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Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

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Nuclear pore disruption in flo mutants.(A–C) Confocal projections through the posterior intestine of 75 hpf wild type (A), flo (B), and elys morpholino injected larvae (C), following anti-FG nucleoporin immunostainings with mAb414 (green; DAPI–blue). There is a dramatic reduction of nuclear pores in the flo and morpholino injected larvae. Inset shows higher magnification of localized regions of the DAPI-stained image. (D–F) Identical findings are evident in the retina of these larvae. Note apparent cytoplasmic accumulation of the immunoreactive FG-nucleoporins in flo and morpholino injected larvae. (G,H) Normal nuclear distribution of FG nucleoporins in wild type and flo skeletal muscle. (I) Western blot showing levels of FG nucleoporin proteins relative to beta-actin in nuclear (nucl) and cytoplasmic (cyto) extracts derived from the intestine of 75 hpf flo and sibling wild type larvae.
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pgen-1000240-g004: Nuclear pore disruption in flo mutants.(A–C) Confocal projections through the posterior intestine of 75 hpf wild type (A), flo (B), and elys morpholino injected larvae (C), following anti-FG nucleoporin immunostainings with mAb414 (green; DAPI–blue). There is a dramatic reduction of nuclear pores in the flo and morpholino injected larvae. Inset shows higher magnification of localized regions of the DAPI-stained image. (D–F) Identical findings are evident in the retina of these larvae. Note apparent cytoplasmic accumulation of the immunoreactive FG-nucleoporins in flo and morpholino injected larvae. (G,H) Normal nuclear distribution of FG nucleoporins in wild type and flo skeletal muscle. (I) Western blot showing levels of FG nucleoporin proteins relative to beta-actin in nuclear (nucl) and cytoplasmic (cyto) extracts derived from the intestine of 75 hpf flo and sibling wild type larvae.

Mentions: NPC disassembly caused by ELYS knockdown in HeLa cells leads to redistribution of nucleoporins from the nuclear envelope to the cytoplasm [20]–[21]. Immunohistochemical analyses of zebrafish nucleoporins using a monoclonal antibody that recognizes nucleoporins with a highly conserved FG domain [37] showed typical nuclear staining in all wild type zebrafish tissues. By contrast, a dramatic reduction of nuclear FG-nucleoporins was evident in flo intestinal and retinal epithelial cells (Figure 4A–4F). A normal FG-nucleoporin immunostaining pattern was clearly evident in flo skeletal muscle (Figure 4G–4H), pronephric duct epithelia and other non-proliferative tissues (data not shown). The flo retinal NPC defects were evident as early as 36 hpf, and the intestinal NPC defects could be seen at 48 hpf (Figure S5). FG-nucleoporin immunostainings from flo and elys morpholino injected larvae were nearly identical and closely resembled those of ELYS-deficient HeLa and U2OS cells [20]–[21] thus suggesting partial cytoplasmic redistribution of these nucleoporins. Western analyses of nuclear and cytoplasmic fractions of flo intestinal proteins confirmed these findings (Figure 4I). Similarly, ultrastructural analyses showed a marked reduction in the number of identifiable NPCs in the flo intestinal epithelial cells (Figure 5) and also showed that the flo mutation had no effect on nuclear envelope formation or stability, as was reported in ELYS-deficient mammalian cells [21]. Together, these data confirm a role for zebrafish Elys in NPC assembly.


Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Nuclear pore disruption in flo mutants.(A–C) Confocal projections through the posterior intestine of 75 hpf wild type (A), flo (B), and elys morpholino injected larvae (C), following anti-FG nucleoporin immunostainings with mAb414 (green; DAPI–blue). There is a dramatic reduction of nuclear pores in the flo and morpholino injected larvae. Inset shows higher magnification of localized regions of the DAPI-stained image. (D–F) Identical findings are evident in the retina of these larvae. Note apparent cytoplasmic accumulation of the immunoreactive FG-nucleoporins in flo and morpholino injected larvae. (G,H) Normal nuclear distribution of FG nucleoporins in wild type and flo skeletal muscle. (I) Western blot showing levels of FG nucleoporin proteins relative to beta-actin in nuclear (nucl) and cytoplasmic (cyto) extracts derived from the intestine of 75 hpf flo and sibling wild type larvae.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570612&req=5

pgen-1000240-g004: Nuclear pore disruption in flo mutants.(A–C) Confocal projections through the posterior intestine of 75 hpf wild type (A), flo (B), and elys morpholino injected larvae (C), following anti-FG nucleoporin immunostainings with mAb414 (green; DAPI–blue). There is a dramatic reduction of nuclear pores in the flo and morpholino injected larvae. Inset shows higher magnification of localized regions of the DAPI-stained image. (D–F) Identical findings are evident in the retina of these larvae. Note apparent cytoplasmic accumulation of the immunoreactive FG-nucleoporins in flo and morpholino injected larvae. (G,H) Normal nuclear distribution of FG nucleoporins in wild type and flo skeletal muscle. (I) Western blot showing levels of FG nucleoporin proteins relative to beta-actin in nuclear (nucl) and cytoplasmic (cyto) extracts derived from the intestine of 75 hpf flo and sibling wild type larvae.
Mentions: NPC disassembly caused by ELYS knockdown in HeLa cells leads to redistribution of nucleoporins from the nuclear envelope to the cytoplasm [20]–[21]. Immunohistochemical analyses of zebrafish nucleoporins using a monoclonal antibody that recognizes nucleoporins with a highly conserved FG domain [37] showed typical nuclear staining in all wild type zebrafish tissues. By contrast, a dramatic reduction of nuclear FG-nucleoporins was evident in flo intestinal and retinal epithelial cells (Figure 4A–4F). A normal FG-nucleoporin immunostaining pattern was clearly evident in flo skeletal muscle (Figure 4G–4H), pronephric duct epithelia and other non-proliferative tissues (data not shown). The flo retinal NPC defects were evident as early as 36 hpf, and the intestinal NPC defects could be seen at 48 hpf (Figure S5). FG-nucleoporin immunostainings from flo and elys morpholino injected larvae were nearly identical and closely resembled those of ELYS-deficient HeLa and U2OS cells [20]–[21] thus suggesting partial cytoplasmic redistribution of these nucleoporins. Western analyses of nuclear and cytoplasmic fractions of flo intestinal proteins confirmed these findings (Figure 4I). Similarly, ultrastructural analyses showed a marked reduction in the number of identifiable NPCs in the flo intestinal epithelial cells (Figure 5) and also showed that the flo mutation had no effect on nuclear envelope formation or stability, as was reported in ELYS-deficient mammalian cells [21]. Together, these data confirm a role for zebrafish Elys in NPC assembly.

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Show MeSH
Related in: MedlinePlus