RESUMO
Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study, we demonstrate that, with the microarrays for mass spectrometry platform, we are able to observe this variability. We monitor environmentally (2-deoxy-D-glucose) and genetically (ΔPFK2) perturbed Saccharomyces cerevisiae cells at the single-cell, few-cell, and population levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge, give biological insight that is not accessible from population-level metabolomic data.
Assuntos
Glicólise/fisiologia , Metabolômica/métodos , Saccharomyces cerevisiae/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Contagem de Células , Desoxiglucose , Modelos Lineares , Análise em Microsséries/métodos , Saccharomyces cerevisiae/metabolismoRESUMO
Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5' (5'ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5'ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5'ppp dsRNA panhandle as a RIG-I activator.