RESUMO
The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.
Assuntos
Eletricidade , Eletroporação/métodos , Fosfatos de Fosfatidilinositol/deficiência , Animais , Células CHO , Cálcio/metabolismo , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Diglicerídeos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hidrólise , Espaço Intracelular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fatores de TempoRESUMO
The endolysosomal system and autophagy are essential components of macromolecular turnover in eukaryotic cells. The low-abundance signaling lipid PI(3,5)P2 is a key regulator of this pathway. Analysis of mouse models with defects in PI(3,5)P2 biosynthesis has revealed the unique dependence of the mammalian nervous system on this signaling pathway. This insight led to the discovery of the molecular basis for several human neurological disorders, including Charcot-Marie-Tooth disease and Yunis-Varon syndrome. Spontaneous mutants, conditional knockouts, transgenic lines, and gene-trap alleles of Fig4, Vac14, and Pikfyve (Fab1) in the mouse have provided novel information regarding the role of PI(3,5)P2in vivo. This review summarizes what has been learned from mouse models and highlights the utility of manipulating complex signaling pathways in vivo.
Assuntos
Flavoproteínas/genética , Neuropatia Hereditária Motora e Sensorial/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/deficiência , Proteínas Tirosina Fosfatases não Receptoras/genética , Alelos , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Modelos Animais de Doenças , Feminino , Flavoproteínas/metabolismo , Técnicas de Inativação de Genes , Neuropatia Hereditária Motora e Sensorial/metabolismo , Neuropatia Hereditária Motora e Sensorial/patologia , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Fosfatidilinositol 3-Quinases/deficiência , Fosfatases de Fosfoinositídeos , Proteínas Tirosina Fosfatases não Receptoras/deficiênciaAssuntos
Autofagia/genética , Modelos Animais de Doenças , Degeneração Neural/patologia , Doenças Neurodegenerativas/patologia , Fosfatos de Fosfatidilinositol/fisiologia , Animais , Autofagia/fisiologia , Flavoproteínas/genética , Flavoproteínas/metabolismo , Flavoproteínas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/deficiência , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatases de FosfoinositídeosRESUMO
Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P(2) result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P(2) regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P(2) in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P(2) can contribute to inclusion body disease.
Assuntos
Astrócitos/fisiologia , Autofagia , Neurônios/fisiologia , Fosfatos de Fosfatidilinositol/deficiência , Esclerose Lateral Amiotrófica/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Doença de Charcot-Marie-Tooth/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flavoproteínas/genética , Humanos , Corpos de Inclusão/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatases de Fosfoinositídeos , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Regulação para CimaRESUMO
Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.
