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Systematic Comparison of Vesicular Targeting Signals Leads to the Development of Genetically Encoded Vesicular Fluorescent Zn²⁺ and pH Sensors.

ABSTRACT: Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn²⁺ 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 Zn²⁺ 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 Zn²⁺ levels, we developed CgA-Zn²⁺ 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 Zn²⁺ and pH in individual secretory vesicles by leveraging the acceptor fluorescent protein as a pH sensor and found that pH influenced FRET measurements in situ. While unable to characterize vesicular Zn²⁺ at the single-vesicle level, we were able to monitor Zn²⁺ dynamics in populations of vesicles and detected high vesicular Zn²⁺ 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 Zn²⁺ from pM to nM ranges.

SUBMITTER: Pratt EPS

PROVIDER: S-EPMC8444068 | biostudies-literature |

REPOSITORIES: biostudies-literature

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