Pulsed Interleaved Excitation 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.

Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.

Simultaneous pulsed interleaved excitation (PIE) and alternating-laser excitation (ALEX): an optimized excitation pattern for single molecule FRET

Pulsed Interleaved Excitation (PIE) is a well-known technique for synchronizing and/or delaying pulsed lasers to alternately excite donor- and acceptor-labeled species within single-molecule FRET (smFRET) studies. Laser pulses are separated on a nanosecond (ns) time scale to allow for the simultaneous recording of the temporal behavior of the two species that are excited by the corresponding laser. This allows to differentiate between a FRET pair - even with very low energy transfer efficiency - from a molecule with either absent or non-fluorescing acceptor.

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.

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing.

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.

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.

Elucidating the Molecular Binding Mechanism of the Tata-Binding Protein using Pie-Pife

The TATA-box binding protein (TBP) plays an important regulatory role in regulating gene expression by mediating the formation of the pre-initiation complex on promotor DNA. When binding to DNA, TBP melts the double helix and forms a stable complex while bending the dsDNA by ∼90o along the TATA sequence. In vitro single molecule experiments based on single-pair Förster resonance energy transfer (FRET) have recently revealed a dynamic, step-wise binding process via different intermediate conformations of the dsDNA. In vivo, TBP exists in different forms, e.g. as dimerbound to the dsDNA, or as part of different co-activator/deactivator complexes in the subsequent steps of pre-initiation complex formation. These states however are only observed at cellular concentration of TBP in the micromolar range, which is too high for in vitro single molecule fluorescence imaging. In this contribution, we present a new strategy to visualizes unlabelled TBP binding on DNA with changes in DNA conformation at high concentrations: binding of TBP is monitored via protein-induced fluorescence enhancement (PIFE) while single-pair FRET reports on ongoing conformational changes in the DNA/TBP complex simultaneously. We combine this label-free PIFE-FRET approach with pulsed interleaved excitation (PIE) spectroscopy, to investigate conformational dynamics of freely diffusing protein-DNA complexes in solution at the focus of a confocal microscope. Without the need of surface immobilization it can overcome concentration limitations of multi-colour assays by monitoring two parameters, binding and conformation rearrangement at the same.

Line-Scanning Spatial Correlation Spectroscopy for Studying Dynamics in Biomembranes

Biomembranes that are not attached to a solid support fluctuate incessantly and give rise to sizeable intensity fluctuations in fluorescence correlation spectroscopy (FCS) experiments, masking intensity fluctuations due to molecules diffusing within the membrane. Line-scanning FCS (lsFCS) is an elegant approach to avoid these intensity variations by scanning a line many times perpendicularly through the membrane and analyzing temporal intensity pair correlations of the fluorescence from the membrane. Here we have extended this concept to spatial correlations by displacing each scan line from the previous one by a constant amount and analyzing spatial intensity pair correlations between lines, as is done in raster image correlation spectroscopy (RICS). In line-scanning spatial correlation spectroscopy (lsSCS), the effective observation area is enlarged with respect to lsFCS, resulting in better sampling of sparse, slowly moving molecules and averaging of the resulting parameters over a larger region. The fraction of photobleached molecules is also reduced, which is advantageous for precise determination of area densities of molecules. For cross-correlation analysis, we perform dual-color lsSCS experiments using pulsed interleaved excitation (PIE).

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.

Pulsed interleaved excitation-based line-scanning spatial correlation spectroscopy (PIE-lsSCS)

We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIE-lsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells.

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.

Monitoring of nucleophosmin oligomerization in live cells

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.

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.

Structural Dynamics of Single Metabotropic Glutamate Receptors in Solution

Metabotropic glutamate receptors (mGluRs) are multidomain proteins belonging to class C G-protein coupled receptors (GPCR). There are essential in controlling synaptic transmission, and as such are important drug targets for the treatment of several disorders including pain, Parkinson's disease, schizophrenia etc.We recently introduced the use of single molecule FRET combined with Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to investigate the allosteric transitions associated with mGluR activation. We demonstrated that the isolated ligand binding (VFT) domain oscillates between a resting and an active state in a time range of 50-100 µs∗. Here, we extend these investigation to the full length receptor solubilized in detergent and reconstituted in liposomes. Our result confirm the general mechanism observed for the VFT domain, and decipher the role of the transmembrane domain, that slightly slows down the receptor dynamics, while stabilizing its active state solely in the presence of a full agonist.∗Olofsson et al., Nature Comm., 2015

Studying the Function of BAP in the Nucleotide Cycle of BIP by spFRET using MFD-PIE

Heat shock proteins, like Hsp70 and Hsp90, act as chaperones, a class of proteins that assist nascent proteins in reaching their final functional structure in the appropriate location within the cell. Understanding the underlying mechanisms of chaperone-assisted protein folding is important for the development of treatment for various disorders such as Alzheimer or Parkinson's disease. BiP, the Hsp70 localized in the endoplasmic reticulum (ER), consists of two domains that are connected by a short linker, the nucleotide binding domain (NBD) and the substrate binding domain (SBD). The SBD contains a flexible, alpha helical lid that can open or close the binding pocket of the SBD. When a client protein enters the ER, its charged regions are initially protected by BiP to avoid non-specific interactions. One of the regulating factors in this process is the BiP associated protein, BAP, a nucleotide exchange factor (NEF) of BiP. BAP controls the binding and release of nascent proteins from BiP by accelerating the ATP/ADP exchange. In order to gain insights into the function of BAP, we employed solution based single-pair Forster Resonance Energy Transfer (spFRET) experiments using a Multiparameter Fluorescence Detection (MFD) setup combined with Pulsed Interleaved Excitation (PIE). BiP was fluorescently labeled with the FRET pair Atto532 and Atto647. Our results suggest that the N-terminal domain of BAP interacts with the lid of BiP and pushing the two BiP domains apart from each other. Furthermore we could detect how BAP functioning as a NEF. Without BAP, the lid opens twice during one nucleotide cycle, first upon binding of ATP and secondly when phosphate is released. By adding BAP, the lid stays in its open conformation during hydrolysis and the release of phosphate and ADP is enhanced.

Structural Dynamics of the Full-Length Metabotropic Glutamate Receptors by Single-Molecule FRET

G protein-coupled receptors (GPCRs) constitute the most abundant protein family in mammalian genome. Indeed, GPCRs are the target for about 30% of the drugs on the market. In human particularly, there are 22 genes encoding class C GPCRs, which consists of GABAb receptor, calcium sensing receptor, retinoic acid-inducible receptors (orphan), taste receptors, metabotropic glutamate receptors, and a few additional orphan receptors. Metabotropic glutamate receptors (mGluRs) are involved in controlling synaptic transmission, and involved in various CNS disorders including pain, Parkinson's disease, schizophrenia etc... They are naturally homodimers, and each protomer consists of a heptahelical transmembrane domain linked to a bilobate Venus flytrap domain (VFT) by a Cystein-rich domain. Understanding the conformational changes of mGluRs is essential to decipher the allosteric transition associated with their activation. Crystallographic studies suggested that in the inactive and active states the second lobes of the VFTs are distant and close, respectively. However certain ambiguities and discrepancies about these two states have been observed by X-ray crystallography. For the first time in GPCR activation mechanism studies, we proposed structural dynamics investigations by single-molecule FRET (sm-FRET) in solution. Our results show that the isolated mGluR VFTs oscillate between the resting and active states in a time range of 50-100μs (Olofsson et al. Nat. Comm. 2014). Following the success of the powerful sm-FRET methodology on isolated VFTs, we employ here Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to study the full-length receptors in order to gain additional insights into mGluR activation. Our current results confirm the structural dynamics obtained by the mGluRs VFT, and suggest a stabilizing role of the transmembrane domain. Further studies on allosteric modulation and G protein effect are on-going.

Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy

Stimulated Emission Depletion (STED) microscopy has evolved into an established imaging method offering super-resolution well beyond 50 nm. Whereas STED is now available in many laboratories, it is still in the focus of research to push the boundaries of its capabilities and applications. Time-resolved STED microscopy using time correlated single photon counting (TCSPC), is advantageous for many applications and promises further development for increased resolution and less photo-damage.Here, we show the application of established methods (e.g. gSTED) as well as emerging applications of time-resolved STED. We employ pulsed interleaved excitation (PIE), where the STED laser is pulsed at half the frequency of the excitation laser, such that STED and confocal data is taken practically at the same time. By using this approach, single molecule STED experiments can be carried out while the confocal control-experiment is performed simultaneously, allowing to account for measurement artifacts due to the high power of the STED laser. We will show examples from single molecule imaging, where blinking and bleaching are monitored using the confocal data. Furthermore, we will present STED-FCS data, where the confocal data allows insight into changes of the sample due to the STED laser. Since the control experiment for the influence of the STED laser is performed at the same time as the STED measurement, experimental parameters can be adjusted online to give highest resolution while ascertaining that the relevant information drawn from the experiment is not affected.Furthermore, we will present how electronically delaying the STED laser with respect to the excitation laser can increase resolution with no increase in photo-bleaching. By setting the arrival of the STED laser with an accuracy of about 20 ps, experimental conditions for fluorophores with different fluorescence lifetimes can be adjusted.

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.