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CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans.

Gaggioli V, Zeiser E, Rivers D, Bradshaw CR, Ahringer J, Zegerman P - J. Cell Biol. (2014)

Bottom Line: We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos.By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans.These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

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Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, 2 Department of Genetics, and 3 Department of Zoology, University of Cambridge, Cambridge CB2 1QN, England, UK.

ABSTRACT
Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

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C. elegans sld-2 is essential. (A) Mean embryonic lethality after sld-2(RNAi) by injection into young adult N2 or RNAi-sensitive we9 worms at 15°C (n = 11). Embryo lethality is scored as the number of unhatched eggs relative to the total number of F1 eggs. (B) Mean embryonic lethality for wild-type N2 and div-1(or148ts) worms with and without sld-2(RNAi) injection at 21.5°C, 0–24 and 24–48 h after egg laying. n = 20 except for sld-2(RNAi) in which n = 51. (C) The timing of cell division in early embryos, extruded from N2 worms with or without injection of sld-2 RNAi, was analyzed by time-lapse differential interference contrast (DIC) imaging. The lengths of time after P0 cell nuclear envelope breakdown until the onset of nuclear envelope breakdown in the AB blastomere or the P1 blastomere are shown. n = 9. (D) Images of the early C. elegans embryo from we9 worms with or without sld-2 (RNAi) by injection. SLD-2 was visualized by anti–SLD-2 immunofluorescence using affinity-purified Ab 5058. An arrow indicates DNA stuck in the plane of cytokinesis—the cut phenotype. (E) Differential interference contrast images showing C. elegans embryos of the same age, from mothers of the we9 strain either uninjected or injected with sld-2 (RNAi). Arrows indicate large undifferentiated cells. Error bars are SEMs. Bars, 10 µM.
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fig2: C. elegans sld-2 is essential. (A) Mean embryonic lethality after sld-2(RNAi) by injection into young adult N2 or RNAi-sensitive we9 worms at 15°C (n = 11). Embryo lethality is scored as the number of unhatched eggs relative to the total number of F1 eggs. (B) Mean embryonic lethality for wild-type N2 and div-1(or148ts) worms with and without sld-2(RNAi) injection at 21.5°C, 0–24 and 24–48 h after egg laying. n = 20 except for sld-2(RNAi) in which n = 51. (C) The timing of cell division in early embryos, extruded from N2 worms with or without injection of sld-2 RNAi, was analyzed by time-lapse differential interference contrast (DIC) imaging. The lengths of time after P0 cell nuclear envelope breakdown until the onset of nuclear envelope breakdown in the AB blastomere or the P1 blastomere are shown. n = 9. (D) Images of the early C. elegans embryo from we9 worms with or without sld-2 (RNAi) by injection. SLD-2 was visualized by anti–SLD-2 immunofluorescence using affinity-purified Ab 5058. An arrow indicates DNA stuck in the plane of cytokinesis—the cut phenotype. (E) Differential interference contrast images showing C. elegans embryos of the same age, from mothers of the we9 strain either uninjected or injected with sld-2 (RNAi). Arrows indicate large undifferentiated cells. Error bars are SEMs. Bars, 10 µM.

Mentions: Knockdown of sld-2 by RNAi results in embryonic lethality, and in an RNAi-sensitive worm background (we9), this embryonic lethality approaches 100% after 24 h, confirming that sld-2 is an essential gene in C. elegans (Fig. 2 A). Consistent with a role for sld-2 in DNA replication, sld-2 RNAi results in synthetic lethality with a temperature-sensitive mutant in the B subunit of DNA polymerase α (div-1) at the semipermissive temperature (Fig. 2 B; Encalada et al., 2000). Replication defects in C. elegans embryos are characterized by a long delay in the division of the P1 blastomere after division of the AB blastomere in the second embryonic cell cycle (Encalada et al., 2000). In accordance with a role for sld-2 in DNA replication, RNAi of sld-2 produces a robust P1 division delay (Fig. 2 C). Further analysis of the embryonic phenotype after knockdown of sld-2 revealed that chromosomes are not effectively segregated before division (the “cut” phenotype; Fig. 2 D), and embryos arrest division with abnormally large and undifferentiated cells (Fig. 2 E). These defects are all characteristic of DNA replication mutants (Hirano et al., 1986; Encalada et al., 2000).


CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans.

Gaggioli V, Zeiser E, Rivers D, Bradshaw CR, Ahringer J, Zegerman P - J. Cell Biol. (2014)

C. elegans sld-2 is essential. (A) Mean embryonic lethality after sld-2(RNAi) by injection into young adult N2 or RNAi-sensitive we9 worms at 15°C (n = 11). Embryo lethality is scored as the number of unhatched eggs relative to the total number of F1 eggs. (B) Mean embryonic lethality for wild-type N2 and div-1(or148ts) worms with and without sld-2(RNAi) injection at 21.5°C, 0–24 and 24–48 h after egg laying. n = 20 except for sld-2(RNAi) in which n = 51. (C) The timing of cell division in early embryos, extruded from N2 worms with or without injection of sld-2 RNAi, was analyzed by time-lapse differential interference contrast (DIC) imaging. The lengths of time after P0 cell nuclear envelope breakdown until the onset of nuclear envelope breakdown in the AB blastomere or the P1 blastomere are shown. n = 9. (D) Images of the early C. elegans embryo from we9 worms with or without sld-2 (RNAi) by injection. SLD-2 was visualized by anti–SLD-2 immunofluorescence using affinity-purified Ab 5058. An arrow indicates DNA stuck in the plane of cytokinesis—the cut phenotype. (E) Differential interference contrast images showing C. elegans embryos of the same age, from mothers of the we9 strain either uninjected or injected with sld-2 (RNAi). Arrows indicate large undifferentiated cells. Error bars are SEMs. Bars, 10 µM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3926958&req=5

fig2: C. elegans sld-2 is essential. (A) Mean embryonic lethality after sld-2(RNAi) by injection into young adult N2 or RNAi-sensitive we9 worms at 15°C (n = 11). Embryo lethality is scored as the number of unhatched eggs relative to the total number of F1 eggs. (B) Mean embryonic lethality for wild-type N2 and div-1(or148ts) worms with and without sld-2(RNAi) injection at 21.5°C, 0–24 and 24–48 h after egg laying. n = 20 except for sld-2(RNAi) in which n = 51. (C) The timing of cell division in early embryos, extruded from N2 worms with or without injection of sld-2 RNAi, was analyzed by time-lapse differential interference contrast (DIC) imaging. The lengths of time after P0 cell nuclear envelope breakdown until the onset of nuclear envelope breakdown in the AB blastomere or the P1 blastomere are shown. n = 9. (D) Images of the early C. elegans embryo from we9 worms with or without sld-2 (RNAi) by injection. SLD-2 was visualized by anti–SLD-2 immunofluorescence using affinity-purified Ab 5058. An arrow indicates DNA stuck in the plane of cytokinesis—the cut phenotype. (E) Differential interference contrast images showing C. elegans embryos of the same age, from mothers of the we9 strain either uninjected or injected with sld-2 (RNAi). Arrows indicate large undifferentiated cells. Error bars are SEMs. Bars, 10 µM.
Mentions: Knockdown of sld-2 by RNAi results in embryonic lethality, and in an RNAi-sensitive worm background (we9), this embryonic lethality approaches 100% after 24 h, confirming that sld-2 is an essential gene in C. elegans (Fig. 2 A). Consistent with a role for sld-2 in DNA replication, sld-2 RNAi results in synthetic lethality with a temperature-sensitive mutant in the B subunit of DNA polymerase α (div-1) at the semipermissive temperature (Fig. 2 B; Encalada et al., 2000). Replication defects in C. elegans embryos are characterized by a long delay in the division of the P1 blastomere after division of the AB blastomere in the second embryonic cell cycle (Encalada et al., 2000). In accordance with a role for sld-2 in DNA replication, RNAi of sld-2 produces a robust P1 division delay (Fig. 2 C). Further analysis of the embryonic phenotype after knockdown of sld-2 revealed that chromosomes are not effectively segregated before division (the “cut” phenotype; Fig. 2 D), and embryos arrest division with abnormally large and undifferentiated cells (Fig. 2 E). These defects are all characteristic of DNA replication mutants (Hirano et al., 1986; Encalada et al., 2000).

Bottom Line: We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos.By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans.These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, 2 Department of Genetics, and 3 Department of Zoology, University of Cambridge, Cambridge CB2 1QN, England, UK.

ABSTRACT
Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

Show MeSH
Related in: MedlinePlus