Brandon L. Scott

N-Way FRET Microscopy of Multiple Protein-Protein Interactions in Live Cells

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells

Powerful new methods have extended FRET microscopy to the imaging of three or more interacting proteins inside living cells. Here, we compared widely available fluorescent proteins to find the best trio for 3-Way FRET imaging. We focused on readily available cyan, yellow, and red proteins that have high quantum yields, large extinction coefficients and good photostability, which defined these candidate proteins: CyPet/mTFP1/mTurqoise2, mCitrine/YPet, and TagRFP/TagRFPt/mRuby2/mCherry. By taking advantage of the high structural similarity across the fluorescent proteins, we generated structurally similar, but photophysically distinct donor/acceptor and triple fluorophore fusion proteins and measured their FRET efficiencies inside living cells. Surprisingly, their published photophysical parameters and calculated Förster distances did not predict the best combinations of FPs. Using cycloheximide to inhibit protein synthesis, we found that the different FP maturation rates had a strong effect on the FRET efficiency. This effect was pronounced when comparing rapidly maturing yellow and slowly maturing red FPs. We found that red FPs with inferior photophysics gave superior FRET efficiencies because of faster maturation rates. Based on combined metrics for the FRET efficiency, fluorophore photophysics and fluorophore maturation we determined that Turqoise2, YPet and Cherry were the best available FPs for live cell 3-Way FRET measurements.

Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.

FRETting about the affinity of bimolecular protein-protein interactions: Affinity from Live-Cell FRET

Fluorescence resonance energy transfer (FRET) is a powerful tool to study macromolecular interactions such as protein–protein interactions (PPIs). Fluorescent protein (FP) fusions enable FRET‐based PPI analysis of signaling pathways and molecular structure in living cells. Despite FRETs importance in PPI studies, FRET has seen limited use in quantifying the affinities of PPIs in living cells. Here, we have explored the relationship between FRET efficiency and PPI affinity over a wide range when expressed from a single plasmid system in Escherichia coli. Using live‐cell microscopy and a set of 20 pairs of small interacting proteins, belonging to different structural folds and interaction affinities, we demonstrate that FRET efficiency can reliably measure the dissociation constant (KD) over a range of mM to nM. A 10‐fold increase in the interaction affinity results in 0.05 unit increase in FRET efficiency, providing sufficient resolution to quantify large affinity differences (> 10‐fold) using live‐cell FRET. This approach provides a rapid and simple strategy for assessment of PPI affinities over a wide range and will have utility for high‐throughput analysis of protein interactions.

TIRF imaging of Fc gamma receptor microclusters dynamics and signaling on macrophages during frustrated phagocytosis

BackgroundRecent evidence indicates that in addition to the T-cell receptor, microclustering is an important mechanism for the activation of the B-cell receptor and the mast cell Fcε-receptor. In macrophages and neutrophils, particles opsonized with immunoglobulin G (IgG) antibodies activate the phagocytic Fcγ-receptor (FcγR) leading to rearrangements of the actin cytoskeleton. The purpose of this study was to establish a system for high-resolution imaging of FcγR microclustering dynamics and the recruitment of the downstream signaling machinery to these microclusters.MethodsWe developed a supported lipid bilayer platform with incorporated antibodies on its surface to study the formation and maturation of FcγR signaling complexes in macrophages. Time-lapse multicolor total internal reflection microscopy was used to capture the formation of FcγR-IgG microclusters and their assembly into signaling complexes on the plasma membrane of murine bone marrow derived macrophages.ResultsUpon antibody binding, macrophages formed FcγR-IgG complexes at the leading edge of advancing pseudopods. These complexes then moved toward the center of the cell to form a structure reminiscent of the supramolecular complex observed in the T-cell/antigen presenting cell immune synapse. Colocalization of signaling protein Syk with nascent clusters of antibodies indicated that phosphorylated receptor complexes underwent maturation as they trafficked toward the center of the cell. Additionally, imaging of fluorescent BtkPH domains indicated that 3′-phosphoinositides propagated laterally away from the FcγR microclusters.ConclusionWe demonstrate that surface-associated but mobile IgG induces the formation of FcγR microclusters at the pseudopod leading edge. These clusters recruit Syk and drive the production of diffusing PI(3,4,5)P3 that is coordinated with lamellar actin polymerization. Upon reaching maximal extension, FcγR microclusters depart from the leading edge and are transported to the center of the cellular contact region to form a synapse-like structure, analogous to the process observed for T-cell receptors.

Correlated fluorescence-atomic force microscopy studies of the clathrin mediated endocytosis in SKMEL cells

Clathrin-mediated endocytosis (CME) is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. Currently, there are two models describing membrane bending during the formation of clathrin cages: the first involves the deposition of all clathrin molecules to the plasma membrane, forming a flat lattice prior to membrane bending, whereas in the second model, membrane bending happens simultaneously as the clathrin arrives to the site to form a clathrin-coated cage. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorophores (actin filaments labeled with green phalloidin and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. An extensive statistical survey of many hundreds of CME events, at various stages of progression, are observed via this method, allowing inferences about the dominant mechanisms active in CME in SKMEL cells. Results indicate a mixed model incorporating aspects of both the aforementioned mechanisms for CME.