AggFluor: Fluorogenic Toolbox Enables Direct Visualization of the Multi-Step Protein Aggregation Process in Live Cells

Abstract

Aberrantly processed or mutant proteins misfold and assemble into a variety of soluble oligomers and insoluble aggregates, a process that is associated with an increasing number of diseases that are not curable or manageable. Herein, we present a chemical toolbox, AggFluor, that allows for live cell imaging and differentiation of complex aggregated conformations in live cells. Based on the chromophore core of green fluorescent proteins, AggFluor is comprised of a series of molecular rotor fluorophores that span a wide range of viscosity sensitivity. As a result, these compounds exhibit differential turn-on fluorescence when incorporated in either soluble oligomers or insoluble aggregates. This feature allows us to develop, for the first time, a dual-color imaging strategy to distinguish unfolded protein oligomers from insoluble aggregates in live cells. Furthermore, we have demonstrated how small molecule proteostasis regulators can drive formation and disassembly of protein aggregates in both conformational states. In summary, AggFluor is the first set of rationally designed molecular rotor fluorophores that evenly cover a wide range of viscosity sensitivities. This set of fluorescent probes not only change the status quo of current imaging methods to visualize protein aggregation in live cells but also can be generally applied to study other biological processes that involve local viscosity changes with temporal and spatial resolutions.

Rational Design of Crystallization‐Induced‐Emission Probes To Detect Amorphous Protein Aggregation in Live Cells

“…Another challenge for selective detection of amorphous aggregates is how to achieve selectivity over other organelles in the complex cellular milieu as they accumulate intracellularly rather than the extracellular space for amyloid aggregates. Although AIEgens exhibit appealing performance in detecting amyloid aggregated proteins, they failed to detect cellular amorphous ones without the covalent linkage to proteins exemplified by the seminal works from Hong and Zhang et al [34–38] . Sensors of inherent selective binding towards amorphous aggregated proteins, however, were seldom reported.…”

Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors

“…[55][56][57] Zhang et al introduced chemical tag strategy to the eld, enabling visualization of aggregation process of a specic protein-of-interest in live cells. [58][59][60][61][62] Inspired by these seminal works, through sortase mediated site specic bio-conjugation, we rst demonstrated four environment-sensitive uorophores with spectral coverage from 500 nm to 620 nm can serve as protein aggregation sensors. Two of these sensors (CCVJ and Mero) exhibited minimal uorescence crosstalk and detected the co-aggregation of two different proteins.…”

Covalent Probes for Aggregated Protein Imaging via Michael Addition

“…This is probably due to the fact that the local chemical reactivity within aggregated proteins has not been fully explored. Recently,Z hang [43,44] and Hong [45] et al embarked on the survey of the physical environment inside aggregated proteins,b ut their work primarily revealed the changes in polarity and viscosity upon protein aggregation using environment-sensitive probes.H owever,i ts till remains elusive how chemically reactive proteins are at their aggregated state. Therefore,c urrent powerful chemical sensing and bioconjugation strategies [46][47][48] may provide hints to address this question.…”

“…This is probably due to the fact that the local chemical reactivity within aggregated proteins has not been fully explored. Recently, Zhang [43, 44] and Hong [45] et al. embarked on the survey of the physical environment inside aggregated proteins, but their work primarily revealed the changes in polarity and viscosity upon protein aggregation using environment‐sensitive probes.…”