Assuntos
Atrofia , Microvilosidades , ATPases Vacuolares Próton-Translocadoras , Animais , Microvilosidades/patologia , Microvilosidades/metabolismo , Atrofia/patologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Regulação para Baixo , Humanos , Camundongos , Intestinos/patologia , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismoRESUMO
Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.
Assuntos
Actinas , Miosina Tipo II , Oócitos , Proteínas Serina-Treonina Quinases , Polos do Fuso , Animais , Masculino , Camundongos , Citoesqueleto de Actina , Proteínas do Citoesqueleto , Miosina Tipo II/metabolismo , Rotação , Feminino , Proteínas Serina-Treonina Quinases/metabolismo , Polos do Fuso/metabolismo , AnáfaseRESUMO
Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.
Assuntos
Actomiosina , Corpos Polares , Humanos , Animais , Camundongos , Corpos Polares/metabolismo , Actomiosina/metabolismo , Blastocisto , Cromossomos , Meiose , Oócitos/metabolismo , Fuso Acromático/genética , Miosina Tipo I/genética , Miosina Tipo I/metabolismoRESUMO
Polo-like kinase I (Plk1) is a highly conserved seronine/threonine kinase essential in meiosis and mitosis for spindle formation and cytokinesis. Here, through temporal application of Plk1 inhibitors, we identify a new role for Plk1 in the establishment of cortical polarity essential for highly asymmetric cell divisions of oocyte meiosis. Application of Plk1 inhibitors in late metaphase I abolishes pPlk1 from spindle poles and prevents the induction of actin polymerization at the cortex through inhibition of local recruitment of Cdc42 and Neuronal Wiskott-Aldrich Syndrome protein (N-WASP). By contrast, an already established polar actin cortex is insensitive to Plk1 inhibitors, but if the polar cortex is first depolymerized, Plk1 inhibitors completely prevent its restoration. Thus, Plk1 is essential for establishment but not maintenance of cortical actin polarity. These findings indicate that Plk1 regulates recruitment of Cdc42 and N-Wasp to coordinate cortical polarity and asymmetric cell division.
Assuntos
Actinas , Meiose , Oócitos , Actinas/genética , Actinas/fisiologia , Meiose/genética , Meiose/fisiologia , Oócitos/fisiologia , Polimerização , Proteínas Serina-Treonina Quinases , Quinase 1 Polo-LikeRESUMO
How multiple actin networks coexist in a common cytoplasm while competing for a shared pool of monomers is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here, we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner upon meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread elongation of microvilli, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.
Assuntos
Fatores de Despolimerização de Actina , Actinas , Fatores de Despolimerização de Actina/genética , Animais , Homeostase , Meiose , Camundongos , Microvilosidades , Oócitos , Fuso AcromáticoRESUMO
Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II-arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.
Assuntos
Segregação de Cromossomos , Meiose , Oócitos/citologia , Fuso Acromático , Actinas/metabolismo , Animais , Feminino , Camundongos , Proteína cdc42 de Ligação ao GTP , Proteína rhoA de Ligação ao GTPRESUMO
Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H(2)O(2) to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass.
Assuntos
Catalase/genética , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Podospora/enzimologia , Madeira/metabolismo , Sequência de Bases , Southern Blotting , Análise por Conglomerados , Deleção de Genes , Perfilação da Expressão Gênica , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Oxirredução , Fenótipo , Filogenia , Podospora/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Fúngicas/metabolismo , Oxirredução , Peroxissomos/metabolismo , Podospora/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Mitocôndrias/metabolismo , Ácido Oleico/metabolismo , Peroxissomos/genética , Podospora/citologia , Podospora/genética , Podospora/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
BACKGROUND: The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. RESULTS: We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown. CONCLUSION: The features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.
Assuntos
Evolução Molecular , Genoma Fúngico , Podospora/genética , Sequência de Bases , Carbono/metabolismo , Etiquetas de Sequências Expressas , Duplicação Gênica , Dados de Sequência Molecular , Neurospora crassa/genética , Podospora/metabolismoRESUMO
RIP (Repeat-Induced point Mutation) and PR (Premeiotic Recombination) are two developmentally regulated processes in filamentous ascomycetes. RIP detects and mutates duplicated DNA sequences, while PR results in deletion of the interstitial sequence between cis-duplicated DNA sequences. These two silencing processes take place between fertilization and premeiotic replication, a period during which the mating-type genes play an active role in several developmental processes. Previous studies have shown that mutations in the mating-type genes affect the development of the fruiting body. This study provides evidence that mutations in the mating-type genes reduce the frequency of RIP and PR. It establishes that alleles which have the more stringent effect on fruiting-body development, have also the strongest effect on RIP and PR frequencies. We propose two models for the relation between mating-type genes and RIP and PR, one based on the direct control of RIP and PR by mating-type regulatory proteins, the other based on an indirect effect through the control of a development step during which RIP and PR take place.
Assuntos
Genes Fúngicos Tipo Acasalamento/genética , Mutação , Podospora/genética , Mutação Puntual , Sequência de Bases , Cruzamentos Genéticos , DNA Fúngico/genética , Modelos Genéticos , Dados de Sequência Molecular , Podospora/crescimento & desenvolvimento , Recombinação Genética/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
PEX5, PEX7 and PEX2 are involved in the peroxisomal matrix protein import machinery. PEX5 and PEX7 are the receptors for the proteins harbouring, respectively, a PTS1 and a PTS2 peroxisomal targeting sequence and cycle between the cytoplasm and the peroxisome. PEX2 belongs to the RING-finger complex located in the peroxisomal membrane and acts in protein import downstream of PEX5 and PEX7; it is therefore required for the import of both PTS1 and PTS2 proteins. We have shown previously that PEX2 deficiency leads to an impairment of meiotic commitment in the filamentous fungus Podospora anserina. Here we report that both PEX5 and PEX7 receptors are dispensable for this commitment but are needed for normal sexual cycle. Data suggest also a new role of PEX2 and/or the RING-finger complex in addition to their role in PTS1 and PTS2 import. Strikingly, Deltapex5 and Deltapex7 single and double knockout strains analyses indicate that Deltapex7 acts as a partial suppressor of Deltapex5 life cycle deficiencies. Moreover, contrary to pex2 mutants, Deltapex5 and Deltapex7 show mitochondrial morphological abnormalities.
Assuntos
Proteínas Fúngicas/metabolismo , Peroxissomos/metabolismo , Podospora/metabolismo , Transporte Biológico/fisiologia , Divisão Celular/fisiologia , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Imunofluorescência , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação/genética , Podospora/genética , Podospora/crescimento & desenvolvimento , Análise de Sequência de DNA , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
The chemokine macrophage inflammatory protein 1beta/CCL4, ligand of the major HIV co-receptor CCR5, is encoded by two genes, Act-2 and Lag-1. Our work focused on R22H, a variant of Lag-1 located near the N-loop, in the 310 turn, a domain essential for interacting with CCR5. We observed that HIV-1-infected patients from the SEROCO cohort, bearing the R22H variant either at the homozygous or heterozygous state, exhibit a worse global survival compared with wild-type homozygous individuals.
Assuntos
Infecções por HIV/genética , HIV-1 , Proteínas Inflamatórias de Macrófagos/genética , Polimorfismo Genético , Quimiocina CCL4 , Estudos de Coortes , Infecções por HIV/imunologia , Infecções por HIV/mortalidade , Heterozigoto , Homozigoto , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores CCR5/metabolismo , Análise de SobrevidaRESUMO
We identified two novel isoforms of the human chemokine receptor CX3CR1, produced by alternative splicing and with N-terminal regions extended by 7 and 32 aa. Expression of the messengers coding these isoforms, compared with that of previously described V28 messengers, is lower in monocytes and NK cells, but higher in CD4(+) T lymphocytes. CX3CR1 and its extended isoforms were expressed in HEK-293 cells and compared for expression, ligand binding, and cellular responses. In steady state experiments, all three CX3CR1 isoforms bound CX3CL1 with similar affinity. In kinetic binding studies, however, k(on) and k(off) were significantly greater for the extended CX3CR1 isoforms, thereby suggesting that the N-terminal extensions may alter the functions induced by CX3CL1. In signaling studies, all three CX3CR1 isoforms mediated agonist-dependent calcium mobilization, but the EC(50) was lower for the extended than for the standard isoforms. In addition, chemotactic responses for these extended isoforms shifted left, also indicating a more sensitive response. Finally, the longer variants appeared to be more potent HIV coreceptors when tested in fusion and infection assays. In conclusion, we identified and characterized functionally two novel isoforms of CX3CR1 that respond more sensitively to CX3CL1 and HIV viral envelopes. These data reveal new complexity in CX3CR1 cell activation and confirm the critical role of the N-terminal domain of the chemokine receptors in ligand recognition and cellular response.