RESUMO
Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.
Assuntos
Proteínas Quinases Ativadas por AMP , Mitocôndrias , Proteínas Quinases Ativadas por AMP/genética , Núcleo Celular , Humanos , Hibridização in Situ Fluorescente , Mitocôndrias/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Understanding signal propagation across biological networks requires to simultaneously monitor the dynamics of several nodes to uncover correlations masked by inherent intercellular variability. To monitor the enzymatic activity of more than two components over short time scales has proven challenging. Exploiting the narrow spectral width of homo-FRET-based biosensors, up to three activities can be imaged through fluorescence polarization anisotropy microscopy. We introduce Caspase Activity Sensor by Polarization Anisotropy Multiplexing (CASPAM) a single-plasmid triple-modality reporter of key nodes of the apoptotic network. Apoptosis provides an ideal molecular framework to study interactions between its three composing pathways (intrinsic, extrinsic, and effector). We characterized the biosensor performance and demonstrated the advantages that equimolar expression has in both simplifying experimental procedure and reducing observable variation, thus enabling robust data-driven modeling. Tools like CASPAM become essential to analyze molecular pathways where multiple nodes need to be simultaneously monitored.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Anisotropia , Caspases/genética , Microscopia de FluorescênciaRESUMO
In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.