Conformational coupling across the plasma membrane in activation of the EGF receptor.

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

The ß2-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 ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR 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 ß2AR 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 ß2AR 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-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer.

Oncogenic mutations within the epidermal growth factor receptor (EGFR) are found in 15 to 30% of all non-small-cell lung carcinomas. The term exon 19 deletion (ex19del) is collectively used to refer to more than 20 distinct genomic alterations within exon 19 that comprise the most common EGFR mutation subtype in lung cancer. Despite this heterogeneity, clinical treatment decisions are made irrespective of which EGFR ex19del variant is present within the tumor, and there is a paucity of information regarding how individual ex19del variants influence protein structure and function. Herein, we identified allele-specific functional differences among ex19del variants attributable to recurring sequence and structure motifs. We built all-atom structural models of 60 ex19del variants identified in patients and combined molecular dynamics simulations with biochemical and biophysical experiments to analyze three ex19del mutations (E746_A750, E746_S752 > V, and L747_A750 > P). We demonstrate that sequence variation in ex19del alters oncogenic cell growth, dimerization propensity, enzyme kinetics, and tyrosine kinase inhibitor (TKI) sensitivity. We show that in contrast to E746_A750 and E746_S752 > V, the L747_A750 > P variant forms highly active ligand-independent dimers. Enzyme kinetic analysis and TKI inhibition experiments suggest that E746_S752 > V and L747_A750 > P display reduced TKI sensitivity due to decreased adenosine 5'-triphosphate Km. Through these analyses, we propose an expanded framework for interpreting ex19del variants and considerations for therapeutic intervention.

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

The Oligomeric State of Vasorin in the Plasma Membrane Measured Non-Invasively by Quantitative Fluorescence Fluctuation Spectroscopy.

Vasorin (VASN), a transmembrane protein heavily expressed in endothelial cells, has garnered recent interest due to its key role in vascular development and pathology. The oligomeric state of VASN is a crucial piece of knowledge given that receptor clustering is a frequent regulatory mechanism in downstream signaling activation and amplification. However, documentation of VASN oligomerization is currently absent. In this brief report, we describe the measurement of VASN oligomerization in its native membranous environment, leveraging a class of fluorescence fluctuation spectroscopy. Our investigation revealed that the majority of VASN resides in a monomeric state, while a minority of VASN forms homodimers in the cellular membrane. This result raises the intriguing possibility that ligand-independent clustering of VASN may play a role in transforming growth factor signaling.

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.

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.

Covalently Immobilizing Interferon-γ Drives Filopodia Production through Specific Receptor-Ligand Interactions Independently of Canonical Downstream Signaling.

Immobilizing a signaling protein to guide cell behavior has been employed in a wide variety of studies. This approach draws inspiration from biology, where specific, affinity-based interactions between membrane receptors and immobilized proteins in the extracellular matrix guide many developmental and homeostatic processes. Synthetic immobilization approaches, however, do not necessarily recapitulate the in vivo signaling system and potentially lead to artificial receptor-ligand interactions. To investigate the effects of one example of engineered receptor-ligand interactions, we focus on the immobilization of interferon-γ (IFN-γ), which has been used to drive differentiation of neural stem cells (NSCs). To isolate the effect of ligand immobilization, we transfected Cos-7 cells with only interferon-γ receptor 1 (IFNγR1), not IFNγR2, so that the cells could bind IFN-γ but were incapable of canonical signal transduction. We then exposed the cells to surfaces containing covalently immobilized IFN-γ and studied membrane morphology, receptor-ligand dynamics, and receptor activation. We found that exposing cells to immobilized but not soluble IFN-γ drove the formation of filopodia in both NSCs and Cos-7, showing that covalently immobilizing IFN-γ is enough to affect cell behavior, independently of canonical downstream signaling. Overall, this work suggests that synthetic growth factor immobilization can influence cell morphology beyond enhancing canonical cell responses through the prolonged signaling duration or spatial patterning enabled by protein immobilization. This suggests that differentiation of NSCs could be driven by canonical and non-canonical pathways when IFN-γ is covalently immobilized. This finding has broad implications for bioengineering approaches to guide cell behavior, as one ligand has the potential to impact multiple pathways even when cells lack the canonical signal transduction machinery.

Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.

Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (fc) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of fc values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

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.

E-cadherin junction formation involves an active kinetic nucleation process.

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins.

G protein-coupled receptors (GPCRs) detect a wide variety of physical and chemical signals and transmit that information across the cellular plasma membrane. Dimerization is a proposed modulator of GPCR signaling, but the structure and stability of class A GPCR dimerization have been difficult to establish. Here we investigated the dimerization affinity and binding interface of human cone opsins, which initiate and sustain daytime color vision. Using a time-resolved fluorescence approach, we found that human red cone opsin exhibits a strong propensity for dimerization, whereas the green and blue cone opsins do not. Through mutagenesis experiments, we identified a dimerization interface in the fifth transmembrane helix of human red cone opsin involving amino acids I230, A233, and M236. Insights into this dimerization interface of red cone opsin should aid ongoing investigations of the structure and function of GPCR quaternary interactions in cell signaling. Finally, we demonstrated that the same residues needed for dimerization are also partially responsible for the spectral tuning of red cone opsin. This last observation has the potential to open up new lines of inquiry regarding the functional role of dimerization for red cone opsin.

Protein Trapping in Plasmonic Nanoslit and Nanoledge Cavities: The Behavior and Sensing.

A novel plasmonic nanoledge device was presented to explore the geometry-induced trapping of nanoscale biomolecules and examine a generation of surface plasmon resonance (SPR) for plasmonic sensing. To design an optimal plasmonic device, a semianalytical model was implemented for a quantitative analysis of SPR under plane-wave illumination and a finite-difference time-domain (FDTD) simulation was used to study the optical transmission and refractive index (RI) sensitivity. In addition, total internal reflection fluorescence (TIRF) imaging was used to visualize the migration of fluorescently labeled bovine serum albumin (BSA) into the nanoslits; and fluorescence correlation spectroscopy (FCS) was further used to investigate the diffusion of BSA in the nanoslits. Transmission SPR measurements of free prostate specific antigen (f-PSA), which is similar in size to BSA, were performed to validate the trapping of the molecules via specific binding reactions in the nanoledge cavities. The present study may facilitate further development of single nanomolecule detection and new nanomicrofluidic arrays for effective detection of multiple biomarkers in clinical biofluids.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

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.

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.

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells.

T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell-nanopatterned supported lipid bilayer system to quantitatively probe the actin-TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.