A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors

Abstract

Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells

“…Given the close coupling of the Na + /K + pump to neuronal glycolysis, it will be exciting to measure how changes in Na + /K + pump activity affect the flux of other glycolytic metabolites using biosensors that report lactate ( Koveal et al, 2022 ; San Martín et al, 2013 ), pyruvate ( San Martín et al, 2014 ), and glucose ( Díaz-García et al, 2019 ), or how changes in Na + /K + pump activity affect cellular energy status (i.e., [ATP] [ Imamura et al, 2009 ] and ATP:ADP [ Tantama et al, 2013 ]). These investigations will ultimately provide a more complete picture of the relationship between ion homeostasis and metabolism.…”

A red fluorescent genetically encoded biosensor for extracellular L-lactate

“…To help to surmount this obstacle, we have now expanded the color spectrum of eLACCO1.1 by engineering a red fluorescent biosensor, R-eLACCO2. To the best of our knowledge, no red fluorescent biosensors for extracellular or intracellular [25][26][27][28] L-lactate have been reported. R-eLACCO2 is a first-in-class red fluorescent biosensor for minimally-invasive imaging of the dynamics of extracellular L-lactate, potentially enabling an orthogonal readout of distinct and multiple metabolic events.…”

“…Inspired by the tremendous impact that genetically encoded biosensors have had in various areas of biology and physiology, we and others have developed biosensors for the detection of intracellular [25][26][27][28] and extracellular L-lactate 29 . For imaging of extracellular L-lactate, we recently reported a genetically encoded green fluorescent biosensor, designated eLACCO1.1, that was developed using an extensive directed evolution and an intensive effort to target it to the extracellular environment 29 .…”

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells

“…Given the close coupling of the Na + /K + pump to neuronal glycolysis, it will be exciting to measure how changes in Na + /K + pump activity affect the flux of other glycolytic metabolites using biosensors that report lactate (Koveal et al, 2022;San Martín et al, 2013), pyruvate (San Martín et al, 2014, and glucose (Díaz-García et al, 2019), or how changes in Na + /K + pump activity affect cellular energy status (i.e., [ATP] (Imamura et al, 2009) and ATP:ADP (Tantama et al, 2013)). These investigations will ultimately provide a more complete picture of the relationship between ion homeostasis and metabolism.…”