Systematic Comparison of Vesicular Targeting Signals Leads to the Development of Genetically Encoded Vesicular Fluorescent Zn and pH Sensors.

Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn and pH, in living cells. However, vesicular sensors have a tendency to mislocalize and are susceptible to the acidic intraluminal pH. In this study, we performed a systematic comparison of five different vesicular proteins to target the fluorescent protein mCherry and a Zn Förster resonance energy transfer (FRET) sensor to secretory vesicles. We found that motifs derived from vesicular cargo proteins, including chromogranin A (CgA), target vesicular puncta with greater efficacy than transmembrane proteins. To characterize vesicular Zn levels, we developed CgA-Zn FRET sensor fusions with existing sensors ZapCY1 and eCALWY-4 and characterized subcellular localization and the influence of pH on sensor performance. We simultaneously monitored Zn and pH in individual secretory vesicles by leveraging the acceptor fluorescent protein as a pH sensor and found that pH influenced FRET measurements . While unable to characterize vesicular Zn at the single-vesicle level, we were able to monitor Zn dynamics in populations of vesicles and detected high vesicular Zn in MIN6 cells compared to lower levels in the prostate cancer cell line LnCaP. The combination of CgA-ZapCY1 and CgA-eCALWY-4 allows for measurement of Zn from pM to nM ranges.