PHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps

Anyuan Liu,Fudong Xue,Pingyong Xu,Liangyi Chen,Jiajia Liu,Lin Yuan,Xiaoshuai Huang,Wenting He,Yanrui Yang

doi:10.1038/s41467-021-21666-7

Abstract

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).

Highlights

PH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events
This effect is directly related to the pH sensitivity of each FP, a property that is controlled by two main factors: pKa and the apparent Hill coefficient[5]
We propose a photostable red pH-sensitive FP that is developed from mScarlet-I for the simultaneous detection of vesicle docking and fusion

Summary

Introduction

PH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. The patch-clamp electrophysiological technique can effectively detect the total exocytosis and/or endocytosis signals of all vesicles in a single cell at very high temporal resolution. Among the available FPs, SEP has a nearly optimal pKa (7.2) and the highest nH (1.90)[5] As such, it is highly pH sensitive, and it shows the greatest change in fluorescence intensity upon transfer to the extracellular environment. PHuji exhibits pronounced photo-switching behavior wherein its fluorescence decreases to 35% of the initial value in less than 2 s of continuous illumination[5] This renders the protein unsuitable for quantitative imaging of exocytosis. The docked vesicles that appear near the plasma membrane are visualized using an electron microscope[12] This instrument cannot be used to simultaneously detect the dynamic stages of docking and fusion in living cells. PHuji cannot be used to reliably monitor the docking and fusion kinetics of vesicle exocytosis due to its photo-switching behavior[5]

Methods

Results

Conclusion