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Event Details

  • Tuesday, April 10, 2018
  • 18:05 - 18:15

Alejandro San Martin - Development of Optogenetics Tools to Measure Metabolic Flux at Subcellular Resolution in Intact Cells

Metabolites are the nodes of a complex network which is highly conserved throughout life. Understanding of local fluxes within the metabolic grid is constrained by technological limitations, mainly spatiotemporal resolution. For example, it has been known for a while that the first enzyme of glycolysis is attached to mitochondria, but we do not know what is the functional meaning of this location. Current models of cellular metabolism assume that all mitochondria in a cell behave equally. Yet mitochondria within a given cell differ in terms of morphology, age, location, connectivity, motility, and crosstalk with plasma membrane and organelles. Are they all similar in terms of their metabolism? Technologies are emerging that are capable of measuring metabolites with subcellular resolution in living cells. Genetically-encoded nanosensors are proteins build by the fusion of a ligand binding moiety with a fluorescent protein. The binding of the test molecule produces a conformational change that affect the spectroscopy properties of the fluorescent partner. Being genetically-encoded, they can be expressed in single-cells and targeted to subcellular localizations such as mitochondria. Here we introduce PyronicSF, a single-wavelength nanosensor for pyruvate. This sensor undergoes a maximal fluorescent change of 250% and detects pyruvate between 0.03 and 12 mM, spanning the physiological range in mammalian cells. PyronicSF was insensitive to pH and a panel of molecules structurally related to pyruvate. In combination with pharmacological inhibition of the mitochondrial pyruvate carrier (MPC) it was possible to obtain real-time, reversible measurements of the rate of pyruvate consumption in single mitochondria. Our results show that it is possible to characterize metabolic flux in individual organelles. We envisage that single-mitochondria pyruvate consumption measurements will allow to go deep into the understanding of how subcellular localization, morphology, aging and motility can modulate mitochondrial function.