RESUMO
Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.
Assuntos
Anoikis/fisiologia , Gliotoxina/metabolismo , Transdução de Sinais/fisiologia , Fatores de Virulência/metabolismo , Amidas , Animais , Anoikis/genética , Linhagem Celular , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 7/genética , MAP Quinase Quinase 7/metabolismo , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Piridinas , Transdução de Sinais/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2- or Bcl-xL-overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-ß but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose/fisiologia , Caspase 8/fisiologia , Efeito Citopatogênico Viral/fisiologia , Mitocôndrias/fisiologia , Vírus da Floresta de Semliki/fisiologia , Animais , Caspase 3/metabolismo , RNA Helicases DEAD-box/fisiologia , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas/fisiologia , Fibroblastos/virologia , Células HEK293/virologia , Células HeLa/virologia , Humanos , Helicase IFIH1 Induzida por Interferon , Camundongos , Mitocôndrias/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Viral/genética , Transdução de Sinais , Replicação Viral , Proteína bcl-X/metabolismoRESUMO
In this chapter, capillary electrophoresis (CE) is demonstrated to be a useful technique for the determination of alkaloids in microsamples of single plant cells. A single cell sampling technique with microcapillaries that includes extraction of sample volumes in the pl range from single cells, division into aliquots, addition of internal standard, and injection into the CE capillary is described. The danger of contamination and evaporation of such low sample volumes has been avoided by handling them under an inert protective layer of silicone oil. For the determination of alkaloids in cell samples, CE with direct ultraviolet detection using a high concentration of citric acid as background electrolyte provides sufficient sensitivity.
