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Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.

Lee HO, Zacharek SJ, Xiong Y, Duronio RJ - Mol. Biol. Cell (2010)

Bottom Line: In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration.We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation.These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

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DupΔPIP arrests the cell cycle in interphase. (A and B) DupFL-GFP (A) or DupΔPIP-GFP (B)-expressing dupa1  cells stained with anti-CycA (red) and anti-GFP (green). (C and D) DupFL-GFP– (C) or DupΔPIP-GFP– (D) expressing WT cells stained with anti-GFP (green, C and D), anti-CycA (red, C′ and D′), and anti-Dlg (white, C″ and D″). Note the larger cell size in the left side of D″ relative to the right side, indicating cell cycle arrest caused by DupΔPIP expression.
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Figure 7: DupΔPIP arrests the cell cycle in interphase. (A and B) DupFL-GFP (A) or DupΔPIP-GFP (B)-expressing dupa1 cells stained with anti-CycA (red) and anti-GFP (green). (C and D) DupFL-GFP– (C) or DupΔPIP-GFP– (D) expressing WT cells stained with anti-GFP (green, C and D), anti-CycA (red, C′ and D′), and anti-Dlg (white, C″ and D″). Note the larger cell size in the left side of D″ relative to the right side, indicating cell cycle arrest caused by DupΔPIP expression.

Mentions: To distinguish between these two possibilities, we assessed whether cell division occurred by first examining cell size. Each epidermal cell division during Drosophila embryogenesis results in a reduction in cell size (Lehner and O'Farrell, 1989). Thus, if the Dup transgenes were able to support progression through mitosis and cell division, then the cells would be smaller than the dup neighbors. To assess cell size, embryos were stained for the membrane protein Discs large (Dlg), and the size of the cells within and outside the domain of Dup transgene expression was quantified. Although DupFL-expressing cells were approximately half the size of their dup mutant neighbors (Figure 6, C″ and E), DupΔPIP-expressing cells remained the same size as their neighbors (Figures 6D″ and E). This finding suggests that DupFL can rescue the dup cell phenotype and support completion of the cell cycle, whereas DupΔPIP-expressing dup cells remain in interphase and do not enter mitosis. To test this assertion, we detected cyclin A protein, which should accumulate in cells arrested in interphase of cycle 16 but not in cells that divide and enter the following G1 phase of cycle 17 (Lehner and O'Farrell, 1989). The DupΔPIP-expressing cells accumulate high levels of cyclin A (Figure 7B), whereas the DupFL cells do not (Figure 7A). Together these data indicate that DupFL transgenic protein provides normal Dup function and rescues the replication and cell cycle defect of dup cells, whereas DupΔPIP does not.


Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.

Lee HO, Zacharek SJ, Xiong Y, Duronio RJ - Mol. Biol. Cell (2010)

DupΔPIP arrests the cell cycle in interphase. (A and B) DupFL-GFP (A) or DupΔPIP-GFP (B)-expressing dupa1  cells stained with anti-CycA (red) and anti-GFP (green). (C and D) DupFL-GFP– (C) or DupΔPIP-GFP– (D) expressing WT cells stained with anti-GFP (green, C and D), anti-CycA (red, C′ and D′), and anti-Dlg (white, C″ and D″). Note the larger cell size in the left side of D″ relative to the right side, indicating cell cycle arrest caused by DupΔPIP expression.
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Related In: Results  -  Collection

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Figure 7: DupΔPIP arrests the cell cycle in interphase. (A and B) DupFL-GFP (A) or DupΔPIP-GFP (B)-expressing dupa1 cells stained with anti-CycA (red) and anti-GFP (green). (C and D) DupFL-GFP– (C) or DupΔPIP-GFP– (D) expressing WT cells stained with anti-GFP (green, C and D), anti-CycA (red, C′ and D′), and anti-Dlg (white, C″ and D″). Note the larger cell size in the left side of D″ relative to the right side, indicating cell cycle arrest caused by DupΔPIP expression.
Mentions: To distinguish between these two possibilities, we assessed whether cell division occurred by first examining cell size. Each epidermal cell division during Drosophila embryogenesis results in a reduction in cell size (Lehner and O'Farrell, 1989). Thus, if the Dup transgenes were able to support progression through mitosis and cell division, then the cells would be smaller than the dup neighbors. To assess cell size, embryos were stained for the membrane protein Discs large (Dlg), and the size of the cells within and outside the domain of Dup transgene expression was quantified. Although DupFL-expressing cells were approximately half the size of their dup mutant neighbors (Figure 6, C″ and E), DupΔPIP-expressing cells remained the same size as their neighbors (Figures 6D″ and E). This finding suggests that DupFL can rescue the dup cell phenotype and support completion of the cell cycle, whereas DupΔPIP-expressing dup cells remain in interphase and do not enter mitosis. To test this assertion, we detected cyclin A protein, which should accumulate in cells arrested in interphase of cycle 16 but not in cells that divide and enter the following G1 phase of cycle 17 (Lehner and O'Farrell, 1989). The DupΔPIP-expressing cells accumulate high levels of cyclin A (Figure 7B), whereas the DupFL cells do not (Figure 7A). Together these data indicate that DupFL transgenic protein provides normal Dup function and rescues the replication and cell cycle defect of dup cells, whereas DupΔPIP does not.

Bottom Line: In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration.We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation.These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

Show MeSH
Related in: MedlinePlus