
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© The Company of Biologists Ltd 2005
