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Development 130 (6): 1089-1099
Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C. elegans
Takashi Hirose*, ,
Yoshiya Nakano,
Yasuko Nagamatsu,
Takashi Misumi,
Hiromitsu Ohta, and
Yasumi Ohshima
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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