First published online 16 August 2006
doi: 10.1242/dev.02534
Development 133, 3597-3606 (2006)
Published by The Company of Biologists 2006
Deleted in cancer 1 (DICE1) is an essential protein controlling the topology of the inner mitochondrial membrane in C. elegans
Sung Min Han1,*,
Tae Hoon Lee1,*,
Ji Young Mun2,*,
Moon Jeong Kim1,
Ekaterini A. Kritikou3,
Se-Jin Lee1,
Sung Sik Han2,
Michael O. Hengartner3 and
Hyeon-Sook Koo1,
1 Department of Biochemistry, College of Science, Yonsei University, Seoul
120-749, Korea.
2 School of Life Sciences and Biotechnology, Korea University, Seoul 136-701,
Korea.
3 Institute of Molecular Biology, University of Zurich, 8057 Zurich,
Switzerland.
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Fig. 1. Evolutionary conservation of DICE1. (A) Amino acid sequence
alignment of DICE1 homologs in C. elegans, human, mouse and fly. The
striped boxes represent von Willebrand factor type A motifs, and the gray and
white boxes are homologous and nonconserved regions, respectively. The
percentages refer to identical amino acids in the human homolog and the other
proteins. (B) The amino acid sequences of the human and C.
elegans DICE1 homologs were compared using the Vector NTI Advance program
(Invitrogen). Dark and light gray backgrounds of letters signify identical and
similar amino acids, respectively.
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Fig. 2. Abnormal morphogenesis and tissue-specific gene expression in
dic-1 (RNAi) embryos. RNA interference was carried out
from the L4 stage by feeding bacteria producing double-stranded RNA.
(A) Time lapse microscopy of embryonic development using Nomarski
optics. Developmental stage and time post fertilization (in minutes) are
indicated for each wild-type embryo. dic-1(RNAi) embryos
laid at the same time as the wild-type embryos were arrested after the bean
cell stage but before the comma stage. (B) Embryos obtained after
RNAi of dic-1 in wild-type N2 and ced-3(n717),
after longer incubation than in A. Cavities are marked with arrowheads.
(C) Wild-type embryos at the threefold stage and
dic-1(RNAi) embryos at the same time post fertilization,
showing Psnap-25::gfp expression in neurons,
ajm-1::gfp expression in the junctions of intestinal cells and
unc-15::gfp expression in muscle cells. Scale bars: 10 µm.
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Fig. 3. Increased apoptosis in dic-1(RNAi) embryos.
(A) Apoptotic cell corpses (arrowheads) were counted in 380 cell or
bean cell stage embryos of wild type N2 strain with or without dic-1
knockdown. This procedure was repeated with ced-3(n717) strain.
(B) TUNEL-positive cells in wild-type and dic-1(RNAi)
embryos were scored with a fluorescence microscope at the same embryonic
stages as in A. Scale bars: 10 µm. Error bars indicate standard errors of
the mean (s.e.m.).
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Fig. 4. Increased apoptosis and cell cavity formation in
dic-1(RNAi) gonads. (A) Apoptotic cell corpses
were counted in the germline with Nomarski optics after feeding C.
elegans worms with dsRNA encoding dic-1 for 48 hours from the L4
stage. Germ cell corpses in the loop region of the gonad are indicated by the
boxed area. The effects of apoptosis regulators ced-3(lf),
ced-3(n717); ced-4(lf), ced-4(n1162); cep-1(lf) and
cep-1(gk138) on the induction of apoptosis by
dic-1(RNAi) are plotted in the graph. Error bars indicate
standard errors. (B) RNAi was performed from the L1 stage and
resulted in various phenotypes in the gonad, including undersized gonads,
defective oocyte development and cavities. Cavities (brackets) were first
observed in the young adult stage gonad but later expanded to the distal tip
region of the gonad at the adult stage. ced-3(lf), ced-3(n717). V,
vulva; Ds, distal tip region; Pr, proximal region. Scale bars: 25 µm.
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Fig. 5. Immunolocalization of DIC-1 in mitochondria. (A-C)
Immunolocalization of DIC-1 in embryos and germline by fluorescence
microscopy. (A) Wild-type and dic-1(RNAi) embryos.
(B) In wild-type gonads, DIC-1 was restricted to the cytoplasm from the
distal tip cells to the oocytes. (C) Co-localization of DIC-1 with
cytochrome c oxidase subunit 1 within mitotic cells of the gonad. (D)
Electron micrograph (left) showing immunogold particles (arrowheads) in
mitochondria: the bracketed area is enlarged in the right micrograph. Scale
bars: 10 µm in A; 50 µm in B; 25 µm in C; 0.2 µm in D.
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Fig. 6. Subcellular localization of GFP-tagged DIC-1 in embryonic and muscle
cells by fluorescence microscopy. (A) Expression of GFP-tagged
DIC-1 under the control of the hsp-16.2 promoter was induced by heat
shock in N2 worms and spotted expression was observed in the cytoplasm of
early embryos. (B) GFP-tagged DIC-1 was expressed under the control of
the myo-3 promoter in N2 worms and expression was observed in the
mitochondria of body-wall muscle cells. N, nucleus. Scale bars: 10 µm in A;
5 µm in B.
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Fig. 7. Disrupted morphology and inner structure of mitochondria in
DIC-1(RNAi) muscle cells observed by fluorescence microscopy and
cryo-electron microscopy. (A) The musculature of
dic-1(RNAi) worms shows abnormal morphology of active
mitochondria stained with Mitotracker Red. (B) The mitochondrial matrix
visualized with a mitochondrial matrix-targeting sequence::GFP construct
controlled by the myo-3 promoter. (C) Electron micrographs of
mitochondria (enclosed by brackets) in wild-type and
dic-1(RNAi) muscle cells. Scale bars: 10 µm in A; 5 µm
in B; 0.2 µm in C.
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Fig. 8. L3 stage growth arrest of dic-1(tm1615). (A)
Schematic representation of the dic-1 gene and the region deleted in
the dic-1(tm1615) mutation. (B) A dic-1(tm1615) L3
larva with cavities in all tissues observed with Nomarski optics. Cavities are
marked with arrowheads. Scale bar: 50 µm.
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© The Company of Biologists Ltd 2006
