RESUMO
Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
Assuntos
Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.
Assuntos
Endocitose/efeitos dos fármacos , Melanoma/metabolismo , Modelos Biológicos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ressonância de Plasmônio de Superfície/métodos , Transferrina/farmacologia , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Vesículas Transportadoras/efeitos dos fármacosRESUMO
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P(2) and PI(3,4,5)P(3) in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P(2) accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P(2) depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P(2) contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P(3) clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P(3) beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.
Assuntos
Escherichia coli Enteropatogênica/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Linhagem Celular , Escherichia coli Enteropatogênica/genética , Células Epiteliais/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/metabolismoRESUMO
The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.
Assuntos
Fase G1/fisiologia , Fase de Repouso do Ciclo Celular/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Antineoplásicos/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Nocodazol/metabolismo , Oócitos/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Xenopus laevisRESUMO
We have previously described two nucleolar proteins, named p14 and p21, in MMTV-induced T cell lymphomas. These proteins were identified by a monoclonal antibody (M-66) generated from a nontumorigenic, immunogenic variant of S49 T cell lymphoma. While p14 was common to several MMTV-derived T cell lymphomas, p21 was found only in highly tumorigenic variants of S49 cells. Here we report that p14 is the leader peptide of the MMTV env precursor. The epitope recognized by M-66 contains a putative nuclear localization signal. Actinomycin D was found to induce redistribution of p14/p21 from the nucleolus to the nucleoplasm. p14 coimmunoprecipitated and colocalized with the cellular protein, B23. Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (HTLV).