Resonance Energy Transfer Imaging Research Articles

Conformational dynamics of SARS-CoV-2 Omicron spike trimers during fusion activation at single molecule resolution

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron entry involves spike (S)glycoprotein-mediated fusion of viral and late endosomal membranes. Here, using single-molecule Förster resonance energy transfer (sm-FRET) imaging and biochemical measurements, we directly visualized conformational changes of individual spike trimers on the surface of SARS-CoV-2 Omicron pseudovirions during fusion activation. We observed that the S2 domain of the Omicron spike is a dynamic fusion machine. S2 reversibly interchanges between the pre-fusion conformation and two previously undescribed intermediate conformations. Acidic pH shifts the conformational equilibrium of S2 toward an intermediate conformation and promotes the membrane hemi-fusion reaction. Moreover, we captured conformational reversibility in the S2 domain, which suggests that spike can protect itself from pre-triggering. Furthermore, we determined that Ca2+ directly promotes the S2 conformational change from an intermediate conformation to post-fusion conformation. In the presence of a target membrane, low pH and Ca2+ stimulate the irreversible transition to S2 post-fusion state and promote membrane fusion.

Interferometric excitation fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a well-established technique with numerous imaging applications. Yet, one of the limitations of FLIM is that it only provides information about the emitting state. Here, we present an extension of FLIM by interferometric measurement of fluorescence excitation spectra. Interferometric Excitation Fluorescence Lifetime Imaging Microscopy (ixFLIM) reports on the correlation of the excitation spectra and emission lifetime, providing the correlation between the ground-state absorption and excited-state emission. As such, it extends the applicability of FLIM and removes some of its limitations. We introduce ixFLIM on progressively more complex systems, directly compare it to standard FLIM, and apply it to quantitative resonance energy transfer imaging from a single measurement.

SARS-CoV-2 spike fusion peptide trans interaction with phosphatidylserine lipid triggers membrane fusion for viral entry.

The role of lipids in the host cell target membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is not clear. We do not know whether SARS-CoV-2 spike protein has any specificity in terms of lipid for membrane fusion reaction. Here, using in vitro reconstitution of membrane fusion assay and single-molecule fluorescence resonance energy transfer imaging of SARS-CoV-2 spike trimers on the surface of the virion, we have demonstrated that phosphatidylserine (PS) lipid plays a key role in SARS-CoV-2 spike-mediated membrane fusion reaction for entry. Membrane-externalized PS lipid strongly promotes spike-mediated membrane fusion and COVID-19 infection. Blocking externalized PS lipid with PS-binding protein or in the absence of PS, SARS-CoV-2 spike-mediated fusion is strongly inhibited. Therefore, PS is an important target for restricting viral entry and intervening spike, and PS interaction presents new targets for COVID-19 interventions.

Single-molecule imaging reveals allosteric stimulation of SARS-CoV-2 spike receptor binding domain by host sialic acid.

Conformational dynamics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S) mediate exposure of the binding site for the cellular receptor, angiotensin-converting enzyme 2 (ACE2). The N-terminal domain (NTD) of S binds terminal sialic acid (SA) moieties on the cell surface, but the functional role of this interaction in virus entry is unknown. Here, we report that NTD-SA interaction enhances both S-mediated virus attachment and ACE2 binding. Through single-molecule Förster resonance energy transfer imaging of individual S trimers, we demonstrate that SA binding to the NTD allosterically shifts the S conformational equilibrium, favoring enhanced exposure of the ACE2-binding site. Antibodies that target the NTD block SA binding, which contributes to their mechanism of neutralization. These findings inform on mechanisms of S activation at the cell surface.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or –mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Nanoencapsulated deltamethrin combined with indoxacarb: An effective synergistic association against aphids.

Widespread pesticide use for decades has caused environmental damage, biodiversity loss, serious human and animal health problems, and resistance to insecticides. Innovative strategies are needed to reduce treatment doses in pest management and to overcome insecticide resistance. In the present study, combinations of indoxacarb, an oxadiazine insecticide, with sublethal concentrations of deltamethrin encapsulated in lipid nanocapsules, have been tested on the crop pest Acyrthosiphon pisum. In vivo toxicological tests on A. pisum larvae have shown a synergistic effect of nanoencapsulated deltamethrin with a low dose of indoxacarb. Furthermore, the stability of deltamethrin nanoparticles has been demonstrated in vitro under different mimicking environmental conditions. In parallel, the integrity and stability of lipid nanoparticles in the digestive system of aphid larvae over time have been observed by Förster Resonance Energy Transfer (FRET) imaging. Thus, the deltamethrin nanocapsules/indoxacarb synergistic association is promising for the development of future formulations against pest insects to reduce insecticide doses.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

Development of a Compensated Förster Resonance Energy Transfer Imaging for Improved Assessment of the Intrapulmonary Distribution of Polymeric Nanoparticles

Inhalation-based drug delivery systems have gained attention as potential therapeutic options for various respiratory diseases. Among these systems, nanoparticles are being explored as drug carriers because of their ability to deliver therapeutic agents directly to the lungs. It is essential to accurately evaluate the intrapulmonary behavior of nanoparticles to optimize drug delivery and achieve selective targeting of lung lesions. Prior research used the Förster resonance energy transfer (FRET) phenomenon to study the in vivo behavior of nanoparticles as drug carriers. In this study, image reconstruction involving bleed-through compensation was used to quantitatively assess the behavior of FRET nanoparticles in the lungs. When the nanoparticles for FRET fluorescence imaging, which employed 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) as the donor and as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodide (DiR) the acceptor, were administered to mouse lungs, whole-body in vivo imaging could not compensate for the influence of respiration and heartbeat. However, ex vivo imaging of excised lungs enabled the quantitative evaluation of the time–concentration profiles and distribution of nanoparticles within the lungs. This imaging technique is particularly useful for the development of inhalable nanoparticles that specifically target the lesions and exhibit controlled-release capabilities within the lungs.

Inverse agonism of lysophospholipids with cationic head groups at Gi-coupled receptor GPR82

GPR82 is an orphan G protein-coupled receptor (GPCR) that has been implicated in lipid storage in mouse adipocytes. However, the intracellular signaling as well as the specific ligands of GPR82 remain unknown. GPR82 is closely related to GPR34, a GPCR for the bioactive lipid molecule lysophosphatidylserine. In this study, we screened a lipid library using GPR82-transfected cells to search for ligands that act on GPR82. By measuring cyclic adenosine monophosphate levels, we found that GPR82 is an apparently constitutively active GPCR that leads to Gi protein activation. In addition, edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), an artificial lysophospholipid with a cationic head group that exerts antitumor activity, inhibited the Gi protein activation by GPR82. Two endogenous lysophospholipids with cationic head groups, lysophosphatidylcholine (1-oleoyl-sn-glycero-3-phosphocholine) and lysophosphatidylethanolamine (1-oleoyl-sn-glycero-3-phosphoethanolamine), also exhibited GPR82 inhibitory activity, albeit weaker than edelfosine. Förster resonance energy transfer imaging analysis consistently demonstrated that Gi protein-coupled GPR82 has an apparent constitutive activity that is edelfosine-sensitive. Consistent data were obtained from GPR82-mediated binding analysis of guanosine-5'-O-(3-thiotriphosphate) to cell membranes. Furthermore, in GPR82-transfected cells, edelfosine inhibited insulin-induced extracellular signal-regulated kinase activation, like compounds that function as inverse agonists at other GPCRs. Therefore, edelfosine is likely to act as an inverse agonist of GPR82. Finally, GPR82 expression inhibited adipocyte lipolysis, which was abrogated by edelfosine. Our findings suggested that the cationic lysophospholipids edelfosine, lysophosphatidylcholine and lysophosphatidylethanolamine are novel inverse agonists for Gi-coupled GPR82, which is apparently constitutively active, and has the potential to exert lipolytic effects through GPR82.

Single-molecule FRET imaging and deep learning reveal concentration dependence of aggregation pathways during Aβ aggregation.

Protein aggregation into amyloid fibrils is the hallmark of several devasting neurodegenerative diseases. Gaining an understanding of disease etiology hinges on our ability to understand the molecular mechanics of how soluble monomers assemble to form insoluble fibrils consisting of thousands of constituent monomers. Although amyloid fibril formation is a highly specific self-assembly process, growth patterns and resultant fibril morphologies are highly dependent on solution conditions. Using fluorescence lifetime imaging and deep learning, we have recently shown that amyloid assembly occurs via heterogeneous aggregation pathways resulting in a mixture of co-present mature fibrils with unique morphologies and physicochemical properties (Meng et al., PNAS_2022_e2116736119). Bulk biophysical methods are unable to fully characterize these mixtures of fibril polymorphs. Here, we further develop and use Förster resonance energy transfer (FRET) imaging to monitor the entire aggregation pathway of the Alzheimer's Disease related peptide amyloid β 42 (Aβ42) at the single fibril level in real time. We incubated a mixture of donor-labeled, acceptor-labeled, and unlabeled Aβ42 monomers, which resulted in the formation of fibrils with diverse FRET efficiency values, indicating structural heterogeneity. Single-fibril images reveal that increasing monomer concentration promotes the formation of more homogeneous fibrils. Fibrils formed at lower concentrations show assemble via highly heterogeneous pathways. Deep learning methods (https://github.com/hoisunglab/FNet) enable segmentation of single fibrils within images of highly overlapping fibrils, allowing for quantitative analysis of the aggregation process in terms of fibril growth rate and photon density for each fibril over the course of fibril assembly.

Bad is essential for Bcl-xL-enhanced Bax shuttling between mitochondria and cytosol.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

The number of SNAP25 molecules changing conformation preceding a fusion event increases with vesicle size.

Regulated vesicular exocytosis is governed by the SNARE complex, consisting of synaptobrevin2, SNAP25 and syntaxin. However, the number of copies of SNARE complexes that are cooperating in membrane fusion is highly debated. To estimate the number of SNARE complexes changing conformation preceding a single fusion event we used a SNAP25-based SNARE Complex Reporter (SCORE2) incorporating mCeruelan-3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. We demonstrated that all parameters of exocytosis in SNAP25 knockout cells are completely rescued by SCORE2 (SNARE Complex REporter2) and unchanged compared to wild type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging of SCORE2 conformational changes we detected a rapid FRET increase specifically associated with individual fusion events. This conformational change precedes fusion by ∼60 ms. Calibrated by the SCORE2 overexpression factor, we estimate that in wt cells ∼7 copies of SNAP25 change conformation preceding a fusion event. This number increased by ∼20% in L-Dopa-loaded vesicles which exhibited 1.67 fold larger quantal size and presumably 21% larger diameter. The number of SNAP25 copies undergoing a conformational change increases with vesicle size in parallel to the estimated area of the vesicle-plasma membrane contact zone. The results suggest that the FRET change of SCORE2 reports the docking of the vesicle, which is associated with a conformational of all SNAP25 copies in the contact zone.

Ribosome hyper-swivel head domain motions are required for translocation and resetting.

Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism.1 We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.1Nishima, et al., Nucleic Acids Research, Volume 50, 8302-8320, 2022.

Transthyretin binds soluble endoglin and increases its uptake by hepatocytes: A possible role for transthyretin in preeclampsia?

BackgroundPreeclampsia is a common but life-threatening condition of pregnancy. It is caused by poor placentation resulting in release of trophoblast material (including soluble endoglin (sEng)) into the maternal circulation leading to maternal vascular dysfunction and to the life-threatening condition of eclampsia. The only cure is early delivery, which can have lifelong consequences for the premature child. The thyroid hormone binding protein transthyretin is dysregulated in preeclampsia, however it is not known if this plays a role in disease pathology. We hypothesised that transthyretin may bind sEng and abrogate its negative effects by removing it from the maternal serum. MethodsThe effect of transthyretin on hepatocyte uptake of Alexa-labelled sEng was measured using live cell imaging. Interactions between transthyretin, and sEng were investigated using molecular modelling, direct binding on CnBr Sepharose columns, confocal imaging, and measurement of fluorescence resonance energy transfer. ResultsTransthyretin directly bound to sEng and increased its uptake by hepatocytes. This uptake was altered in the presence of transforming growth factor-β1 (TGF-β1). Molecular modelling predicted that transthyretin and TGF-β1 bind at the same site in sEng and may compete for binding. Endocytosed transthyretin and endoglin entered cells together and co-localised inside hepatocyte cells. ConclusionTransthyretin can bind sEng and increase its uptake from the extracellular medium. This suggests that increasing transthyretin levels or developing drugs that normalise or mimic transthyretin, may provide treatment options to reduce sEng induced vascular dysfunction.

Dual-channel structured illumination super-resolution quantitative fluorescence resonance energy transfer imaging

The Structured illumination (SI)-based super resolution fluorescence resonance energy transfer (SR-FRET) imaging technique, known as SISR-FRET, enables the investigation of molecular structures and functions in cellular organelles by resolving sub-diffraction FRET signals within living cells. The FRET microscopy offers unique advantages for quantitatively detecting dynamic interactions and spatial distribution of biomolecules within living cells. The spatial resolution of conventional FRET microscopy is limited by the diffraction limit, and it can only capture the average behavior of these events within the resolution limits of conventional fluorescence microscopy. The SISR-FRET performs sequential linear reconstruction of the three-channel SIM images followed by FRET quantitative analysis by using a common localization mask-based filtering approach. This two-step process ensures the fidelity of the reconstructed SR-FRET signals while effectively removing false-positive FRET signals caused by SIM artifacts. However, the slow imaging speed resulting from the switching of excitation-emission channels in SISR-FRET imaging limits its application in fast imaging scenarios. To address this issue, this study proposes a dual-channel structured illumination super-resolution quantitative FRET imaging system and method. By incorporating an FRET dual-channel imaging and registration module into the imaging pathway, the spatial switching and channel multiplexing of the SISR-FRET excitation-emission channels are achieved. Combining the image reconstruction algorithm with channel sub-pixel registration correction, the dual-channel SISR-FRET technique enhances the temporal resolution by 3.5 times while preserving the quantitative super-resolution FRET analysis. Experimental results are obtained by using a multi-color SIM system to perform super-resolution imaging of living cells expressing mitochondria outer membrane FRET standard plasmids. These experiments validate the improved spatial and temporal resolution of dual-channel SISR-FRET and the fidelity of FRET quantification analysis. In summary, this research presents a novel dual-channel structured illumination super-resolution FRET imaging system and method. It overcomes the limitations of slow imaging speed in SISR-FRET by realizing the spatial switching and channel multiplexing of excitation-emission channels. The proposed technique enhances the temporal resolution while maintaining quantitative analysis of super-resolution FRET. Experimental validation demonstrates the increased spatial and temporal resolution of dual-channel SISR-FRET and the accuracy of FRET quantification analysis. This advancement contributes to the study of molecular structures and functions in cellular organelles, providing valuable insights into the intricate mechanisms of living cells.

Three-Fluorophore FRET Enables the Analysis of Ternary Protein Association in Living Plant Cells.

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids.

The cytoplasmic domain of receptor tyrosine kinases (RTKs) plays roles as a kinase and a protein scaffold; however, the allocation of these two functions is not fully understood. Here, we analyzed the assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied members of RTKs, by combining single-pair fluorescence resonance energy transfer (FRET) imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (T654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias toward oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of T654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which T654 phosphorylation causes a switch of the major function of EGFR from kinase-activating dimers to scaffolding oligomers.