# Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors
## 2 Cells Sense Intracellular Metabolites and Transduce This Information into Cellular Responses  
Cells adapt to dynamic environmental conditions. Upon changes in nutrient availability, eukaryotic and prokaryotic cells reshape their molecular machinery to optimize the uptake and metabolism of particular carbon sources. Membrane-bound protein sensors can mediate the detection of the extracellular concentration of specific nutrients. At least six different types of membrane receptors are involved in chemotaxis in _E. coli_ in response to a broad spectrum of extracellular nutrients, including amino acids, sugars, and nucleotides [8]. By coupling motility to nutrient-sensing, bacteria are more suited for nutrient scavenging in limiting conditions. In yeast, membrane receptors Snf3 and Rgt2 sense low and high glucose concentrations, respectively, and initiate intracellular signal transduction cascades that lead to the induction of membrane transporters with different affinities [9], thereby adjusting glucose uptake to its actual extracellular concentration. However, the occurrence in mammals of homologous systems that sense extracellular levels of glucose or amino acids through membrane-bound receptors could not be proven so far. Cells can take up multiple nutrient species, which are ultimately utilized through a reduced set of metabolic pathways. Asthis diverse set of carbon, nitrogen, and sulfur sources is funneled intracellularly into a few metabolic intermediates, the generation of dedicated specific membrane-bound sensors might be an unjustified evolutionary effort. In fact, evidence accumulated over decades indicates that, instead, cells sense nutrient availability through intracellular sensors [3; 4; 10; 11; 12]. Intracellular sensors bind specific metabolites or second messengers, which then generate a downstream effect that allows cells to adapt to changes in nutrient availability. For instance, \(\alpha\)-proteobacteria sense nitrogen scarcity by monitoring the intracellular concentration of glutamine and 2-oxoglutate. The balance between these two metabolites ultimately controls the activity of glutamine synthase, the enzyme that catalyzes the first step of ammonium assimilation [13]. In eukaryotes, nitrogen availability is also sensed by intracellular molecules. The TOR/mTOR and GCN2-GCN4/ATF4 pathways, which are conserved from yeast to mammalian cells, sense amino acid availability and, in turn, control cell metabolism and mediate growth decisions and general stress response [14]. Interestingly, the intracellular signal that relays amino acid scarcity through GCN2 in eukaryotes (uncharged tRNAs) also triggers a general stress response that inhibits protein translation and stops cell growth in bacteria [15]. Therefore, intracellular nutrient sensing and signaling could converge into a few specific metabolites across evolution. One such example is the glycolytic intermediate fructose-1,6-bisphosphate (FBP). FBP levels change in cells grown under different nutritional regimes and respond to dynamic perturbations of glycolysis across a spectrum of bacterial and yeast species [4; 10]. FBP not only correlates with glycolytic flux but also transduces it into cellular responses, and then it is often referred to as a _flux-signaling metabolite_[3; 16]. Evidence of glycolytic flux sensing through FBP has been found in bacteria, yeast, and mammalian cells. For instance, in Gram-positive bacteria, carbon catabolite repression (CCR) is mediated by the CcpA transcription factor [17]. When glucose is available, FBP stabilizes CcpA binding to DNA, which represses genes involved in the uptake and metabolism of alternative carbon sources [18; 19]. In addition, FBP induces the expression of glycolytic enzymes by prompting the dissociation of the CggR repressor from the promoter of the _gapA_ operon [20; 21; 22]. In yeast, high FBP concentration favors fermentative over oxidative metabolism by activating the H-Ras homolog Ras2 [23; 24].