Localizing protein-protein interactions by bimolecular fluorescence complementation in planta

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein–protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein–protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.     

Genome-wide YFP fluorescence complementation screen identifies new regulators for telomere signaling in human cells

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ～12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.     

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation

Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.     

Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells

Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a bright-yellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.     

WAVE forms hetero- and homo-oligomeric complexes at integrin junctions in Drosophila visualized by bimolecular fluorescence complementation

Dynamic actin polymerization drives a variety of morphogenetic events during metazoan development. Members of the WASP/WAVE protein family are central nucleation-promoting factors. They are embedded within regulatory networks of macromolecular complexes controlling Arp2/3-mediated actin nucleation in time and space. WAVE (Wiskott-Aldrich syndrome protein family verprolin-homologous protein) proteins are found in a conserved pentameric heterocomplex that contains Abi, Kette/Nap1, Sra-1/CYFIP, and HSPC300. Formation of the WAVE complex contributes to the localization, activity, and stability of the various WAVE proteins. Here, we established the Bimolecular Fluorescence Complementation (BiFC) technique in Drosophila to determine the subcellular localization of the WAVE complex in living flies. Using different split-YFP combinations, we are able to visualize the formation of the WAVE-Abi complex in vivo. We found that WAVE also forms dimers that are capable of forming higher order clusters with endogenous WAVE complex components. The N-terminal WAVE homology domain (WHD) of the WAVE protein mediates both WAVE-Abi and WAVE-WAVE interactions. Detailed localization analyses show that formation of WAVE complexes specifically takes place at basal cell compartments promoting actin polymerization. In the wing epithelium, hetero- and homooligomeric WAVE complexes co-localize with Integrin and Talin suggesting a role in integrin-mediated cell adhesion. RNAi mediated suppression of single components of the WAVE and the Arp2/3 complex in the wing further suggests that WAVE-dependent Arp2/3-mediated actin nucleation is important for the maintenance of stable integrin junctions.     

Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique

Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.     

A high-throughput screen identifies a new natural product with broad-spectrum antibacterial activity

Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples.     

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with >/=50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with K(d) 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.     

A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.     

A novel functional module detection algorithm for protein-protein interaction networks

Background  

The sparse connectivity of protein-protein interaction data sets makes identification of functional modules challenging. The purpose of this study is to critically evaluate a novel clustering technique for clustering and detecting functional modules in protein-protein interaction networks, termed STM.     

A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.