Dataset Information

Photostable and Orthogonal Solvatochromic Fluorophores for Simultaneous In Situ Quantification of Multiple Cellular Signaling Molecules.

ABSTRACT: Ratiometric fluorescence sensors are powerful tools for direct quantification of diverse biological analytes. To overcome a shortage of solvatochromic fluorophores crucial for in situ ratiometric imaging of biological targets, we prepared and characterized a small library of modular fluorophores with diverse spectral properties. Among them, WCB and WCR showed excellent spectral properties, including high photostability, brightness, and solvatochromism, and are ideally suited for dual ratiometric imaging due to their spectral orthogonality. By conjugating WCB and WCR with protein-based lipid sensors, we were able to achieve robust simultaneous in situ quantitative imaging of two metabolically linked signaling lipids, phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate in live cells. This study shows that any combination of signaling molecules can be simultaneously quantified in a spatiotemporally resolved manner by ratiometric imaging with finely tuned solvatochromic fluorophores.

SUBMITTER: Sharma A

PROVIDER: S-EPMC7909721 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Photostable and Orthogonal Solvatochromic Fluorophores for Simultaneous <i>In Situ</i> Quantification of Multiple Cellular Signaling Molecules.

Sharma Ashutosh A Sun Jiachen J Singaram Indira I Ralko Arthur A Lee Daesung D Cho Wonhwa W

ACS chemical biology 20200625 7

Ratiometric fluorescence sensors are powerful tools for direct quantification of diverse biological analytes. To overcome a shortage of solvatochromic fluorophores crucial for <i>in situ</i> ratiometric imaging of biological targets, we prepared and characterized a small library of modular fluorophores with diverse spectral properties. Among them, WCB and WCR showed excellent spectral properties, including high photostability, brightness, and solvatochromism, and are ideally suited for dual rati ...[more]

PMID: 32525312

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Project description:Proper three-dimensional structures are essential for maintaining the functionality of proteins and for avoiding pathological consequences of improper folding. Misfolding and aggregation of proteins have been both associated with neurodegenerative disease. Therefore, a variety of fluorogenic tools that respond to both polarity and viscosity have been developed to detect protein aggregation. However, the rational design of highly sensitive fluorophores that respond solely to polarity has remained elusive. In this work, we demonstrate that electron-withdrawing heteroatoms with (d-p)-π* conjugation can stabilize lowest unoccupied molecular orbital (LUMO) energy levels and promote bathochromic shifts. Guided by computational analyses, we have devised a novel series of xanthone-based solvatochromic fluorophores that have rarely been systematically studied. The resulting probes exhibit superior sensitivity to polarity but are insensitive to viscosity. As proof of concept, we have synthesized protein targeting probes for live-cell confocal imaging intended to quantify the polarity of misfolded and aggregated proteins. Interestingly, our results reveal several layers of protein aggregates in a way that we had not anticipated. First, microenvironments with reduced polarity were validated in the misfolding and aggregation of folded globular proteins. Second, granular aggregates of AgHalo displayed a less polar environment than aggregates formed by folded globular protein represented by Htt-polyQ. Third, our studies reveal that granular protein aggregates formed in response to different types of stressors exhibit significant polarity differences. These results show that the solvatochromic fluorophores solely responsive to polarity represent a new class of indicators that can be widely used for detecting protein aggregation in live cells, thus paving the way for elucidating cellular mechanisms of protein aggregation as well as therapeutic approaches to managing intracellular aggregates.

Dataset Information

Photostable and Orthogonal Solvatochromic Fluorophores for Simultaneous In Situ Quantification of Multiple Cellular Signaling Molecules.

Publications

Photostable and Orthogonal Solvatochromic Fluorophores for Simultaneous <i>In Situ</i> Quantification of Multiple Cellular Signaling Molecules.

Similar Datasets

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