Ankle2-dependent mechanisms of nuclear reassembly and physiological responses to defects in this process
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- Mitosis
- Mitose, réassemblage nucléaire, PP2A-Ankle2, BAF, défauts nucléaire, apoptose, P53
- Apoptose
- Nuclear reassembly
- PP2A-Ankle2
- BAF
- Nuclear defects
- Apoptosis
- P53
- Réassemblage nucléaire
- Défauts nucléaire
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Résumé
In eukaryotes, chromosomes are enclosed within a nucleus that is delimited by the nuclear envelope (NE). During mitosis, the NE breaks down to allow the segregation of chromosomes on a microtubule-based spindle and then reforms to ensure chromosomal integrity in daughter cells. However, the molecular mechanisms that control nuclear reassembly (NR) in telophase are not fully understood. Moreover, how cells respond to NR defects has not yet been investigated. Barrier-to-Autointegration Factor (BAF) is a dimeric protein that binds to DNA and NE proteins during interphase. Upon mitotic entry, BAF is phosphorylated by NHK1, inducing its dissociation from the NE and DNA. At mitotic exit, NE reformation requires BAF dephosphorylation to ensure the formation of a single nucleus in daughter cells. Loss of BAF results in nuclear defects, including micronucleation and lamin abnormalities. Studies in C. elegans and human cells have shown that Ankle2 is required for BAF recruitment during NR at the end of mitosis. During my PhD, we found that this function of Ankle2 is also conserved in Drosophila. By Identifying Ankle2 interaction partners, we showed that Ankle2 forms a complex with the structural and catalytic subunits of Protein Phosphatase 2A (PP2A). Taking advantage of the GAL4-UAS system, we disrupted the PP2A-Ankle2-BAF axis by expressing RNA Interference (RNAi) in developing wing discs. Partial depletion of Ankle2 and BAF in Drosophila imaginal wing discs leads to increased apoptosis and severe morphological defects in adult wings. Interestingly, artificially blocking apoptosis in Ankle2-depleted wing discs strongly enhances wing defects and nuclear abnormalities. This suggests that cells with nuclear defects trigger apoptosis, and the removal of these defective cells by apoptosis promotes normal tissue development. Additionally, we found that apoptosis resulting from Ankle2 depletion is P53-independent. However, when NR is compromised due to Ankle2 loss, P53-dependent cell cycle control becomes essential. At the molecular level, we demonstrated that Ankle2 physically interacts with PP2A and Vap33 (a resident protein of the endoplasmic reticulum) through its ankyrin repeat region and FFAT motifs, respectively. Using time-lapse microscopy, we demonstrated that the interaction of Ankle2 with Vap33 is required for its telophase localization, whereas the interaction of Ankle2 with PP2A is required for inducing the essential functions of BAF during NR. Furthermore, in vitro and in vivo rescue experiments allowed us to conclude that the interaction of Ankle2 with PP2A, but not with Vap33 is essential for its function in NR during late mitosis, as well as in the context of early embryonic and wing development. Moreover, we provided strong evidence that Ankle2 serves as a novel regulatory subunit of PP2A. Using a phosphoproteomics approach, we also identified several potential substrates of PP2A-Ankle2, suggesting that Ankle2-dependent phosphoregulation may play a role in other cellular mechanisms. Our study provides a deeper understanding of the mechanisms by which PP2A-Ankle2 contributes to NR at the end of mitosis. Additionally, inactivating the PP2A-Ankle2-BAF axis has allowed us to analyze the biological response to NR defects in vivo.
In eukaryotes, chromosomes are enclosed within a nucleus that is delimited by the nuclear envelope (NE). During mitosis, the NE breaks down to allow the segregation of chromosomes on a microtubule-based spindle and then reforms to ensure chromosomal integrity in daughter cells. However, the molecular mechanisms that control nuclear reassembly (NR) in telophase are not fully understood. Moreover, how cells respond to NR defects has not yet been investigated. Barrier-to-Autointegration Factor (BAF) is a dimeric protein that binds to DNA and NE proteins during interphase. Upon mitotic entry, BAF is phosphorylated by NHK1, inducing its dissociation from the NE and DNA. At mitotic exit, NE reformation requires BAF dephosphorylation to ensure the formation of a single nucleus in daughter cells. Loss of BAF results in nuclear defects, including micronucleation and lamin abnormalities. Studies in C. elegans and human cells have shown that Ankle2 is required for BAF recruitment during NR at the end of mitosis. During my PhD, we found that this function of Ankle2 is also conserved in Drosophila. By Identifying Ankle2 interaction partners, we showed that Ankle2 forms a complex with the structural and catalytic subunits of Protein Phosphatase 2A (PP2A). Taking advantage of the GAL4-UAS system, we disrupted the PP2A-Ankle2-BAF axis by expressing RNA Interference (RNAi) in developing wing discs. Partial depletion of Ankle2 and BAF in Drosophila imaginal wing discs leads to increased apoptosis and severe morphological defects in adult wings. Interestingly, artificially blocking apoptosis in Ankle2-depleted wing discs strongly enhances wing defects and nuclear abnormalities. This suggests that cells with nuclear defects trigger apoptosis, and the removal of these defective cells by apoptosis promotes normal tissue development. Additionally, we found that apoptosis resulting from Ankle2 depletion is P53-independent. However, when NR is compromised due to Ankle2 loss, P53-dependent cell cycle control becomes essential. At the molecular level, we demonstrated that Ankle2 physically interacts with PP2A and Vap33 (a resident protein of the endoplasmic reticulum) through its ankyrin repeat region and FFAT motifs, respectively. Using time-lapse microscopy, we demonstrated that the interaction of Ankle2 with Vap33 is required for its telophase localization, whereas the interaction of Ankle2 with PP2A is required for inducing the essential functions of BAF during NR. Furthermore, in vitro and in vivo rescue experiments allowed us to conclude that the interaction of Ankle2 with PP2A, but not with Vap33 is essential for its function in NR during late mitosis, as well as in the context of early embryonic and wing development. Moreover, we provided strong evidence that Ankle2 serves as a novel regulatory subunit of PP2A. Using a phosphoproteomics approach, we also identified several potential substrates of PP2A-Ankle2, suggesting that Ankle2-dependent phosphoregulation may play a role in other cellular mechanisms. Our study provides a deeper understanding of the mechanisms by which PP2A-Ankle2 contributes to NR at the end of mitosis. Additionally, inactivating the PP2A-Ankle2-BAF axis has allowed us to analyze the biological response to NR defects in vivo.
In eukaryotes, chromosomes are enclosed within a nucleus that is delimited by the nuclear envelope (NE). During mitosis, the NE breaks down to allow the segregation of chromosomes on a microtubule-based spindle and then reforms to ensure chromosomal integrity in daughter cells. However, the molecular mechanisms that control nuclear reassembly (NR) in telophase are not fully understood. Moreover, how cells respond to NR defects has not yet been investigated. Barrier-to-Autointegration Factor (BAF) is a dimeric protein that binds to DNA and NE proteins during interphase. Upon mitotic entry, BAF is phosphorylated by NHK1, inducing its dissociation from the NE and DNA. At mitotic exit, NE reformation requires BAF dephosphorylation to ensure the formation of a single nucleus in daughter cells. Loss of BAF results in nuclear defects, including micronucleation and lamin abnormalities. Studies in C. elegans and human cells have shown that Ankle2 is required for BAF recruitment during NR at the end of mitosis. During my PhD, we found that this function of Ankle2 is also conserved in Drosophila. By Identifying Ankle2 interaction partners, we showed that Ankle2 forms a complex with the structural and catalytic subunits of Protein Phosphatase 2A (PP2A). Taking advantage of the GAL4-UAS system, we disrupted the PP2A-Ankle2-BAF axis by expressing RNA Interference (RNAi) in developing wing discs. Partial depletion of Ankle2 and BAF in Drosophila imaginal wing discs leads to increased apoptosis and severe morphological defects in adult wings. Interestingly, artificially blocking apoptosis in Ankle2-depleted wing discs strongly enhances wing defects and nuclear abnormalities. This suggests that cells with nuclear defects trigger apoptosis, and the removal of these defective cells by apoptosis promotes normal tissue development. Additionally, we found that apoptosis resulting from Ankle2 depletion is P53-independent. However, when NR is compromised due to Ankle2 loss, P53-dependent cell cycle control becomes essential. At the molecular level, we demonstrated that Ankle2 physically interacts with PP2A and Vap33 (a resident protein of the endoplasmic reticulum) through its ankyrin repeat region and FFAT motifs, respectively. Using time-lapse microscopy, we demonstrated that the interaction of Ankle2 with Vap33 is required for its telophase localization, whereas the interaction of Ankle2 with PP2A is required for inducing the essential functions of BAF during NR. Furthermore, in vitro and in vivo rescue experiments allowed us to conclude that the interaction of Ankle2 with PP2A, but not with Vap33 is essential for its function in NR during late mitosis, as well as in the context of early embryonic and wing development. Moreover, we provided strong evidence that Ankle2 serves as a novel regulatory subunit of PP2A. Using a phosphoproteomics approach, we also identified several potential substrates of PP2A-Ankle2, suggesting that Ankle2-dependent phosphoregulation may play a role in other cellular mechanisms. Our study provides a deeper understanding of the mechanisms by which PP2A-Ankle2 contributes to NR at the end of mitosis. Additionally, inactivating the PP2A-Ankle2-BAF axis has allowed us to analyze the biological response to NR defects in vivo.
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