A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions.

Shiqi Mao,Yantao Yang,Antony K Chen,Yachen Ying,Zhao Ma

doi:10.1021/acsnano.1c03242

Shiqi Mao, Yantao Yang + Show 3 more

Open Access

https://doi.org/10.1021/acsnano.1c03242

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Journal: ACS nano	Publication Date: Aug 24, 2021
Citations: 8	License type: CC BY 4.0

Affiliation: Peking University

Abstract

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition. We showed that when BAC-BiFC is designed to image PPIs regulating Human Immunodeficiency Virus type 1 (HIV-1) assembly, the fluorescence ratio could decrease with decreasing probe quantity, and ratios approaching the limit of detection could allow physiologically relevant characterization of the assembly process on the nanoscale by single-molecule localization microscopy (SMLM). With much improved clarity, previously undescribed features of HIV-1 assembly were revealed.

Highlights

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein−protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level
In this study we showed that when the reference FP is appended to one of the fusion protein pair, the fluorescence ratio of the BiFC signal to the reference signal can serve as a means to correct variations in probe transfection and enable tuning of transfection protocols to result in an optimal expression level at which the contribution of nonspecific PPIs to the overall BiFC signal becomes marginal compared with specific PPIs, leading to accurate characterization of specific PPIs with a spatial resolution down to the nanometer scale
Emerging evidence has shown that, alternative to mediating RNA interference (RNAi), AGO2 is usurped by the virus to facilitate Gag oligomerization.[14−17] We showed that by transfecting cells with different quantities of the BAC-BiFC constructs and assessing the corresponding fluorescence ratios on a single-cell basis, optimal transfection conditions could be identified that allow Gag−Gag interaction or Gag−AGO2 interaction events to be illuminated with much improved clarity on the nanoscale by single-molecule localization microscopy (SMLM)

Summary

Introduction

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein−protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. The second PPI studied occurs between Gag and argonaute-2 (AGO2), a host cellular protein that is best known for its ability to orchestrate RNA interference (RNAi).[12,13] Emerging evidence has shown that, alternative to mediating RNAi, AGO2 is usurped by the virus to facilitate Gag oligomerization.[14−17] We showed that by transfecting cells with different quantities of the BAC-BiFC constructs and assessing the corresponding fluorescence ratios on a single-cell basis, optimal transfection conditions could be identified that allow Gag−Gag interaction or Gag−AGO2 interaction events to be illuminated with much improved clarity on the nanoscale by single-molecule localization microscopy (SMLM) With such high definition, previously undescribed nanoscale organizations of the HIV-1 biogenesis process were reported

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