Bimolecular Fluorescence Complementation Signals Research Articles

Abstract 169: BiFC-FRET: A Novel Tool for Visualizing SR-BI Oligomerization in Vivo

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high-density lipoprotein (HDL) and mediates the selective uptake of HDL-cholesteryl esters (CE) into the liver for excretion. As cholesterol flux from peripheral tissues to the liver is a critical step for cholesterol disposal via reverse cholesterol transport, a better understanding of the SR-BI/HDL complex and mechanisms that enhance this interaction will aid in developing strategies for preventing cardiovascular diseases such as atherosclerosis. Previous fluorescence resonance energy transfer (FRET) data from our laboratory revealed that SR-BI exists as a dimer in live cells. Although dimers and higher order oligomers are detected in vitro, live cell FRET analysis is limited to interactions between two molecules only. Our in vitro data also reveals several mutant receptors that are unable to mediate selective uptake of HDL-CE are also unable to form higher order oligomers, suggesting that a functional SR-BI complex consists of greater than two molecules. Therefore, we hypothesize that SR-BI function correlates with higher order oligomer formation (i.e. a hydrophobic channel). To test this hypothesis, we have developed an assay that couples bimolecular fluorescence complementation (BiFC) with FRET, which will help us visualize, for the first time, higher order oligomer formation in vivo. Wild-type SR-BI, Cys-less-SR-BI, and ΔLeuZip-SR-BI were cloned into pBiFC vectors containing either the N-terminal half of Venus (VN), the C-terminal half of Venus (VC) [fluorescence acceptor halves], or full-length Cerulean [fluorescence donor]. Total cell expression and cell surface expression of each construct was confirmed by protein immunoblotting and flow cytometry, respectively. To confirm that addition of the BiFC tags did not alter protein function, fusion proteins were transiently expressed in COS7 cells and assayed for selective uptake efficiency. All wild-type tagged SR-BI constructs displayed similar levels of selective uptake efficiency as untagged SR-BI. To confirm the presence of dimers and validate the BiFC methodology, COS7 cells were transiently transfected with (i) SRBI-VN alone, (ii) SR-BI-VC alone, or (iii) both SR-BI-VN and SR-BI-VC, and we measured Venus fluorescence two days post-transfection. Co-expression of SR-BI-VN and SR-BI-VC in COS7 cells resulted in reconstitution of Venus fluorescence and a positive BiFC signal following excitation at 514 nm, while single transfections of either SR-BI-VN or SR-BI-VC displayed no detectable fluorescence as expected. BiFC was also observed for Cys-less-SR-BI and ΔLeuZip-SR-BI, confirming that these mutants are still able to dimerize, despite being unable to mediate selective uptake. Therefore, we have successfully validated the use of BiFC to confirm the presence of SR-BI dimers in cells. As we have confirmed proper cell expression of Cerulean-tagged SR-BI, we now have all the necessary tools to couple BiFC with FRET methodologies to provide the first evidence for the existence of SR-BI oligomers in vivo.

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.

Ligand-Dependent Oligomerization of Dopamine D2and Adenosine A2AReceptors in Living Neuronal Cells

Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

Cell Cycle-dependent Binding of HMGN Proteins to Chromatin

Throughout the cell cycle, the histones remain associated with DNA, but the repertoire of proteins associated with the chromatin fiber continuously changes. The chromatin interaction of HMGNs, a family of nucleosome binding proteins that modulates the structure and activity of chromatin, during the cell cycle is controversial. Immunofluorescence studies demonstrated that HMGNs are not associated with chromatin, whereas live cell imaging indicated that they are present in mitotic chromosomes. To resolve this controversy, we examined the organization of wild-type and mutated HMGN1 and HMGN2 proteins in the cell nucleus by using immunofluorescence studies, live cell imaging, gel mobility shift assays, and bimolecular fluorescence complementation (BiFC). We find that during interphase, HMGNs bind specifically to nucleosomes and form homodimeric complexes that yield distinct BiFC signals. In metaphase, the nucleosomal binding domain of the protein is inactivated, and the proteins associate with chromatin with low affinity as monomers, and they do not form specific complexes. Our studies demonstrate that the mode of binding of HMGNs to chromatin is cell cycle dependent.

Identification of New Fluorescent Protein Fragments for Bimolecular Fluorescence Complementation Analysis Under Physiological Conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.