@article {70380, title = {Improved sensors for fructose-1,6-bisphosphate enable in vivo imaging of glycolysis}, journal = {bioRxiv}, year = {2026}, month = {2026 May 01}, abstract = {

Fructose-1,6-bisphosphate (FBP) is the product of the first committed step of glycolysis, and its concentration is tightly correlated with glycolytic flux. Glycolytic activity varies across tissues and cell types: some tissues, such as the brain, dynamically regulate glycolysis in response to demand, while others, such as the liver have characterized spatial heterogeneity. Here, we report HYlight2, an improved sensor for FBP developed through random whole-gene mutagenesis in E. coli lysate. After four rounds of screening, we isolated HYlight2, which retains its binding affinity while displaying a ΔR/R \~9 in vitro, a three-fold improvement in mammalian cells, and a two-fold improvement in detecting glycolytic responses during stimulated neuronal activity. We further demonstrate its use in vivo to detect altered glycolytic activity in C. elegans neurons, zebrafish pancreatic islets, and mouse liver.

}, doi = {10.64898/2026.04.29.721630}, url = {https://www.biorxiv.org/content/early/2026/05/01/2026.04.29.721630}, author = {Tyler, Juliann and Vishwanath, Anjali Amrapali and Menon, Triveni and Duarah, Tanishqua and Adhikari, Raghabendra and Koberstein, John N. and Feliciano, Daniel and Espinosa-Medina, Isabel and Col{\'o}n-Ramos, Daniel and Tebo, Alison G.} } @article {68915, title = {Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.}, journal = {ACS Sensors}, volume = {8}, year = {2023}, month = {2023 Aug 25}, pages = {3014-3022}, abstract = {

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

}, issn = {2379-3694}, doi = {10.1021/acssensors.3c00484}, author = {Shen, Yi and Wen, Yurong and Sposini, Silvia and Vishwanath, Anjali Amrapali and Abdelfattah, Ahmed S and Schreiter, Eric R and Lemieux, M Joanne and de Juan-Sanz, Jaime and Perrais, David and Campbell, Robert E} }