TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

Yongliang Li,Yujie Sun,Chaoying Sun,Hongchen Zhang,Yong Zeng,Shipeng Shao

doi:10.1038/s42003-021-01896-7

Abstract

Protein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

Highlights

Protein-protein interactions (PPIs) are critical for cellular activity regulation
In order to introduce organic dyes into bimolecular fluorescence complementation (BiFC) for precise and long-term tracking of PPIs in living cells, we examined the possibilities of splitting the HaloTag and explored the performance of the split HaloTag system (TagBiFC) for single-molecule PPI imaging in living cells in real time
When the two fragments are fused with two proteins that can interact, the interaction between the fusion proteins bring the two split halves to proximity spatially and facilitate the association between the fragments of the split HaloTag, generating a self-labeling enzyme that can catalyze the ligation of dyeconjugated substrates to itself

Summary

Introduction

Protein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. We developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. When the nonfluorescent fragments are fused with two proteins that can interact with each other, they can complement to form a whole FP and become fluorescent[4,5,6] This approach enables visualization and detection of the subcellular location and dynamics of specific protein complexes in the living cellular environment. In order to introduce organic dyes into BiFC for precise and long-term tracking of PPIs in living cells, we examined the possibilities of splitting the HaloTag and explored the performance of the split HaloTag system (TagBiFC) for single-molecule PPI imaging in living cells in real time. Compared with the split photoactivatable fluorescent protein (PAFP) mMaple[3], the split

Methods

Results

Conclusion