Protein-protein Interactions In Living Cells Research Articles

Progress in research and application of the biotin ligase BirA and its mutants in pathogen-host interaction

Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.

The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

CellFIE: CRISPR- and Cell Fusion-based Two-hybrid Interaction Mapping of Endogenous Proteins

Numerous genetic methods facilitate the detection of binary protein–protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions.

Protein Direct Interactome: Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa (Adv. Biology 3/2021)

Genetically encoded chemical crosslinking (GECX) via bioreactive unnatural amino acid (Uaa) enables the capture of weak and transient protein-protein interactions in live cells for identification with mass spectrometry. In article number 2000308, Chao Liu, Shang-Tong Li, Kui Li, Lei Wang, Bing Yang, and co-workers develop a new software OpenUaa to analyze Uaa-crosslinked peptides, which uniquely considers all fragment ions for high sensitivity and identify direct protein interactome with the GECX-OpenUaa strategy.

A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein–protein interactions in vivo

Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein–protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems—iRFP-BiFC and IFP1.4-BiFC—the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

Highly Modular Protein Micropatterning Sheds Light on the Role of Clathrin-Mediated Endocytosis for the Quantitative Analysis of Protein-Protein Interactions in Live Cells.

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform.

Resolving Membrane Protein-Protein Interactions in Live Cells with Pulsed Interleaved Excitation Fluorescence Cross-Correlation Spectroscopy.

The cell plasma membrane (PM) contains thousands of proteins that sense and respond to the outside environment. These proteins have evolved sensitivity to a wide variety of physical and chemical signals and act as a delivery system across the PM. Membrane proteins are critical for information flow and decision making in the cell and thus are important targets in drug development. A critical aspect of membrane protein function is the way they interact with other proteins, often through the formation of dimers or small oligomers that regulate function at the protein, cell, and organism levels. Resolving membrane protein interactions in a live cell environment is challenging because of the chemical diversity and spatial heterogeneity of the PM. In this Account, we describe a fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) that is ideally suited to quantify membrane associations in live cells. PIE-FCCS is a two-color fluorescence fluctuation method that can simultaneously measure the concentration, mobility, proximity, and oligomerization state of membrane proteins in situ. It has several advantages over two related approaches, single-molecule tracking (SMT) and Förster resonance energy transfer (FRET), including that it measures all of the properties listed above in a single measurement. Another advantage is that PIE-FCCS is most sensitive at the physiological expression levels for many membrane proteins rather than the very low or high levels typical in other techniques. Here, we review the history of FCCS as it has been applied to study membrane protein interactions in cells. We also describe PIE-FCCS and the advantages it has over biochemical approaches like coimmunoprecipitation (co-IP) and proximity ligation assays (PLA). Finally, we review two classes of membrane proteins that have been studied with FCCS and PIE-FCCS: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). For RTKs, ligand induced dimerization directly regulates the catalytic activity of the kinase, but higher order oligomerization and ligand-independent dimerization can complicate this historically simple paradigm. PIE-FCCS data have resolved a low population of EGFR dimers under basal conditions and assembly into multimers when stimulated with ligand. While GPCRs function primarily as monomers, dimerization has been hypothesized to regulate function for some receptors. PIE-FCCS data have established the dimerization potential of rhodopsin at low densities and were critical for the discovery of a novel dimerization interface in human cone opsins. This Account describes the how FCCS and PIE-FCCS can reveal the details of quaternary interactions in each of these receptor systems.

Split T7 RNA polymerase biosensors to study multiprotein interaction dynamics.

Protein-protein interactions (PPIs) are involved in nearly all cellular processes. PPIs are particularly crucial for mediating selectivity along signaling pathways. Thus, measuring the competitive interplay between PPIs in a cell is important for both understanding fundamental cellular regulation and developing therapeutics targeting those whose dysregulation is associated with disease. A variety of split protein reporter-based tools are available to measure if two proteins interact within a cell and thereby characterize the general determinants of their interactions. PPIs, however, occur within complex networks facilitated by dynamic biophysical nuances that determine activity and selectivity. Evolved, proximity-dependent split T7 RNA polymerase (RNAP) biosensors have recently been used to perform deep mutational scanning of PPI interfaces, and to create synthetic gene circuits. In this chapter, we present the application of proximity-dependent split RNAP biosensors as a method to measure multidimensional PPIs in live cells. Orthogonal split RNAP "tags" encode each interaction in a unique RNA signal, thereby enabling the study of multiple competitive PPIs in live cells. Each unique RNA signal can be quantified via established RNA analysis methods. Herein, we provide advice and protocols to aid other researchers in using the split RNAP biosensor, focusing primarily on how to detect multiple PPIs in mammalian cells, including their dynamic interplay in the presence of small molecule inhibitors.

Temporally gated molecular tools for tracking protein-protein interactions in live cells.

Protein-protein interactions (PPIs) are essential in most biological processes. Even though many methods were designed to detect PPIs, detecting PPIs in a large volume of cells with a temporal resolution remains challenging. Recent development of light gated transcriptional reporters, such as SPARK and iTANGO, enabled detection of PPI in a large population of cells with a temporal resolution on the order of minutes. In this chapter, we discussed in detail the application of SPARK to detect PPIs between the activated β-2 adrenergic receptor (B2AR) and both Gα mimic and β-arrestin2. Because SPARK is a multi-component system, the protein expression level is critical for its optimal performance. We also discussed the detailed protocols for using SPARK with either transfection or lentiviral infection in HEK296T/17 cells.

Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer.

Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.

Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells.

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

Characterization of SPAD Array for Multifocal High-Content Screening Applications

Current instruments used to detect specific protein-protein interactions in live cells for applications in high-content screening (HCS) are limited by the time required to measure the lifetime. Here, a 32 × 1 single-photon avalanche diode (SPAD) array was explored as a detector for fluorescence lifetime imaging (FLIM) in HCS. Device parameters and characterization results were interpreted in the context of the application to determine if the SPAD array could satisfy the requirements of HCS-FLIM. Fluorescence lifetime measurements were performed using a known fluorescence standard; and the recovered fluorescence lifetime matched literature reported values. The design of a theoretical 32 × 32 SPAD array was also considered as a detector for a multi-point confocal scanning microscope.

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

Unraveling the mechanism of cholesterol-mediated regulation of receptor dimerization in plasma membranes in vivo

Membrane cholesterol can alter the signaling pathways of living cells. However, the process that modulates the interaction of receptor proteins is still unclear. We performed single-molecule optical tracking of ligand-induced dimerization of epidermal growth factor receptors (EGFRs) in two cancerous cell lines (HeLa and A431) and one normal endothelial cell line (MCF12A). We discovered that unliganded EGFRs typically reside in non-raft regions of the plasma membrane and can move into raft domains upon ligand binding. This ligand-induced motion could be a common behavior in live cells. We found that the amount of membrane cholesterol significantly affects the stability of EGFR dimers by manipulating the total amount of membrane cholesterol with methyl-β-cyclodextrin and the local concentration of cholesterol with nystatin. The EGFR dimers in the plasma membrane of normal cells are more sensitive to changes in the local concentration of cholesterol compared with the cancer cells. Our methodology can yield useful information for understanding cholesterol-mediated protein-protein interactions in live cells.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.

Bioluminescence resonance energy transfer to detect protein-protein interactions in live cells.

Bioluminescence resonance energy transfer (BRET) is a valuable tool to detect protein-protein interactions. BRET utilizes bioluminescent and fluorescent protein tags with compatible emission and excitation properties, making it possible to examine resonance energy transfer when the tags are in close proximity (<10nm) as a typical result of protein-protein interactions. Here we describe a protocol for detecting BRET from two known protein binding partners (Gαi1 and RGS14) in HEK 293 cells using Renilla luciferase and yellow fluorescent protein tags. We discuss the calculation of the acceptor/donor ratio as well as net BRET and demonstrate that BRET can be used as a platform to investigate the regulation of protein-protein interactions in live cells in real time.

Decoding the Role of Receptor Dimerization in Plexin-Semaphorin Signaling

Plexin-semaphorin signaling plays an important role in the formation of neural networks by acting as the guidance cue for axon growth or retraction. The plexin receptor is a single-pass transmembrane protein with a sema domain at the extracellular N-terminus and a GTP-ase activating protein domain on the cytoplasmic C-terminus. Binding of semaphorin is known to activate the plexin receptor, but the mechanism of how that binding event leads to activation is not understood. We will present our recent work using pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to measure plexin protein-protein interactions in live cells. PIE-FCCS transforms fluctuations in fluorescence intensity (arising mainly from diffusion) into information about a protein's mobility and concentration. By cross-correlating the fluorescence fluctuations in two color channels, PIE-FCCS also quantifies co-localization and co-diffusion with high accuracy. This method has been used to resolve the mechanism of epidermal growth factor receptor (EGFR) activation and inhibition, as well as the dimerization constant of the opsin G protein-coupled receptor. In this work we measure the ligand-free dimerization of plexin receptors in live cell membranes and the response of the receptor to semaphorin-induced activation.