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doi: 10.1242/10.1242/dev.00330

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Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C. elegans

Takashi Hirose*,{dagger}, Yoshiya Nakano{dagger}, Yasuko Nagamatsu, Takashi Misumi, Hiromitsu Ohta and Yasumi Ohshima{ddagger}

Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan
* Present address: Research Institute of Microbial Diseases, Osaka University, Yamadaoka, Suita 565-0871, Japan



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  		Fig. 6. Three-dimensional (3D) images of organs and a cell in the wild type. 3D reconstructed images of intestine (A), hypodermis (B), muscles expressing myo-3p::gfp (C), a body wall muscle cell (D) and a dissected half gonad (E) are shown. The stages are 4-day old (A-D) or 2-day old (E) adults.

 


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  		Fig. 1. Isolation of larger body size mutants. (A) Body volumes of 4-day old adults. The numbers of examined animals are as follows: N2 476, ks16 86, ks60 211, ks61 168, ks62 80, n477 53, n478 17, Ex[myo-3::gfp] 42, ks61/+;Ex[myo-3::gfp] 41, ks60/+ 19, him-5 (e1490) male 10, ks61;him-5 male 15, lon-1(e185) 10, lon-2(e678) 43, lon-3(e2175) 43, daf-4(m63) 13, daf-16(mgDf50) 32, daf-16(mgDf50);ks61 37 (results with two lines), daf-2 (e1370) 67, daf-2 (e1370);ks61 157 (results with three lines), daf-3 (mgDf90) 46, ks61;daf-3 (results with three lines) 74, sma-6 (e1482) 29, sma-6;ks61 52, dbl-1 (nk3) 86 and ks61;dbl-1 32. Error bars indicate standard deviation (s.d.) (B) DIC microphotographs of a 4-day old adult of N2 (top) and a larger body size mutant (ks60, bottom). The pictures were taken in 5-8 parts per animal using an AxioCam CCD camera mounted on Zeiss Axiophot 2 with a 20x/0.5 lens and assembled. Scale bar: 200 µm.

 


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  		Fig. 2. Growth and lifespan of the mutants and wild type. (A) Growth curves. The worms were synchronized by allowing them to hatch for 4 hours and then grown at 20°C. Most worms became adults before 51 hours. Each point represents results on an independent culture plate. Average numbers of the examined animals at a point are as follows: N2, 32; ks60, 31; ks61, 25. Error bar indicates s.d. (B) Lifespan. The first time points represent the day when synchronized L1 larvae were put on NGM plates. Mean lifespan, standard deviation, number of examined animals excluding the missing ones/total number of examined animals, and number of tests performed independently are as follows: N2, 14.5±4.1 days, 207/300(6); ks60, 21.5±8.7 days, 80/150(2); ks61, 22.4±8.2 days, 143/200(4); daf-16(m26), 15.5±3.6 days, 131/150(3); daf-16(m26);ks61, 14.8±3.5 days, 88/150(3).

 


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  		Fig. 3. Identification of the egl-4 gene. (A) Physical map of the egl-4 region, related genomic clones, exon-intron organization of F55A8.2 and structure of mRNAs. Black boxes indicate protein-coding regions. Red boxes represent 5' extensions of exon 8 in mRNAs for a2 and b2. (B) Amino acid sequences of PKGa1 and PKGb1 predicted by the cDNAs. They differ only in the N termini (the sequence of a1 is the upper line). Underlines denote a glycine-rich domain, two cGMP binding domains and a kinase domain. Double underlines denote putative NLS (see Discussion). (C) Schematic of the primary structure of the EGL-4(PKGa1) protein and the mutation sites. Blue box represents the glycine-rich domain.

 


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  		Fig. 4. Expression patterns of egl-4 promoter::gfp/nls fusions. Larvae (A-E,G) and an embryo (F) expressing green fluorescent protein (GFP) under promoter a (A-F) or promoter b (G). B and C are higher magnification images of head neurons (B) and a part of hypodermis (C) of the worm shown in A. Stages are L3 (A-C), L4 (G), young adult (D,E) and embryo (F). A-C, E and G are lateral views, and D is a ventral view. The images were obtained with a Zeiss LSM-410 (A-C,G) or LSM-510 (D-F) confocal microscope. Scale bars: 50 µm (A,D,E,G) and 20 µm (B,F).

 


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  		Fig. 5. Characterization of endogenous EGL-4 proteins. (A) Extracts of wild-type N2 and indicated mutants (10 µg protein each) were separated in a 7.5% polyacrylamide-SDS gel, subjected to western blotting and probed with purified anti-EGL-4 antibodies. (B) Protein kinase activities in extracts from egl-4 (ks16) (lane 1) or N2 (lanes 2-6) were assayed in the absence (lane 2) and presence of 0.1 µM (lane 3), 1 µM (lane 4) or 10 µM (lanes 1, 5, 6) cGMP. For lane 6, the antibodies were preincubated with the antigen GST-EGL-4.

 

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