Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

Abstract

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

“…This property, which is the basis for Bimolecular Fluorescence Complementation (BiFC) and several related protein fragment complementation assays (PCAs), has been exploited to detect protein-protein interactions in living cells and even whole animals [85], although the effectively irreversible nature of BiFC has limited its application in tracking dynamic interactions. In order to generate high-resolution maps of specific protein-protein interactions, specific pairs of fragments have been identified for photoactivatable or photoswitchable proteins and used for imaging protein-protein interactions in superresolution [86–89]. For example, a BiFC-PALM approach was recently developed using PAmCherry1 [88].…”

RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells

“…In addition, fragments of PAGFP have been used to localize and characterize constitutively formed protein dimers (Xia et al, 2014). Moreover, the photoactivatable FP mEos3.2 has also been used to demonstrate BiFC-PALM, predominantly in bacteria, to investigate constitutive PPIs (Liu et al, 2014). Although these PALM-based techniques currently provide greater spatial resolution enhancement than SOFI-based methods under optimal conditions, refSOFI can serve as a complementary strategy, since it offers several advantages that allow outperforming BiFC-PALM depending on the experimental context.…”

“…Fulfilling these distinct requirements in a single fluorescent protein complicates the task of identifying suitable fragments. Recently, super-resolution imaging utilizing BiFC has been demonstrated using PALM (Liu et al, 2014; Nickerson et al, 2014; Xia et al, 2014). Although development of BiFC-PALM represents a valuable advance, live-cell imaging requires adjustments that limit its actual performance.…”

Intranucleus Single-Molecule Imaging in Living Cells

“…2 c ③) (42). Another similar strategy based on background rejection is implemented by bimolecular fluorescence complementation, including mEos3.2 (43), sfGFP (44), and sfCherry (45). Fusing the protein of interest (POI) with the multiple short fragment of FPs (the 11th b-sheet), the remaining of the larger fragment (the 1-10th b-sheet) can bind the array with high affinity (Fig.…”