Selecting for pH sensitive flourescent proteins with library screening methods.

Abstract

Intracellular pH (pHi) is known to regulate diverse cell behaviors including cell migration, proliferation, and metabolism. For most of these behaviors, an increase in pHi from ∼7.2 to ∼7.6 has been shown to be a permissive cue. Our current understanding of pHi dynamics during these cell processes is limited by a lack of bright, red-shifted, photostable pH-sensitive fluorescent protein biosensors. Most existing pH biosensors are incompatible with long-term imaging, in vivo pHi measurements, and deep tissue imaging required to understand physiological pHi dynamics during processes like wound healing, cell-cycle progression, and metabolic adaptation. Here we establish a platform for selection of new red-shifted pH-sensitive fluorescent proteins using both random mutagenesis and rational protein design in combination with library selection strategies. We are applying this platform to develop pH biosensors with improved photophysical properties for imaging 3D organoid models as well as increase the linear dynamic range of existing pH-sensitive fluorescent proteins. New red-shifted pH biosensors can be used to track pHi changes inside cells over long time-lapse experiments, while avoiding cellular autofluorescence and allowing for simultaneous imaging of pH and GFP-targeted proteins of interest. The application of these tools will expand our understanding of pHi dynamics and heterogeneity in both normal cell processes as well as models for diseases where pHi is dysregulated like neurodegeneration (low pHi) and cancer (high pHi).