Real-time monitoring of newly acidified organelles during autophagy enabled by reaction-based BODIPY dyes

Hanzhuang Liu,Zhen Shen,Yinghong Lu,Wenting Song,Delia Gröninger,Zhikuan Zhou,Kin Shing Chan,Kin Shing Chan,Knut Rurack,Lei Zhang

doi:10.1038/s42003-019-0682-1

Abstract

Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes. Herein, we describe a reaction-based boron dipyrromethene (BODIPY) dye containing strongly electron-withdrawing diethyl 2-cyanoacrylate groups at the α-positions. The probe exhibits intense red fluorescence in acidic organelles or the acidified cytosol while exhibiting negligible fluorescence in other regions of the cell. The underlying mechanism is a nucleophilic reaction at the central meso-carbon of the indacene core, resulting in the loss of π-conjugation entailed by dramatic spectroscopic changes of more than 200 nm between its colorless, non-fluorescent leuco-BODIPY form and its red and brightly emitting form. The reversible transformation between red fluorescent BODIPY and leuco-BODIPY along with negligible cytotoxicity qualifies such dyes for rapid and direct intracellular lysosome imaging and cytosolic acidosis detection simultaneously without any washing step, enabling the real-time monitoring of newly acidified organelles during autophagy.

Highlights

Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes
The formally positive charge is partly localized on the bridge head or meso-carbon and the degree of stabilization of the single structures depends on the pattern and electronic properties of substituents at the indacene core[37]
We prepared boron dipyrromethene (BODIPY) 1 substituted with strongly electron-withdrawing groups (EWG) at the α-positions (Fig. 1b, Methods Section)

Summary

Introduction

Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes. A versatile strategy for the design of reaction-based probes is the ′′pro-chromophore′′ approach[4], where a weakly (ideally zero) fluorescent pro-chromophore is transformed into its highly fluorescent parent dye through reaction with the target analyte If this ′′lighting up′′ of the fluorescence is accompanied by a color change from colorless to a visible color and if the reaction itself is reversible, such a dye system belongs to the perhaps oldest class of responsive dyes: dyes that can be switched reversibly between a colorless leuco form and a colored all-π-conjugated form[5]. Compared to ICT, PET or FRET, the transformation between a π-conjugated and a non-π-conjugated state generally offers dramatic spectral changes, minimal background and high output signals Along this strategy, especially dyes based on the rhodamine skeleton have led to a variety of excellent fluorescent probes for super-resolution imaging[12] and the detection of biologically relevant analytes[8,13,14,15]. At pH levels that are characteristic of newly acidified organelles, it is transformed into the typical, all-π-conjugated BODIPY form that shows bright red color and fluorescence

Methods

Results

Conclusion