| Autor: |
Pratt EPS; Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Ave, UCB 596, Boulder, Colorado 80309-0401, United States., Anson KJ; Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Ave, UCB 596, Boulder, Colorado 80309-0401, United States., Tapper JK; Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Ave, UCB 596, Boulder, Colorado 80309-0401, United States., Simpson DM; Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Ave, UCB 596, Boulder, Colorado 80309-0401, United States., Palmer AE; Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Ave, UCB 596, Boulder, Colorado 80309-0401, United States. |
| Abstrakt: |
Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn 2+ 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 2+ 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 2+ levels, we developed CgA-Zn 2+ 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 2+ 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 Zn 2+ at the single-vesicle level, we were able to monitor Zn 2+ dynamics in populations of vesicles and detected high vesicular Zn 2+ 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 2+ from pM to nM ranges. |
