Quantitative fluorescent biosensors to study brain metabolism
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rosen-pcrosen-phd-biology-2026-thesis .pdf
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8.89 MB
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Author(s)
Rosen, Paul C.
Advisor(s)
Lourido, Sebastian
Yellen, Gary
Date Issued
February 2026
Publisher
Massachusetts Institute of Technology
Abstract
Genetically encoded biosensors that have changes in fluorescence lifetime can enable robust measurements of metabolite levels in single living cells over time, even in tissue contexts. However, these sensors are uncommon, which restricts the approach to the small number of metabolites for which lifetime-readout sensors have been engineered. Furthermore, lifetimereadout sensors are poorly understood at a molecular level, which makes it difficult to build new sensors. Using X-ray crystallography and other biophysical approaches, I defined the molecular mechanisms by which three fluorescence-lifetime readout sensors (LiLac, RoTq-On, and RoTqOff) transform changes in analyte binding into changes in fluorescence. The changes in fluorescence lifetime in these sensors reflect how the chromophore is rigidified by sealing. I also engineered new, robust lifetime-readout sensors for thiol-disulfide redox and citrate. I imaged these sensors in the cytosol of neurons in acute mouse brain slices. Most cortical neurons are highly reduced at baseline, but ~10% of them are stably more oxidized; citrate dynamics in neurons upon stimulation are consistent with the idea that citrate contributes to the inhibition of neuronal glycolysis. Together, the work provides (1) a biophysical basis for understanding how lifetime-readout biosensors translate changes in ligand binding into changes in fluorescence, (2) several new lifetime-readout sensors for metabolites, and (3) demonstrations of how these sensors can quantify otherwise invisible molecules in the context of the mouse brain.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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