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The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans.

Liu HL, Osmani AH, Osmani SA - PLoS ONE (2015)

Bottom Line: How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood.We suggest the term "reboot regulation" to define this mode of cell cycle regulation.The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210, United States of America.

ABSTRACT
How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term "reboot regulation" to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

No MeSH data available.


Related in: MedlinePlus

Δsrc1 nuclei undergo architectural modifications from G1 that are typical of mitosis.(A) Δsrc1 spores were germinated in the presence of 10 mM HU to arrest them in interphase. NLS-DsRed was transported within the nucleus in Δsrc1 cells and Nup49 located around their nuclear periphery as typical of interphase Wt cells. (B-C) The localization of Nup2, NLS-DsRed, Bop1, and Nup49 were monitored in the first mitosis of Δsrc1 cells after release from G2 arrest imposed by the nimT23ts allele. (B) As occurs during normal mitosis, Nup2-GFP translocates to condensed chromatin and NLS-DsRed escapes from the nucleus during mitosis in Δsrc1 cells. However, soon after the two G1 nuclei are established and accumulate NLS-DsRed, with Nup2 now around their nuclear periphery, the nucleus indicated by the white arrowhead becomes mitotic-like with Nup2 locating to chromatin and losing its nuclear transport capacity. These effects were then largely reversed at the 21’ time point with the nucleus re-importing NLS-DsRed again. (C) As occurs during normal mitosis, Nup49 disperses from NPCs and returns to NPCs of the two new G1 nuclei generated during mitosis (0–8’). The nucleolus, marked by Bop1-GFP, then disassembles with the released nucleolar proteins being reimported into the new G1 nuclei. This process occurs normally in the absence of Src1 and is completed apparently normally (time point 17’). However the G1 nuclei are not normal as the nucleus to the right undergoes transitions typical of mitosis including the movement of Nup49 into the nucleus, presumably onto chromatin, and the disassembly of Bop1 after it is apparently expelled to the cytoplasm (indicated by a white arrowhead). This nucleolus then disassembles and Bop1 is imported into the transport competent daughter nucleus.
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pone.0132489.g005: Δsrc1 nuclei undergo architectural modifications from G1 that are typical of mitosis.(A) Δsrc1 spores were germinated in the presence of 10 mM HU to arrest them in interphase. NLS-DsRed was transported within the nucleus in Δsrc1 cells and Nup49 located around their nuclear periphery as typical of interphase Wt cells. (B-C) The localization of Nup2, NLS-DsRed, Bop1, and Nup49 were monitored in the first mitosis of Δsrc1 cells after release from G2 arrest imposed by the nimT23ts allele. (B) As occurs during normal mitosis, Nup2-GFP translocates to condensed chromatin and NLS-DsRed escapes from the nucleus during mitosis in Δsrc1 cells. However, soon after the two G1 nuclei are established and accumulate NLS-DsRed, with Nup2 now around their nuclear periphery, the nucleus indicated by the white arrowhead becomes mitotic-like with Nup2 locating to chromatin and losing its nuclear transport capacity. These effects were then largely reversed at the 21’ time point with the nucleus re-importing NLS-DsRed again. (C) As occurs during normal mitosis, Nup49 disperses from NPCs and returns to NPCs of the two new G1 nuclei generated during mitosis (0–8’). The nucleolus, marked by Bop1-GFP, then disassembles with the released nucleolar proteins being reimported into the new G1 nuclei. This process occurs normally in the absence of Src1 and is completed apparently normally (time point 17’). However the G1 nuclei are not normal as the nucleus to the right undergoes transitions typical of mitosis including the movement of Nup49 into the nucleus, presumably onto chromatin, and the disassembly of Bop1 after it is apparently expelled to the cytoplasm (indicated by a white arrowhead). This nucleolus then disassembles and Bop1 is imported into the transport competent daughter nucleus.

Mentions: To define the role of mitosis in generating Δsrc1 transport incompetent nuclei we germinated Δsrc1 spores from heterokaryons in the presence of hydroxyurea (HU), an inhibitor of DNA replication, to arrest them in interphase before their first mitosis [51]. Δsrc1 spores germinated in HU were able to transport NLS-DsRed into their single interphase nuclei (Fig 5A). We also arrested Δsrc1 cells in late G2 before entry into mitosis using the nimT23ts mutation that prevents activation of Cdk1 [45]. At the G2 arrest point of nimT23tsΔsrc1 nuclei were transport competent (example in Fig 5B, 0’). These results indicate that nuclear transport is active in Δsrc1 cells before they undergo mitosis and that generation of transport incompetent nuclei depends upon passage through mitosis. To investigate this we followed the distribution of Nup2 and NLS-DsRed during the first cell cycle in Δsrc1 cells released from the nimT23 G2 arrest into mitosis. During entry into mitosis Nup2 moved from the nuclear periphery onto chromatin and NLS-DsRed dispersed in Δsrc1 cells; as occurs during wildtype mitosis (Fig 5B, 3’ to 4’30”). DNA segregation then occurred and as both daughter nuclei exited mitosis Nup2 relocated back to the nuclear periphery and nuclear transport was reestablished, as expected during exit from mitosis into G1 (Fig 5B 10’ 30”). Subsequently, however, there was a defect in some G1 nuclei as they surprisingly reverted back to a more mitotic-like state indicated by the dispersal of NLS-DsRed from nuclei (Fig 5B, 16’30” nucleus indicated by arrowhead).


The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans.

Liu HL, Osmani AH, Osmani SA - PLoS ONE (2015)

Δsrc1 nuclei undergo architectural modifications from G1 that are typical of mitosis.(A) Δsrc1 spores were germinated in the presence of 10 mM HU to arrest them in interphase. NLS-DsRed was transported within the nucleus in Δsrc1 cells and Nup49 located around their nuclear periphery as typical of interphase Wt cells. (B-C) The localization of Nup2, NLS-DsRed, Bop1, and Nup49 were monitored in the first mitosis of Δsrc1 cells after release from G2 arrest imposed by the nimT23ts allele. (B) As occurs during normal mitosis, Nup2-GFP translocates to condensed chromatin and NLS-DsRed escapes from the nucleus during mitosis in Δsrc1 cells. However, soon after the two G1 nuclei are established and accumulate NLS-DsRed, with Nup2 now around their nuclear periphery, the nucleus indicated by the white arrowhead becomes mitotic-like with Nup2 locating to chromatin and losing its nuclear transport capacity. These effects were then largely reversed at the 21’ time point with the nucleus re-importing NLS-DsRed again. (C) As occurs during normal mitosis, Nup49 disperses from NPCs and returns to NPCs of the two new G1 nuclei generated during mitosis (0–8’). The nucleolus, marked by Bop1-GFP, then disassembles with the released nucleolar proteins being reimported into the new G1 nuclei. This process occurs normally in the absence of Src1 and is completed apparently normally (time point 17’). However the G1 nuclei are not normal as the nucleus to the right undergoes transitions typical of mitosis including the movement of Nup49 into the nucleus, presumably onto chromatin, and the disassembly of Bop1 after it is apparently expelled to the cytoplasm (indicated by a white arrowhead). This nucleolus then disassembles and Bop1 is imported into the transport competent daughter nucleus.
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Related In: Results  -  Collection

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pone.0132489.g005: Δsrc1 nuclei undergo architectural modifications from G1 that are typical of mitosis.(A) Δsrc1 spores were germinated in the presence of 10 mM HU to arrest them in interphase. NLS-DsRed was transported within the nucleus in Δsrc1 cells and Nup49 located around their nuclear periphery as typical of interphase Wt cells. (B-C) The localization of Nup2, NLS-DsRed, Bop1, and Nup49 were monitored in the first mitosis of Δsrc1 cells after release from G2 arrest imposed by the nimT23ts allele. (B) As occurs during normal mitosis, Nup2-GFP translocates to condensed chromatin and NLS-DsRed escapes from the nucleus during mitosis in Δsrc1 cells. However, soon after the two G1 nuclei are established and accumulate NLS-DsRed, with Nup2 now around their nuclear periphery, the nucleus indicated by the white arrowhead becomes mitotic-like with Nup2 locating to chromatin and losing its nuclear transport capacity. These effects were then largely reversed at the 21’ time point with the nucleus re-importing NLS-DsRed again. (C) As occurs during normal mitosis, Nup49 disperses from NPCs and returns to NPCs of the two new G1 nuclei generated during mitosis (0–8’). The nucleolus, marked by Bop1-GFP, then disassembles with the released nucleolar proteins being reimported into the new G1 nuclei. This process occurs normally in the absence of Src1 and is completed apparently normally (time point 17’). However the G1 nuclei are not normal as the nucleus to the right undergoes transitions typical of mitosis including the movement of Nup49 into the nucleus, presumably onto chromatin, and the disassembly of Bop1 after it is apparently expelled to the cytoplasm (indicated by a white arrowhead). This nucleolus then disassembles and Bop1 is imported into the transport competent daughter nucleus.
Mentions: To define the role of mitosis in generating Δsrc1 transport incompetent nuclei we germinated Δsrc1 spores from heterokaryons in the presence of hydroxyurea (HU), an inhibitor of DNA replication, to arrest them in interphase before their first mitosis [51]. Δsrc1 spores germinated in HU were able to transport NLS-DsRed into their single interphase nuclei (Fig 5A). We also arrested Δsrc1 cells in late G2 before entry into mitosis using the nimT23ts mutation that prevents activation of Cdk1 [45]. At the G2 arrest point of nimT23tsΔsrc1 nuclei were transport competent (example in Fig 5B, 0’). These results indicate that nuclear transport is active in Δsrc1 cells before they undergo mitosis and that generation of transport incompetent nuclei depends upon passage through mitosis. To investigate this we followed the distribution of Nup2 and NLS-DsRed during the first cell cycle in Δsrc1 cells released from the nimT23 G2 arrest into mitosis. During entry into mitosis Nup2 moved from the nuclear periphery onto chromatin and NLS-DsRed dispersed in Δsrc1 cells; as occurs during wildtype mitosis (Fig 5B, 3’ to 4’30”). DNA segregation then occurred and as both daughter nuclei exited mitosis Nup2 relocated back to the nuclear periphery and nuclear transport was reestablished, as expected during exit from mitosis into G1 (Fig 5B 10’ 30”). Subsequently, however, there was a defect in some G1 nuclei as they surprisingly reverted back to a more mitotic-like state indicated by the dispersal of NLS-DsRed from nuclei (Fig 5B, 16’30” nucleus indicated by arrowhead).

Bottom Line: How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood.We suggest the term "reboot regulation" to define this mode of cell cycle regulation.The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210, United States of America.

ABSTRACT
How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term "reboot regulation" to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

No MeSH data available.


Related in: MedlinePlus