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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

Src1 is essential.(A) Conidia (asexual spores) from wildtype control (pyrG+) or Δsrc1::pyrG deletion primary transformants were replica streaked onto nonselective (+UU) and selective (-UU) plates and grown for 40 hours for photography. Conidia that are not able to grow on–UU media are from primary Δsrc1::pyrG / src1 pyrG89 heterokaryon transformants indicating src1 to be essential. (B) Bright field image of the limited growth of conidia from Δsrc1::pyrG / src1 pyrG89 heterokaryons on selective media for 24 and 72 h. Scale bar 10 μm. (C) Diagnostic PCR using DNA isolated from a wild-type strain (Wt.), from a putative heterokaryon grown in +UU media (stops selection for Δsrc1), and from a putative heterokaryon growing in-UU selective media. The deletion allele carried in the heterokaryotic transformant is rapidly lost upon propagation without selection indicating the transformant is not a diploid but a heterokaryon. (D) The degree of growth of both wildtype and Δsrc1 cells were compared at 12, 15, and 18 hours at 23.5°C by measuring the average length of germlings. (E) Δsrc1 germlings fixed and stained with DAPI to reveal nuclear defects, including condensed DNA, odd numbers of nuclei (indicated by white arrowheads), and an unusual pattern of nuclear transport evidenced by only one nucleus being able to accumulate NLS-DsRed (indicated by arrow). Scale bar 5 μm.
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pone.0132489.g003: Src1 is essential.(A) Conidia (asexual spores) from wildtype control (pyrG+) or Δsrc1::pyrG deletion primary transformants were replica streaked onto nonselective (+UU) and selective (-UU) plates and grown for 40 hours for photography. Conidia that are not able to grow on–UU media are from primary Δsrc1::pyrG / src1 pyrG89 heterokaryon transformants indicating src1 to be essential. (B) Bright field image of the limited growth of conidia from Δsrc1::pyrG / src1 pyrG89 heterokaryons on selective media for 24 and 72 h. Scale bar 10 μm. (C) Diagnostic PCR using DNA isolated from a wild-type strain (Wt.), from a putative heterokaryon grown in +UU media (stops selection for Δsrc1), and from a putative heterokaryon growing in-UU selective media. The deletion allele carried in the heterokaryotic transformant is rapidly lost upon propagation without selection indicating the transformant is not a diploid but a heterokaryon. (D) The degree of growth of both wildtype and Δsrc1 cells were compared at 12, 15, and 18 hours at 23.5°C by measuring the average length of germlings. (E) Δsrc1 germlings fixed and stained with DAPI to reveal nuclear defects, including condensed DNA, odd numbers of nuclei (indicated by white arrowheads), and an unusual pattern of nuclear transport evidenced by only one nucleus being able to accumulate NLS-DsRed (indicated by arrow). Scale bar 5 μm.

Mentions: We replaced the coding region of src1 with the pyrG nutritional marker to determine the phenotype. Using heterokaryon rescue [47] we determined that src1 is essential (Fig 3A and 3C) and after src1 deletion heterokaryons were generated that had two types of genetically distinct nuclei; nuclei with Src1 intact but which are pyrG- and nuclei that have Src1 deleted (ΔSrc1) and are pyrG+ (Fig 3C). Both nuclear types are therefore required for heterokaryotic growth when selection for pyrG+ is imposed. Because the asexual spores generated from heterokaryons contain a single nucleus, the src1 + pyrG- nuclei and Δsrc1 + pyrG+ nuclei are segregated into individual spores. By germinating spores from the heterokaryon on medium selective for pyrG+ (hence ΔSrc1) we determined that cells lacking Src1 are able to break dormancy and undergo polarized growth at a comparable rate to normal cells during the first 15 hours of their growth (Fig 3D). The Δsrc1 cells failed to continue normal growth resulting in a terminal arrest phenotype as germling cells (Fig 3B). Visualization of nuclei after DAPI staining showed that a significant percentage of Δsrc1 cells (52%) contained odd numbers of nuclei (Fig 3E). This suggests that some nuclei had undergone mitotic division while others had not, something rarely seen in wildtype cells in which the nuclear number doubles during each para-synchronous mitosis. In addition, Δsrc1 nuclei often contained condensed chromatin of uneven intensity (data not shown) suggestive of mitotic DNA segregation defects.


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)

Src1 is essential.(A) Conidia (asexual spores) from wildtype control (pyrG+) or Δsrc1::pyrG deletion primary transformants were replica streaked onto nonselective (+UU) and selective (-UU) plates and grown for 40 hours for photography. Conidia that are not able to grow on–UU media are from primary Δsrc1::pyrG / src1 pyrG89 heterokaryon transformants indicating src1 to be essential. (B) Bright field image of the limited growth of conidia from Δsrc1::pyrG / src1 pyrG89 heterokaryons on selective media for 24 and 72 h. Scale bar 10 μm. (C) Diagnostic PCR using DNA isolated from a wild-type strain (Wt.), from a putative heterokaryon grown in +UU media (stops selection for Δsrc1), and from a putative heterokaryon growing in-UU selective media. The deletion allele carried in the heterokaryotic transformant is rapidly lost upon propagation without selection indicating the transformant is not a diploid but a heterokaryon. (D) The degree of growth of both wildtype and Δsrc1 cells were compared at 12, 15, and 18 hours at 23.5°C by measuring the average length of germlings. (E) Δsrc1 germlings fixed and stained with DAPI to reveal nuclear defects, including condensed DNA, odd numbers of nuclei (indicated by white arrowheads), and an unusual pattern of nuclear transport evidenced by only one nucleus being able to accumulate NLS-DsRed (indicated by arrow). Scale bar 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492595&req=5

pone.0132489.g003: Src1 is essential.(A) Conidia (asexual spores) from wildtype control (pyrG+) or Δsrc1::pyrG deletion primary transformants were replica streaked onto nonselective (+UU) and selective (-UU) plates and grown for 40 hours for photography. Conidia that are not able to grow on–UU media are from primary Δsrc1::pyrG / src1 pyrG89 heterokaryon transformants indicating src1 to be essential. (B) Bright field image of the limited growth of conidia from Δsrc1::pyrG / src1 pyrG89 heterokaryons on selective media for 24 and 72 h. Scale bar 10 μm. (C) Diagnostic PCR using DNA isolated from a wild-type strain (Wt.), from a putative heterokaryon grown in +UU media (stops selection for Δsrc1), and from a putative heterokaryon growing in-UU selective media. The deletion allele carried in the heterokaryotic transformant is rapidly lost upon propagation without selection indicating the transformant is not a diploid but a heterokaryon. (D) The degree of growth of both wildtype and Δsrc1 cells were compared at 12, 15, and 18 hours at 23.5°C by measuring the average length of germlings. (E) Δsrc1 germlings fixed and stained with DAPI to reveal nuclear defects, including condensed DNA, odd numbers of nuclei (indicated by white arrowheads), and an unusual pattern of nuclear transport evidenced by only one nucleus being able to accumulate NLS-DsRed (indicated by arrow). Scale bar 5 μm.
Mentions: We replaced the coding region of src1 with the pyrG nutritional marker to determine the phenotype. Using heterokaryon rescue [47] we determined that src1 is essential (Fig 3A and 3C) and after src1 deletion heterokaryons were generated that had two types of genetically distinct nuclei; nuclei with Src1 intact but which are pyrG- and nuclei that have Src1 deleted (ΔSrc1) and are pyrG+ (Fig 3C). Both nuclear types are therefore required for heterokaryotic growth when selection for pyrG+ is imposed. Because the asexual spores generated from heterokaryons contain a single nucleus, the src1 + pyrG- nuclei and Δsrc1 + pyrG+ nuclei are segregated into individual spores. By germinating spores from the heterokaryon on medium selective for pyrG+ (hence ΔSrc1) we determined that cells lacking Src1 are able to break dormancy and undergo polarized growth at a comparable rate to normal cells during the first 15 hours of their growth (Fig 3D). The Δsrc1 cells failed to continue normal growth resulting in a terminal arrest phenotype as germling cells (Fig 3B). Visualization of nuclei after DAPI staining showed that a significant percentage of Δsrc1 cells (52%) contained odd numbers of nuclei (Fig 3E). This suggests that some nuclei had undergone mitotic division while others had not, something rarely seen in wildtype cells in which the nuclear number doubles during each para-synchronous mitosis. In addition, Δsrc1 nuclei often contained condensed chromatin of uneven intensity (data not shown) suggestive of mitotic DNA segregation defects.

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