RESUMO
The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.
Assuntos
DNA/imunologia , Herpesvirus Humano 8/imunologia , Imunidade Inata , RNA Longo não Codificante/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/imunologia , Autoantígeno Ku/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/imunologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/imunologia , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/imunologia , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição , TransfecçãoRESUMO
Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.
Assuntos
Acetiltransferases/metabolismo , Megacariócitos/metabolismo , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Quinases Ativadas por p21/metabolismo , Acetilação , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Inibidores de Histona Desacetilases/farmacologia , Fígado/citologia , Megacariócitos/citologia , Camundongos , Microtúbulos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , XenopusRESUMO
BACKGROUND: Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation. RESULTS: Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bß3integrin signaling in D723H cells is sufficient to induce ß1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and ß1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of ß1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation. CONCLUSIONS: We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.
Assuntos
Plaquetas/citologia , Megacariócitos/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Plaquetas/metabolismo , Megacariócitos/citologia , CamundongosRESUMO
While our knowledge of bivalve gametogenesis has progressed in recent times, more molecular markers are needed in order to develop tissue imaging. Here, we identified stem cell and mitotic markers to further characterize oyster early gametogenesis, mainly through immunofluorescence microscopy. Intense alkaline phosphatase activity, a non-specific marker for stem cells, was detected on the outer edge of the gonad ducts at the post-spawning stage, suggesting an abundance of undifferentiated cells very early during the sexual cycle. This observation was confirmed using an antibody against Sox2, a transcription factor specific for stem or germline cells, which labeled cells in the gonad duct inner mass and ciliated epithelium early during the initial oyster sexual cycle. Moreover, Vasa, a cytoplasmic marker for germline cells, was also detected in the gonad acini and duct cells, thus confirming that germline cells were abundant early on. In addition, the binding of the minichromosome maintenance MCM6 protein to chromatin indicated the gonad acini and duct cells were engaged in the cell cycle. DNA replication was indeed confirmed by an abundant in vivo incorporation of BrdU into the duct cell chromatin. Finally, proliferation of acini and duct cells was demonstrated by the chromatin-bound Ser10-phosphorylated histone H3, a mitotic marker. The markers for the cell cycle and mitosis used here thus indicate that acini and duct cells were already actively dividing early during the oyster sexual cycle. In addition, together with the stem cell markers, these data reveal that the epithelium delimiting the duct outer edge contains a dynamic population of undifferentiated cells.
Assuntos
Crassostrea/fisiologia , Gametogênese , Mitose/fisiologia , Células-Tronco/metabolismo , Animais , Biomarcadores/análise , Microscopia de FluorescênciaRESUMO
Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills.
Assuntos
Diferenciação Celular , Brânquias/metabolismo , Hematopoese , Hemócitos/fisiologia , Ostreidae/fisiologia , Células-Tronco/fisiologia , Animais , DNA/biossíntese , Brânquias/anatomia & histologia , Brânquias/citologia , Hemócitos/citologia , Ostreidae/citologia , Fatores de Transcrição SOXB1/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Immunocytochemical analysis of meiotic proteins on mouse chromosome spreads is one method of choice to study prophase I chromosome organization and homologous recombination. In recent decades, the development of microscopic approaches led to the production of a large number of images that monitor fluorescent proteins, defined as fluorescent objects, and a major challenge facing the community is the deep analysis of these fluorescent objects (measurement of object length, intensity, distance between objects, as well as foci identification, counting, and colocalization). We propose a set of tools designed from the macro language of the widely used image analysis software ImageJ (Schindelin et al., Nat Methods 9: 676-682, 2012), embedded in the "MeiQuant" macro, which are specifically designed for analyzing objects in the field of meiosis. Our aim is to propose a unified evolutive common tool for image analysis, with a specific focus on mouse prophase I meiotic events.
Assuntos
Meiose , Prófase Meiótica I , Animais , Camundongos , Prófase , CromossomosRESUMO
During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.
Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Meiose , Camundongos Knockout , Rad51 Recombinase , Espermatócitos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Animais , Masculino , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Camundongos , Espermatócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Dano ao DNA , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Cromatina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genéticaRESUMO
Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Reprodutibilidade dos Testes , SoftwareRESUMO
This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment. For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).
Assuntos
Núcleo Celular/química , DNA , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae , DNA/análise , DNA/química , DNA/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Sondas Moleculares/análise , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologiaRESUMO
Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1. ATAT1-depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1, by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/enzimologia , Células Epiteliais/enzimologia , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/enzimologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Células LLC-PK1 , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/genética , Mitose , Transdução de Sinais , Fuso Acromático/genética , Suínos , Quinase 1 Polo-LikeRESUMO
Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss, eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that ß-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation, and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control of CDC42 activity to ensure efficient bone resorption.
RESUMO
BACKGROUND INFORMATION: N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N-cadherin-dependent cell-cell contacts in myoblasts remain unexplored. RESULTS: In the present study, we show that N-cadherin-dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N-cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N-cadherin-dependent cell contacts, characterized by the immobilization of a pool of N-cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F-actin filaments. We also demonstrated that the formation of N-cadherin-dependent cell-cell contacts requires a co-ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N-cadherin-dependent cell-cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N-cadherin-dependent cell contact formation, since, via ROCK (Rho-associated kinase) signalling and myosin 2 activation, it allows the stabilization of N-cadherin at the cell-cell contact sites. CONCLUSIONS: We have shown that Rac1 and RhoA have opposite effects on N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.
Assuntos
Caderinas/metabolismo , Mioblastos/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Caderinas/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , HumanosRESUMO
In the original version of this Article, the affiliation details for Antoine Aze, Michalis Fragkos, Stéphane Bocquet, Julien Cau and Marcel Méchali incorrectly omitted 'CNRS and the University of Montpellier'. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
Tubulin glutamylation is a reversible posttranslational modification that accumulates on stable microtubules (MTs). While abnormally high levels of this modification lead to a number of disorders such as male sterility, retinal degeneration, and neurodegeneration, very little is known about the molecular mechanisms underlying the regulation of glutamylase activity. Here, we found that CSAP forms a complex with TTLL5, and we demonstrate that the two proteins regulate their reciprocal abundance. Moreover, we show that CSAP increases TTLL5-mediated glutamylation and identify the TTLL5-interacting domain. Deletion of this domain leads to complete loss of CSAP activating function without impacting its MT binding. Binding of CSAP to TTLL5 promotes relocalization of TTLL5 toward MTs. Finally, we show that CSAP binds and activates all of the remaining autonomously active TTLL glutamylases. As such, we present CSAP as a major regulator of tubulin glutamylation and associated functions.
Assuntos
Proteínas de Transporte/metabolismo , Glutamatos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Upon fertilisation, the sperm pronucleus acquires the competence to replicate the genome through a cascade of events that link chromatin remodelling to nuclear envelope formation. The factors involved have been partially identified and are poorly characterised. Here, using Xenopus laevis egg extracts we show that RNAs are required for proper nuclear envelope assembly following sperm DNA decondensation. Although chromatin remodelling and pre-replication complex formation occur normally, RNA-depleted extracts show a defect in pre-RC activation. The nuclear processes affected by RNA-depletion included ELYS recruitment, which accounts for the deficiency in nuclear pore complex assembly. This results in failure in chromatin relaxation as well as in the import and proper nuclear concentration of the S-phase kinases necessary for DNA replication activation. Our results highlight a translation-independent RNA function necessary for the parental genome progression towards the early embryonic cell cycle programme.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Membrana Nuclear/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Extratos Celulares/química , Extratos Celulares/farmacologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Masculino , Membrana Nuclear/genética , Poro Nuclear/genética , Poro Nuclear/metabolismo , Óvulo/citologia , Óvulo/metabolismo , RNA/genética , Espermatozoides/metabolismo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.
Assuntos
Mitose/fisiologia , Proteínas de Xenopus/metabolismo , Quinases Ativadas por p21/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Cromossomos/metabolismo , Feminino , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Oócitos/metabolismo , Fosforilação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Especificidade por Substrato , Xenopus/genética , Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
Cdc42, a Rho family GTPase, is a key regulator of cell polarity. In Saccharomyces cerevisiae, it is required for polarized bud formation and pheromone gradient sensing, while in higher eukaryotes, it participates in asymmetric cell division, directional sensing during migration, and morphogenesis. Using a scratch-induced fibroblast migration assay, we previously showed that Cdc42 controls the polarization of both membrane protrusions and the Golgi/centrosome. We now find that Golgi/centrosome polarity is mediated through activation of the Par6/aPKC complex, as previously described in astrocytes. However, this complex is not involved in Cdc42-dependent polarization of protrusions, which instead is mediated by Pak acting through the Rac guanine nucleotide exchange factor, betaPIX. Pak kinase activity is essential for spatially restricting Rac-dependent actin polymerization to the leading edge of the migrating cells, though it is not required for actin polymerization per se. We conclude that in migrating cells, Cdc42 co-ordinately regulates the polarity of the microtubule and actin cytoskeletons through two distinct pathways.
Assuntos
Actinas/metabolismo , Polaridade Celular , Microtúbulos/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Regulação para Cima/genética , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21 , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The p21-activated kinase (PAK) proteins regulate many cellular events including cell cycle progression, cell death and survival, and cytoskeleton rearrangements. We previously identified X-PAK5 that binds the actin and microtubule networks, and could potentially regulate their coordinated dynamics during cell motility. In this study, we investigated the functional importance of this kinase during gastrulation in Xenopus. X-PAK5 is mainly expressed in regions of the embryo that undergo extensive cell movements during gastrula such as the animal hemisphere and the marginal zone. Expression of a kinase-dead mutant inhibits convergent extension movements in whole embryos and in activin-treated animal cap by modifying behavior of cells. This phenotype is rescued in embryo by adding back X-PAK5 catalytic activity. The active kinase decreases cell adhesiveness when expressed in animal hemisphere and inhibits the calcium-dependent reassociation of cells, while dead X-PAK5 kinase localizes to cell-cell junctions and increases cell adhesion. In addition, endogenous X-PAK5 colocalizes with adherens junction proteins and its activity is regulated by extracellular calcium. Taken together, our results suggest that X-PAK5 regulates convergent extension movements in vivo by modulating the calcium-mediated cell-cell adhesion.