Pulsed-interleaved Excitation Fluorescence Cross-correlation Spectroscopy Research Articles

The β2-adrenergic receptor associates with CXCR4 multimers in human cancer cells

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the β2 adrenergic receptor (β2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-β2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and β2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that β2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-β2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Direct Quantification of Serum Protein Interactions with PEGylated Micelle Nanocarriers.

A large repertoire of nanocarrier (NC) technologies exists, each with highly specified advantages in terms of targetability, stability, and immunological inertness. The characterization of such NC properties within physiological conditions is essential for the development of optimized drug delivery systems. One method that is well established for reducing premature elimination by avoiding protein adsorption on NCs is surface functionalization with poly(ethylene glycol) (PEG), aptly called PEGylation. However, recent studies revealed that some PEGylated NCs have a delayed immune response, indicating the occurrence of protein-NC interactions. Obvious protein-NC interactions, especially in micellar systems, may have been overlooked as many early studies relied on techniques less sensitive to molecular level interactions. More sensitive techniques have been developed, but a major challenge is the direct measurement of interactions, which must be done in situ, as micelle assemblies are dynamic. Here, we report the use of pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to interrogate the interactions between two PEG-based micelle models and serum albumin protein to compare protein adsorption differences based on linear or cyclic PEG architectures. First, by measuring micelle diffusion in isolated and mixed solutions, we confirmed the thermal stability of diblock and triblock copolymer micelle assemblies. Further, we measured the co-diffusion of micelles and serum proteins, the magnitudes of which increased with concentration and continued incubation. The results demonstrate that PIE-FCCS is capable of measuring direct interactions between fluorescently labeled NC and serum proteins, even at concentrations 500 times lower than those observed physiologically. This capability showcases the potential utility of PIE-FCCS in the characterization of drug delivery systems in biomimetic conditions.

Resolving the hierarchical network of receptor tyrosine kinase interactions and the effect of oncogenic mutations.

Cells have an array of protein receptors on the plasma membrane that help process environmental cues. The spatial and temporal arrangement of these receptors is critical to function, but the chemical forces driving this organization are not well understood. This talk will focus on receptor tyrosine kinases (RTKs), which are transmembrane proteins that regulate cell growth, proliferation, and differentiation. Several RTKs are oncogenic and are targeted in next generation anti-cancer drug development. The degree to which these oncogenic mutations affect the local network of interactions between RTKs has not been explored beyond a few high-profile examples. We use pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to resolve membrane protein mobility, concentration, and monomer/dimer/oligomer distributions. I will describe ongoing work in my group to resolve the network of interactions between EGFR, HER2, HER3 and HER4, and how this network responds to oncogenic mutations.

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

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.

Investigating the Homotypic and Heterotypic Interactions of ErbB Receptor Tyrosine Kinases

Human epidermal growth factor receptor family (HER1-4 or ErbB1-4) is vital to cellular homeostasis, and any dysregulation of the receptors leads to serious diseases such as cancer. HER family receptors share similar structures: a ligand-binding extracellular domain, transmembrane domain, juxtamembrane domain, kinase domain, and a C-terminal tail. HER2 has an extracellular domain with an intrinsically closed conformation that does not bind to ligands, and HER3 has a catalytically inactive kinase domain. HER family receptors form an asymmetric dimer to activate the downstream signaling upon ligand binding. With their structural features and their roles in tumorigenesis, researchers have vastly studied EGFR homodimerization and HER1-HER2/ HER2-HER3 heterodimerization as therapeutic targets. However, collective understanding on the communication and the regulation of the HER family network is still elusive due to the difficulties of quantitative assessments of direct receptor-receptor interactions. Here we use pulsed interleaved excitation fluorescence cross correlation spectroscopy (PIE-FCCS) to assess the direct homotypic and heterotypic receptor-receptor interactions of the HER family in live cells before and after ligand stimulation. PIE-FCCS allows us to evaluate the receptor-receptor interactions with quantitative dynamic parameters such as diffusion coefficient, density, binding affinity, and degree of protein co-localization. Accompanied by biochemical functional assays, this study will shed light on the regulatory effect of HER family interactions on downstream signaling.

Quantifying Lipid Mobility and Peptide Binding for Gram-Negative and Gram-Positive Model Supported Lipid Bilayers.

Model membranes are a valuable tool to investigate the mechanism of interaction between antibiotic compounds and bacterial membranes. However, the development of supported lipid bilayer (SLB) models for Gram-negative and Gram-positive bacteria has been challenging because of the high charge and spontaneous curvature of the lipids that make up these membranes. Here we describe a method for preparing mimetic Gram-negative inner membrane and Gram-positive membrane SLBs, including asymmetric SLBs (asy-SLBs) that contain a fluorescent tracer only in the upper leaflet of the membrane. We quantified the dynamics of the lipids in these membranes with fluorescence correlation spectroscopy (FCS) and found that lipid diffusion is slower in Gram-negative SLBs/asySLBs than in Gram-positive SLBs/asySLBs. Peptide binding to these membranes was also characterized using colistin, a Gram-negative specific antibiotic. Interactions between colistin and membrane lipids phosphatidylethanolamine (PE) or cardiolipin (TOCL) were probed with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Overall, our data provide unique insight into the diffusion dynamics of lipids in Gram-negative and Gram-positive membranes as well as a novel platform for investigating the mechanism of interaction between antibiotic peptides and bacterial membrane lipids.

Quantifying Associations between an Endogenous Protein Model and mPEG-Pcl Micellar Nanocarriers

A promising technique for drug administration involves the use of polyethylene glycol (PEG)-based nanocarriers, which have demonstrated exceptional aptitude for evading immune responses within the body. Such antifouling properties are ideal for designing delivery systems capable of avoiding early removal by the spleen and liver, allowing prolonged circulation of the drug. However, recent studies have shown that many PEGylated nanocarriers actually trigger an immunogenic response, called the accelerated blood clearance (ABC) phenomenon, upon sequential doses of the carriers. This response suggests the existence of non-specific interactions between the carriers and endogenous proteins, making the degree of these interactions an important metric for developing improved PEGylated nanocarriers. Therefore, we sought to elucidate a baseline for non-specific PEG-protein interactions between mPEG-b-polycaprolactone (PCL) micelles and bovine serum albumin (BSA) using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report the measured mobility and dimerization affinities of nile red labeled mPEG-PCL and fluorescein conjugated BSA.

The Retinitis Pigmentosa-Linked Mutations in Transmembrane Helix 5 of Rhodopsin Disrupt Cellular Trafficking Regardless of Oligomerization State.

G protein-coupled receptors can exist as dimers and higher-order oligomers in biological membranes. The specific oligomeric assembly of these receptors is believed to play a major role in their function, and the disruption of native oligomers has been implicated in specific human pathologies. Computational predictions and biochemical analyses suggest that two molecules of rhodopsin (Rho) associate through the interactions involving its fifth transmembrane helix (TM5). Interestingly, there are several pathogenic loss-of-function mutations within TM5 that face the lipid bilayer in a manner that could potentially influence the dimerization of Rho. Though several of these mutations are known to induce misfolding, the pathogenic defects associated with V209M and F220C Rho remain unclear. In this work, we utilized a variety of biochemical and biophysical approaches to elucidate the effects of these mutations on the dimerization, folding, trafficking, and function of Rho in relation to other pathogenic TM5 variants. Chemical cross-linking, bioluminescence energy transfer, and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy experiments revealed that each of these mutants exhibits a wild type-like propensity to self-associate within the plasma membrane. However, V209M and F220C each exhibit subtle defects in cellular trafficking. Together, our results suggest that the RP pathology associated with the expression of the V209M and F220C mutants could arise from defects in folding and cellular trafficking rather than the disruption of dimerization, as has been previously proposed.

Exploring the Rhodopsin Dimer Interface in Live Cells

In low-light environments, the G protein-coupled receptor (GPCR), rhodopsin, is responsible for detection of light within the retina. Several studies have shown evidence for dimeric, or oligomeric, conformations of rhodopsin within native disc membranes. However, while many oligomeric packing structures have been proposed, the exact dimerization interface of rhodopsin has yet to be firmly establish. Here we investigate rhodopsin dimerization in live cells with fluorescence correlation spectroscopy (FCS) and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). One of the proposed dimerization interfaces is between TM4/5 of rhodopsin. Recently we found evidence for a similar interface in red cone opsin. Based on those previous findings, we have made point mutations along rhodopsin's TM 5, and found significant disruption of the dimerization affinity compared to the wild-type construct. Our results provide new chemical details for the previously proposed packing structure between TM 4 and 5 of rhodopsin. Moreover, our findings further support the idea of conserved dimerization interface motifs shared among class A GPCRs. Such a discovery may provide more general conceptualization of self-association among one of the largest classes of transmembrane receptors, which is essential for properly gauging the potential relationship between dimerization and function.

Probing the Dimerization Affinity of Visual Opsins

Visual opsins are the light sensing proteins at the center of human vision. Rhodopsin is the pigment for low-light vision, and three cone opsins serve as the pigments for daytime image-forming vision. Opsin proteins are part of the G protein-coupled receptor (GPCR) family, and rhodopsin has served as something like a prototype for structural studies of GPCRs. Rhodopsin has also been observed to form dimeric complexes in vitro, in tissue, and in cell cultures, including recent work from our lab using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report on our recent investigation of cone opsin dimerization in a heterologous expression system. PIE-FCCS is used to resolve the relative population of dimeric complexes over a range of concentrations in live cell membranes. Surprisingly, the homodimerization affinities of the cone opsins are not identical to rhodopsin and there are significant differences in the dimerization affinity of the three cone opsins. This raises important questions about the physiological role of opsin dimerization, which has wide-ranging implications for GPCR dimerization in general. To investigate these questions we explored the differential dimerization affinity with mutational studies, in which we identify key residues responsible for dimerization. To connect the dimerization affinity to its functional consequences we also measured the spectral shifts in the absorption profiles of the wild-type and mutant cone opsins regenerated with the retinal chromophore. The results of our work provide critical insight into the molecular basis of color vision. They also will inform the ongoing study of GPCR dimerization in cell signaling.

Single Molecule Diffusion of Phosphatidylinositol Bisphosphate (PIP2) Lipids on Asymmetric Lipid Bilayers under the Influence of Polycationic Macromolecules

Proteins binding with PIP2 lipids in the plasma membrane initiate and regulate many cell signaling events. Previous research suggests that proteins interact with PIP2 lipids through electrostatic interactions between polycationic protein motifs and anionic lipid head groups, leading to potential sequestration of multiple PIP2 lipids. The sequestration of PIP2 lipids will cause a change in the lipid mobility and the spatial distribution of PIP2 in the plasma membrane. We set out to understand the molecular structure and dynamics of the protein-PIP2 interface at single molecule level. In this study, a polycationic polymer, quaternized polyvinylpyridine (QPVP), and Myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were chosen to mimic the membrane binding proteins. The behavior of PIP2 lipids in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid.

Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10-80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Taken together, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Tuning the Mobility Coupling of Quaternized Polyvinylpyridine and Anionic Phospholipids in Supported Lipid Bilayers

Binding of biomacromolecules to anionic lipids in the plasma membrane is a common motif in many cell signaling pathways. Previous work has shown that macromolecules with cationic sequences can form nanodomains with sequestered anionic lipids, which alters the lateral distribution and mobility of the membrane lipids. Such sequestration is believed to result from the formation of a lipid-macromolecule complex. To date, however, the molecular structure and dynamics of the lipid-polymer interface are poorly understood. We have investigated the behavior of polycationic quaternized polyvinylpyridine (QPVP) on supported lipid bilayers doped with phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids using time-resolved fluorescence microscopy, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and colocalization. By labeling the polymer and lipids, we investigated the adsorption-induced translational mobility of lipids and systematically studied the influence of lipid charge density and solution ionic strength. Our results show that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid headgroup and is modulated by the ionic strength of the solution, indicating that electrostatic interactions drive the decrease in lateral mobility of anionic lipids by adsorbed QPVP. At physiological salt concentration we observe that the lipid lateral mobility is weakly influenced by QPVP and that there is no evidence of stable lipid-polymer complexes.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

PIE-FCCS Study of the Effects of Polycationic Macromolecules on Phosphatidylserine and Phosphatidylinositol Phosphate Lipid Mobility

The interaction between proteins and anionic lipids in the plasma membrane is a common motif in cell communication at the plasma membrane. Such interactions can gate membrane protein function and have also been proposed to sequester membrane lipids during quiescent phases of signaling. To date, however, the molecular structure and dynamics of the lipid-protein interface is poorly understood. To isolate these interactions in a biophysical assay, we have investigated the behavior of a polycationic polymer, quaternized polyvinylpyridine (QPVP), on supported lipid bilayers doped with tail-labeled phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids. To measure the mobility and association of the lipid and adsorbed polymer, we use a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and co-localization. With PIE-FCCS we have investigated the polymer adsorption-dependent translational mobility of the lipids and systematically studied the influence of lipid head-group charge and solvent ionic strength. Our results indicate that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid head group and is significantly screened by the ionic strength of the solution. At physiological salt concentration we observe that the lipid lateral mobility is nearly unaffected by mobile, adsorbed polymers and that there is no evidence of stable lipid-polymer complexes.

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.

Time-resolved fluorescence spectroscopy measures clustering and mobility of a G protein-coupled receptor opsin in live cell membranes.

Determining membrane protein quaternary structure is extremely challenging, especially in live cell membranes. We measured the oligomerization of opsin, a prototypical G protein-coupled receptor with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Individual cell measurements revealed that opsin is predominantly organized into dimeric clusters. At low concentrations, we observed that the population of oligomers increased linearly with the square of the individual monomer populations. This finding supports a monomer–dimer equilibrium and provides an experimental measurement of the equilibrium constant.

Monitoring Lipid Anchor Organization in Cell Membranes by PIE-FCCS

This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.