RESUMO
Cell-to-cell heterogeneity in metabolism plays an unknown role in physiology and pharmacology. To functionally characterize cellular variability in metabolism, we treated cells with inhibitors of oxidative phosphorylation (OXPHOS) and monitored their responses with live-cell reporters for ATP, ADP/ATP, or activity of the energy-sensing kinase AMPK. Across multiple OXPHOS inhibitors and cell types, we identified a subpopulation of cells resistant to activation of AMPK and reduction of ADP/ATP ratio. This resistant state persists transiently for at least several hours and can be inherited during cell divisions. OXPHOS inhibition suppresses the mTORC1 and ERK growth signaling pathways in sensitive cells, but not in resistant cells. Resistance is linked to a multi-factorial combination of increased glucose uptake, reduced protein biosynthesis, and G0/G1 cell-cycle status. Our results reveal dynamic fluctuations in cellular energetic balance and provide a basis for measuring and predicting the distribution of cellular responses to OXPHOS inhibition.
Assuntos
Antineoplásicos/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fase G1/efeitos dos fármacos , Glucose/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Activating mutations in RAS are present in ~ 30% of human tumors, and the resulting aberrations in ERK/MAPK signaling play a central role in oncogenesis. However, the form of these signaling changes is uncertain, with activating RAS mutants linked to both increased and decreased ERK activation in vivo. Rationally targeting the kinase activity of this pathway requires clarification of the quantitative effects of RAS mutations. Here, we use live-cell imaging in cells expressing only one RAS isoform to quantify ERK activity with a new level of accuracy. We find that despite large differences in their biochemical activity, mutant KRAS isoforms within cells have similar ranges of ERK output. We identify roles for pathway-level effects, including variation in feedback strength and feedforward modulation of phosphatase activity, that act to rescale pathway sensitivity, ultimately resisting changes in the dynamic range of ERK activity while preserving responsiveness to growth factor stimuli. Our results reconcile seemingly inconsistent reports within the literature and imply that the signaling changes induced by RAS mutations early in oncogenesis are subtle.
Assuntos
Carcinogênese/genética , Genes ras/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Processamento de Imagem Assistida por Computador , Cinética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas , Análise de Célula ÚnicaRESUMO
Single-cell analysis of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) provides a means to perform highly detailed kinetic studies, assess heterogeneity between cells, and distinguish the subcellular localization of ERK activity. We describe here the methods needed to perform such measurements in a cell type of the investigator's choosing. We discuss the selection of appropriate reporters and provide detailed methods for stably introducing reporters, collecting live-cell data, and automatically extracting quantitative information from individual cells.