RESUMO
Microtubule-severing enzymes (MSEs), such as Katanin, Spastin, and Fidgetin play essential roles in cell division and neurogenesis. They damage the microtubule (MT) lattice, which can either destroy or amplify the MT cytoskeleton, depending on the cellular context. However, little is known about how they interact with their substrates. We have identified the microtubule-binding domains (MTBD) required for Katanin function in C. elegans. Katanin is a heterohexamer of dimers containing a catalytic subunit p60 and a regulatory subunit p80, both of which are essential for female meiotic spindle assembly. Here, we report that p80-like(MEI-2) dictates Katanin binding to MTs via two MTBDs composed of basic patches. Substituting these patches reduces Katanin binding to MTs, compromising its function in female meiotic-spindle assembly. Structural alignments of p80-like(MEI-2) with p80s from different species revealed that the MTBDs are evolutionarily conserved, even if the specific amino acids involved vary. Our findings highlight the critical importance of the regulatory subunit (p80) in providing MT binding to the Katanin complex.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Katanina , Microtúbulos , Animais , Feminino , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Katanina/genética , Katanina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Ligação Proteica , Fuso Acromático , Meiose , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismoRESUMO
The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.
Assuntos
Proteínas de Caenorhabditis elegans , Poro Nuclear , Animais , Humanos , Poro Nuclear/genética , Poro Nuclear/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quinase 1 Polo-LikeRESUMO
Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single Caenorhabditis elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required to form a pronuclear envelope scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera et al., 2020). Here, we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation, and conversely, nuclear envelopes remodeling facilitates spindle elongation. We also demonstrate that weakening the pronuclear envelopes via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero , Laminina/genética , Microtúbulos , Mitose , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático , ZigotoRESUMO
Life of sexually reproducing organisms starts with the fusion of the haploid egg and sperm gametes to form the genome of a new diploid organism. Using the newly fertilized Caenorhabditis elegans zygote, we show that the mitotic Polo-like kinase PLK-1 phosphorylates the lamin LMN-1 to promote timely lamina disassembly and subsequent merging of the parental genomes into a single nucleus after mitosis. Expression of non-phosphorylatable versions of LMN-1, which affect lamina depolymerization during mitosis, is sufficient to prevent the mixing of the parental chromosomes into a single nucleus in daughter cells. Finally, we recapitulate lamina depolymerization by PLK-1 in vitro demonstrating that LMN-1 is a direct PLK-1 target. Our findings indicate that the timely removal of lamin is essential for the merging of parental chromosomes at the beginning of life in C. elegans and possibly also in humans, where a defect in this process might be fatal for embryo development.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Laminina/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Genoma Helmíntico , Laminina/metabolismo , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The evolutionarily conserved microtubule (MT)-severing AAA-ATPase enzyme Katanin is emerging as a critical regulator of MT dynamics. In Caenorhabditis elegans, Katanin MT-severing activity is essential for meiotic spindle assembly but is toxic for the mitotic spindle. Here we analyzed Katanin dynamics in C. elegans and deciphered the role of Katanin phosphorylation in the regulation of its activity and stability. Katanin is abundant in oocytes, and its levels drop after meiosis, but unexpectedly, a significant fraction is present throughout embryogenesis, where it is dynamically recruited to the centrosomes and chromosomes during mitosis. We show that the minibrain kinase MBK-2, which is activated during meiosis, phosphorylates Katanin at multiple serines. We demonstrate unequivocally that Katanin phosphorylation at a single residue is necessary and sufficient to target Katanin for proteasomal degradation after meiosis, whereas phosphorylation at the other sites only inhibits Katanin ATPase activity stimulated by MTs. Our findings suggest that cycles of phosphorylation and dephosphorylation fine-tune Katanin level and activity to deliver the appropriate MT-severing activity during development.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Katanina/metabolismo , Microtúbulos/metabolismo , Oócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Katanina/genética , Meiose , Mitose , Fosforilação , Proteínas Tirosina Quinases/genética , Interferência de RNARESUMO
In animal cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity to trigger this event is unclear. Here, we show that both in human cells and Caenorhabditis elegans, the Polo-like kinase 1 (PLK-1) is recruited to the nuclear pore complexes, just prior to NEBD, through its Polo-box domain (PBD). We provide evidence that PLK-1 localization to the nuclear envelope (NE) is required for efficient NEBD. We identify the central channel nucleoporins NPP-1/Nup58, NPP-4/Nup54, and NPP-11/Nup62 as the critical factors anchoring PLK-1 to the NE in C. elegans. In particular, NPP-1, NPP-4, and NPP-11 primed at multiple Polo-docking sites by Cdk1 and PLK-1 itself physically interact with the PLK-1 PBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the NE thereby facilitating phosphorylation of critical downstream targets.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose/fisiologia , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Células HeLa , Humanos , Membrana Nuclear/genética , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/química , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/química , Sequência Conservada , Ciclina B/metabolismo , Dano ao DNA , Embrião não Mamífero/citologia , Ativação Enzimática , Células HeLa , Humanos , Mitose , Fosforilação , Quinase 1 Polo-LikeRESUMO
The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing.
Assuntos
Proteína Quinase CDC2/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinase A/metabolismo , Caenorhabditis elegans/enzimologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Ativação Enzimática , Humanos , Larva/citologia , Larva/enzimologia , Mitose , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Células Sf9 , SpodopteraRESUMO
The small G protein Ras regulates many cell processes, such as gene expression, proliferation, apoptosis, and cell differentiation. Its mutations are associated with one-third of all cancers. Ras functions are mediated, at least in part, by Ral proteins and their downstream effector the Ral-binding protein 1 (RalBP1). RalBP1 is involved in endocytosis and in regulating the dynamics of the actin cytoskeleton. It also regulates early development since it is required for the completion of gastrulation in Xenopus laevis. RalBP1 has also been reported to be the main transporter of glutathione electrophiles, and it is involved in multidrug resistance. Such a variety of functions could be explained by a differential regulation of RalBP1 localization. In this study, we have detected endogenous RalBP1 in the nucleus of interphasic cells. This nuclear targeting is mediated by nuclear localization sequences that map to the N-terminal third of the protein. Moreover, in X. laevis embryos, a C-terminal coiled-coil sequence mediates RalBP1 retention in the nucleus. We have also observed RalBP1 at the level of the actin cytoskeleton, a localization that depends on interaction of the protein with active Ral. During mitosis RalBP1 also associates with the mitotic spindle and the centrosome, a localization that could be negatively regulated by active Ral. Finally, we demonstrate the presence of post-transcriptional and post-translational isoforms of RalBP1 lacking the Ral-binding domain, which opens new possibilities for the existence of Ral-independent functions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Frações Subcelulares/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Processamento de Proteína Pós-Traducional , Processamento Pós-Transcricional do RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The transduction pathways that branch out of fibroblast growth factor signaling are essential for the induction of the mesoderm and the specification of the vertebrate body plan. One of these pathways is thought to control remodeling of the actin cytoskeleton through the Ral binding protein (RLIP also known as RalBP1), an effector of the small G protein Ral. RLIP contains a region of homology with the GTPase-activating protein (GAP) domain involved in the regulation of GTPases of the Rho family. We demonstrate here that the GAP domain of RLIP is responsible for the stability of the actin cytoskeleton in Xenopus laevis embryos. We also demonstrate that the complete N-terminal domain of RLIP containing the mu2 binding domain (mu2BD) and the GAP domain induces disruption of the actin cytoskeleton when targeted to the plasma membrane. Neither domain, however, has any effect on the actin cytoskeleton when individually targeted to the plasma membrane. We also determined that Cdc42-GDP, but neither Rac-GDP nor Rho-GDP, rescues the effect of expression of the membrane-localized Xenopus RLIP on the actin cytoskeleton. We show that the GAP domain of RLIP interacts in vivo with Cdc42-GTP and Cdc42-GDP. Finally, a single mutation (K244A) in the GAP sequence prevented embryos from gastrulating. These results demonstrate that to participate in the control of the actin cytoskeleton, RLIP needs its complete N-terminal region coding for the mu2BD and the GAP domain. We suggest that RLIP, by coordinating two complementary mechanisms, the endocytosis of clathrin-coated pits and the remodeling of cortical actin, participates in the gastrulation process.