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First published online 14 July 2004
doi: 10.1242/dev.01255

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The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span

Molecular Biology Program and Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA

View larger version (67K): [in a new window]   Fig. 1. daf-15 encodes the C. elegans raptor. (A) Physical map of the daf-15 region of chromosome IV (corresponding 0.6 map units). (B) A schematic structure of DAF-15 raptor. The predicted RNC domain, HEAT (Huntingtin, elongation factor 3, the regulatory A subunit of PP2A and Tor1p) repeats and WD40 repeats are indicated. Cosmid C10C5 partially rescues a daf-15 mutant, but lacks the last three exons of daf-15 encoding the last four WD40 repeats. Sequence alterations in m81 and m634 are shown. (C) Alignment of the three blocks of the DAF-15 RNC domain with its orthologs. Identical amino acids are in black boxes. The star in block 1 indicates the G194S change in m81. W440 (black dot) is changed to a TGA stop in m634. The numbers beside the sequences indicate the position of the beginning and ending amino acids in the corresponding protein. The GenBank Accession Number of the daf-15 cDNA sequence is AY396716.

View larger version (143K): [in a new window]   Fig. 2. daf-15p::gfp expression in N2 L4 larvae. Confocal images of selected planes at the same magnification show (A) head, (B) mid-body and (C) tail regions. n, nervous system; p, pharynx; ec, excretory cell; ga, gut autofluorescence; dtc, distal tip cell; m, body wall muscle; int, intestine; hc, hypodermal cell. Scale bar: 20 µm.

View larger version (151K): [in a new window]   Fig. 3. daf-15 and let-363 metabolic defect. Fat accumulation was assayed by Sudan black staining in L3 larvae grown at 20°C; daf-2 dauer larvae were formed constitutively at 25°C. (A) Wild-type N2, (B) daf-2(e1370), (C) daf-15(m81), (D) let-363(h111), (E) daf-16(mgDf47), (F) daf-16(mgDf47); daf-15(m81). All mutants except for the daf-16 single mutant accumulate abnormally high levels of fat relative to wild type. The entire experiment was performed twice, with at least 30 animals observed each time. Fat accumulation in daf-15(m81) and let-363(h111) was also confirmed by Nile red staining (data not shown).

View larger version (15K): [in a new window]   Fig. 4. Age phenotype of daf-15/+ mutants in different genetic backgrounds. Each survival curve depicts one of the two trials presented in Table 1. The life spans were determined from the percentage of worms alive on a given day at 25°C. (A) + dpy-20/daf-15(m81) +, (B) unc-24 +/+ daf-15(m81), (C) unc-24 daf-15(m634)/nT1, (D) daf-2; + dpy-20/daf-15(m81) + and (E) daf-16; + dpy-20/daf-15(m81) +. The daf-15/+ strains show extended longevity, but the m81/+ longevity phenotype is suppressed by daf-16.

View larger version (43K): [in a new window]   Fig. 5. DAF-16 negatively regulates the expression of daf-15. (A) Semi-quantitative RT-PCR shows that expression of daf-15 is negatively regulated by DAF-16. The control rpl-21 gene (encoding the large ribosomal subunit L21 protein) was equally represented in both RNA preparations. The experiment was performed three times using three independent RNA preparations. (B) EMSA indicates DAF-16 binds specifically to the daf-15 IRS in vitro (see text).

View larger version (19K): [in a new window]   Fig. 6. A model for regulation of C. elegans larval development, metabolism and longevity. Proposed wild-type functions are shown, with arrows indicating stimulation of activities and T-bars indicating inhibition, but the steps do not indicate direct protein interactions. Bold arrows represent changes in wild-type activities. Both daf-15 and daf-2 pathways are essential for larval development. Null mutations result in larval lethality. (A) When food is abundant, LET-363/DAF-15 transduces a sufficient nutrient signal to permit growth to the reproductive adult. This signal is also required for DAF-2/insulin signaling to stimulate growth. Disruption of either pathway will cause larval arrest at the second molt. Food availability could also regulate the DAF-2/insulin pathway. (B) When nutrients are limited, the LET-363/DAF-15 signal is insufficient to prevent larval arrest. With concomitant down regulation of the DAF-2 pathway, animals will enter the dauer stage. When DAF-16 activity is high, TOR activity is low, and vice versa. However, in a daf-15 or let-363 mutant TOR activity is low even when insulin/IGF signaling is high, resulting in activation of some target functions, such as autophagy, but the reduced activity of DAF-16 fails to activate other functions needed to complete dauer morphogenesis. daf-15 is epistatic to daf-2 because some targets of TOR that are essential for dauer morphogenesis fail to be activated in the absence of DAF-15 function. Essentially, knockout of LET-363/DAF-15 activity results in dauer-like arrest regardless of DAF-2 signaling because LET-363/DAF-15 is required for both dauer and non-dauer development. TOR regulates autophagy, which is required for dauer morphogenesis and for the increased longevity of daf-2 adults, but TOR activity is also required for maturation to the adult. DAF-16 and LET-363/DAF-15 have other downstream targets not shown here.