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First published online 24 January 2007
doi: 10.1242/dev.02790

Development 134, 867-879 (2007)
Published by The Company of Biologists 2007
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The P-type ATPase CATP-1 is a novel regulator of C. elegans developmental timing that acts independently of its predicted pump function

Anne-Françoise Ruaud and Jean-Louis Bessereau*

ENS, Biologie cellulaire de la synapse; INSERM, U789, Paris, F-75005 France.


Figure 1
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  		Fig. 1. Mutation of catp-1 confers resistance to the nicotinic agonist DMPP. (A) Dose-response sensitivity to DMPP of wild type (WT) and catp-1(kr17) mutants. (B) catp-1 gene structure and constructs used for transgenesis experiments. Triangle, Mos1 insertion; dashes, direct repeats; arrows, inverted repeats; *, STOP codon. (C) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n≥4 independent experiments, N≥96 total individuals, two independent transgenic lines per construct tested). Pcatp-1(br)::catp-1(br): extrachromosomal array carrying the C. briggsae catp-1 genomic region; Pcatp-1(br)::catp-1(el): C. elegans catp-1 cDNA under the control of a C. briggsae catp-1 promoter. Both constructs rescued catp-1(kr17) DMPP resistance. (D-G) catp-1 expression profile. Detection of GFP fluorescence by confocal (D) and epifluorescence microscopy (E) in transgenic larvae expressing Pcatp-1(C. briggsae)::GFP. catp-1 is expressed in the hyp (white arrowheads) and Pn.p cells of the epidermis (D) and in the duct cell of the excretory system (E-G). No GFP expression was detected in the lateral cells of the epidermis (seam cells: black arrowheads). In (G), the GFP image (E) of the duct cell was overlayed on a Nomarski picture of the same field (F) where the excretory duct is visible (black arrow). Scale bars: 20 µm (D); 5 µm (E-G). (H) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n≥3 independent experiments, N≥111 individuals, two independent lines). Expression of a dsRNA targeting catp-1 in the epidermis induces partial DMPP resistance in a sid-1(qt2) background (*P<0.05, Mann-Whitney test).

 

Figure 2
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  		Fig. 2. CATP-1 encodes a cation-transporting ATPase of the P-type family with an ATPase-independent activity. (A) Phylogenetic tree of C. elegans and vertebrate Ca2+-, H+/K+- and Na+/K+-ATPases determined using the ClustalW analysis on full-length sequences. SCA-1, PMR-1, EAT-6, C01G12.8, CATP-1, C02E7.1 and C09H5.2 are C. elegans proteins. (B) Domain structure of human Na+/K+ P-type ATPase {alpha}1 and CATP-1. TM, transmembrane domain (black); P, phosphorylable P-domain (light gray); N, ATP-binding N-domain (dark gray). (C-E) CATP-1 has an ATPase-independent activity. (C,D) Amino-acid sequence comparison among the predicted H+/K+ (HK) and Na+/K+ (NK) P-type ATPase {alpha} subunits. The amino-acid numbers are according to C. elegans CATP-1. Residues identical or similar in more than 50% of the proteins are shaded in black or gray, respectively; residues similar to the identity consensus are also shaded in gray (BOXSHADE 3.21, http://www.ch.embnet.org/software/BOX_form.html). (C) Part 1 of the phosphorylable P-domain. Arrow indicates the phosphorylable aspartate characteristic of P-type ATPases. (D) ATP-binding region of the N-domain. Arrow indicates the arginine equivalent to R544 in pig kidney Na+/K+-ATPase, which is essential for ATP binding (Jacobsen et al., 2002Go). (E) Survival on 0.75 mM DMPP. The D409E mutation disrupts the obligatory phosphorylation site conserved in all P-type ATPases. Error bars represent s.e.m. (n≥3 independent experiments, N≥81 individuals, two independent lines). Two pump-dead mutants of CATP-1 partially rescue catp-1(kr17) DMPP resistance (*P<0.05, Mann-Whitney test).

 

Figure 3
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  		Fig. 3. catp-1 modulates C. elegans L2 developmental rate. (A) catp-1(kr17) specifically delays the timing of L2/L3 molt. During the lethargus period that precedes molting, rythmic contractions of the pharynx (also called pharyngeal pumping) ceases. Each dot represents the percentage of worms pumping at a given time (n>25 individuals). (B) catp-1(kr17) is insensitive to DMPP-induced developmental delay. L2 development was divided into five stages based on seam cell (encircled nuclei) divisions and anchor cell differentiation (Ruaud and Bessereau, 2006Go). Developmental stage was monitored using DIC optics, which did not allow the discrimination between classes 3 and 4. The proportion of worms belonging to each class was scored 32 hours after egg laying (N≥12 individuals). Error bar represents s.e.m., n=3 independent experiments.

 

Figure 4
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  		Fig. 4. catp-1 functions in parallel with UNC-63-nAChR and lipophilic hormone signaling. Survival of wild-type and mutant animals developing on 1 mM DMPP. Error bars represent s.e.m. (n≥3 independent experiments, N≥287 individuals). catp-1(kr17) unc-63(kr13) and catp-1(kr17); daf-12(rh61rh411) double mutants are significantly more DMPP resistant than either single mutant alone (*P<0.05, **P<0.005, Mann-Whitney test).

 

Figure 5
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  		Fig. 5. catp-1 specifically interacts with the daf-2/InsR branch of the dauer pathway to control dauer formation and morphogenesis. (A) Aberrant morphogenesis of catp-1(kr17) dauer larvae induced by exposure to dauer pheromone. Compared with wild-type dauer larvae (upper row), catp-1(kr17) dauer larvae are short (left), with a constricted pharynx (middle), and are trapped in their L2 cuticle (right, arrowheads). Scale bar: dissecting scope, 50 µm (left); Nomarski microscopy, 10 µm (middle and right). (B) Schematic representation of the dauer pathway, adapted from Beckstead and Thummel (Beckstead and Thummel, 2006Go), Gerisch and Antebi (Gerisch and Antebi, 2004Go) and Riddle (Riddle and Albert, 1997Go). (C) Genetic interactions between catp-1(kr17) and mutants of the dauer pathway: dauer formation at 25°C scored by visual inspection. age-1(mg44), daf-7(e1372) and daf-9(dh6) are null alleles. The arrow points to the suppression of constitutive dauer formation in catp-1(kr17); daf-2(m41). * defective dauer morphogenesis. Error bars represent s.e.m. (n≥3 independent experiments, N≥89 individuals). (D) Aberrant morphogenesis of catp-1(kr17); daf-2(m596) dauer larvae. catp-1(kr17); daf-2(m596) dauer larvae (bottom row) are short with a constricted pharynx which appears squeezed in the head (left), abnormal alae (middle) and normal small gonads (right). Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. CATP-1 functions independently of DAF-16/FOXO to modulate DAF-2/InsR signaling. Survival on 0.75 mM DMPP. No dauer larvae were observed in daf-2(e1370) under these experimental conditions. Error bars represent s.e.m. (n≥3 independent experiments, N≥215 individuals) (*P<0.05, Mann-Whitney test).

 

Figure 7
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  		Fig. 7. DAF-2 and CATP-1 interact with the Ras-MAPK signaling pathway. (A) Dauer formation at 25°C scored by visual inspection. Error bars represent s.e.m. (n≥3 independent experiments, N≥72 individuals). let-60(n1046) partially suppresses daf-2(m41) but not daf-2(e1370) constitutive dauer formation (*P<0.05, Mann-Whitney test). (B) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n≥3 independent experiments, N≥136 individuals). mek-2(n1989), and ksr-1(n2526) are significantly more DMPP resistant than N2. let-60(n1046) strongly suppresses catp-1(kr17) and daf-2(m41) DMPP resistance (*P<0.05, **P<0.005, Mann-Whitney test).

 

Figure 8
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  		Fig. 8. A genetic model for CATP-1 action in L2 developmental timing and dauer formation. (A) Control of L2 developmental timing. CATP-1 speeds up both a cell division and a molting timer independently of the UNC-63/nAChR and DAF-12/NHR pathways described in Ruaud and Bessereau (Ruaud and Bessereau, 2006Go). daf-2 mutant DMPP resistance probably results from a similar developmental delay and involves both the DAF-16/FOXO and Ras-MAPK pathways. CATP-1 effect on developmental timing likely involves a Ras-MAPK branch of the daf-2 pathway. Whether catp-1 directly interferes with daf-2/InsR, modulates Ras/MAPK activity and/or functions through a third unidentified parallel pathway remains equally possible at this stage. Dashed lines: hypothetical interactions. (B) Dauer formation. In addition to the DAF-16/FOXO and DAF-12/NHR pathways, DAF-2/InsR controls dauer formation through a Ras-MAPK pathway. CATP-1 is likely to modulate dauer formation by negatively interacting with this Ras-MAPK branch, but could also directly alter daf-2/InsR signaling or work through an uncharacterized parallel pathway (see the text for a full discussion).

 




© The Company of Biologists Ltd 2007