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Novel techniques that enable reagent free detection and analysis of single cells are of great interest for the development of biological and medical sciences, as well as point-of-care health service technologies. Highly sensitive and broadband RF sensors are promising candidates for such a technique. In this work, we present a highly sensitive and tunable RF sensor, which is based on interference processes and built with a 100-nm slotline structure. The highly concentrated RF fields, up to $\sim {\hbox{1.76}}\times {\hbox{10}}^{7} ~{\hbox{V}}/{\hbox{m}}$ , enable strong interactions between giant unilamellar vesicles (GUVs) and fields for high-sensitivity operations. We also provide two modeling approaches to extract cell dielectric properties from measured scattering parameters. GUVs of different molecular compositions are synthesized and analyzed with the RF sensor at $\sim$ 2, $\sim$ 2.5, and $\sim {\hbox{2.8 GHz}}$ with an initial $\vert S _{21} \vert _{\min}$ of $\sim -{\hbox{100 dB}}$ . Corresponding GUV dielectric properties are obtained. A one-dimensional scanning of single GUV is also demonstrated.

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.

Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~ 30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. From the Clinical Editor Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3–4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand Kb-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand Ld-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

Major histocompatibility complex class I (MHC-I) molecules signal infection or transformation by engaging receptors on T lymphocytes. The spatial organization of MHC-I on the plasma membranes is important for this engagement. We and others have shown that MHC-I molecules, like other membrane proteins, are not uniformly distributed, but occur in patches in the plasma membrane. Here, we describe the temporal details of MHC-I patch formation and combine them with the spatial details, which we have described earlier, to yield a comprehensive quantitative description of patch formation. MHC-I is delivered to the plasma membrane in clathrin-coated vesicles, arriving at a rate of ∼2.5×10−3 μm−1 min−1 (or about two arrivals per minute over the whole cell). The vesicles dock and fuse at non-random, apparently targeted, locations on the membrane and the newly delivered MHC-I molecules form patches that are a few hundred nanometers in diameter. The patches are maintained at steady state by a dynamic equilibrium between the rate of delivery and the rate of hindered diffusion of MHC-I molecules out of the patches (caused by components of the actin cytoskeleton).

Lateral heterogeneity of cell membranes has been demonstrated in numerous studies showing anomalous diffusion of membrane proteins; it has been explained by models and experiments suggesting dynamic barriers to free diffusion, that temporarily confine membrane proteins into microscopic patches. This picture, however, comes short of explaining a steady-state patchy distribution of proteins, in face of the transient opening of the barriers. In our previous work we directly imaged persistent clusters of MHC-I, a type I transmembrane protein, and proposed a model of a dynamic equilibrium between proteins newly delivered to the cell surface by vesicle traffic, temporary confinement by dynamic barriers to lateral diffusion, and dispersion of the clusters by diffusion over the dynamic barriers. Our model predicted that the clusters are dynamic, appearing when an exocytic vesicle fuses with the plasma membrane and dispersing with a typical lifetime that depends on lateral diffusion and the dynamics of barriers. In a subsequent work, we showed this to be the case. Here we test another prediction of the model, and show that changing the stability of actin barriers to lateral diffusion changes cluster lifetimes. We also develop a model for the distribution of cluster lifetimes, consistent with the function of barriers to lateral diffusion in maintaining MHC-I clusters.

Here I summarize decades of work using the biophysics of class I MHC molecules to probe the patchiness and heterogeneity of cell surfaces. This program began as a study of membranes generally. MHC molecules were a convenient probe. However, in recent years, it has become clear that the lateral distribution, clustering, of class I MHC molecules in the membrane affects their recognition by effector CTL. This offers the possibility of enhancing or reducing T-cell recognition of targets by altering the clustering of their membrane proteins.

Little is known about the signaling that occurs in an APC during contact with a T cell. In this article we report the concentration of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the APC side of the immunological synapse. In both human and mouse cells, a PI(4,5)P2-specific fluorescent reporter, PH-GFP (where PH is pleckstrin homology), detected an Ag-dependent enrichment of PI(4,5)P2 at the synapse between Ag-specific T cells and APC. When PIP(4,5)P2 was sequestered by a high concentration of PH-GFP reporter, cells were less susceptible to CTL-mediated lysis than control cells. These findings suggest a new regulatory target for modulating immune function that may be exploited for immune escape by pathogens and tumors.

The endoplasmic reticulum (ER) protein Bap31 associates with nascent class I MHC molecules. It appears to mediate the export of class I MHC molecules from the ER and may also be involved in their quality control. In this study, we use Förster resonance energy transfer and quantitative fluorescence imaging to show that in human, HeLa cells, Bap31 clusters with MHC class I (HLA-A2) molecules in the ER, and traffics via export vesicles to the ER/Golgi intermediate compartment. Förster resonance energy transfer between Bap31 and HLA-A2 and forward traffic increases when MHC class I molecules are loaded with a pulse of peptide. The increased forward traffic is blocked by overexpression of Bap29, a partner protein for Bap31, which localizes to the ER. Thus, in HeLa cells, Bap31 is involved in the exit of peptide-loaded MHC class I from the ER, and its function is regulated by its interaction with its homologue, Bap29.

Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids.

A diverse and complex array of lipids plays a vital role in structuring and organizing cell membranes. However, the details of lipid requirements for global membrane organization are poorly understood. One obstacle to this understanding is the difficulty of accurately manipulating the lipid composition of commonly studied mammalian cells. In contrast, the lipid composition of cells of ectotherms changes with changes in environmental temperatures. Thus, comparison of lipid probe diffusion in cells from animals living at different temperatures, together with biochemical analysis, can be used toward understanding membrane organization. We used two dialkyindocarbocyanine iodide (DiI) probes, of differing chain length, to probe lipid organization in terms of their lateral diffusion in eggs of the sea urchin Strongylocentrotus purpuratus. The lateral diffusion of our probes changed in urchins developing in the year of an "El Niño" weather event, which raised the ocean temperature by several degrees, suggesting alterations in membrane domain composition and structure. Indeed the changes in lateral diffusion were correlated with lower levels of unsaturated fatty acids and cholesterol in animals of the "El Niño" year than in animals of the preceding or following years. We found similar trends comparing DiI diffusion in membranes of eggs from 15 degrees C waters with those from 10 degrees C. Our findings establish a new approach for manipulating and studying membrane organization.

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Galphai2 and Lyn via protein-protein and raft interactions. Lyn is activated probably by the Galphai2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLCgamma2 and CD59 clusters revealed that PLCgamma2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP(3)-Ca(2+) signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP(3)-Ca(2+) signal, via PLCgamma2 recruitment. The prolonged, analogue, bulk IP(3)-Ca(2+) signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP(3) bursts, each created by the transient recruitment of PLCgamma2 molecules to STALLed CD59.

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Dietary supplementation with polyunsaturated fatty acids (PUFAs) has immunosuppressive effects; however, the molecular targets of PUFAs and their mode of action remain unclear. One possible target is antigen presentation to T cells through the human leukocyte antigen (HLA) class I pathway. Here we show that incorporation of PUFAs lowers target cell susceptibility to lysis by effector T cells. Treatment of B lymphoblast targets with the omega-6 PUFA arachidonic acid (AA) or omega-3 docosahexaenoic acid lowered their susceptibility to lysis by alloreactive CD8+ T cells by approximately 20-25%. HLA class I surface levels and their rate of endoplasmic reticulum (ER)-Golgi traffic were also reduced by PUFA treatment. Calibration experiments showed that the approximately 15% reduction in surface HLA I was not sufficient to completely account for the decreased lysis. However, PUFAs significantly lowered antigen-presenting cell-T cell conjugate formation, by approximately 30-40%. Taken together, our data show for the first time that an omega-6 and an omega-3 PUFA affect the HLA class I pathway of B lymphoblasts. Our findings suggest that elimination of self- and pathogen-derived peptides by effectors may be compromised by dietary PUFA supplementation. In addition, PUFA-mediated changes in ER-Golgi trafficking point to a new area of PUFA modulation of immune responses.

Fluorescence recovery after photobleaching (FRAP) is a classical biophysical method, that has been used extensively in the study of numerous systems, particularly cell membranes. It consists of irreversibly photobleaching a fluorescently labeled diffusing species, and then following the fluorescence recovery over time. By analyzing the fluorescence recovery dynamics it is possible to extract the diffusion coefficient of the fluorescent species. While the experimental setup is relatively simple, the interpretation of the results is complex, severely limiting the quantitative use of the technique.With the introduction of digital imaging and the Laser Scanning Confocal Microscope (LSCM), a multitude of variations of the technique became possible. While these modern options offer high flexibility and extend the availability of the method, the initial and boundary conditions are too complex to allow a precise closed, analytical solution of the diffusion problem, and thus quantitative extraction of the diffusion coefficient.We have developed a fast algorithm that allows the extraction of diffusion coefficients given a stack of images representing a FRAP experiment, and acquired with any method, for a completely arbitrary geometry of the initially bleached area. The algorithm treats the first post-bleach frame as the initial and boundary conditions, and simulates the diffusion of the fluorescent molecules. The lack of closed analytical solutions for complex geometries is not a limiting factor, since the diffusion equation is effectively solved numerically by iterative simulation.We validated our approach using a well characterized diffusing molecule (DiIC18) and against the well-established analytical procedures for Gaussian (Axelrod, Koppel et al. 1976) and box bleaching geometries (Ellenberg, Siggia et al. 1997). Furthermore, we show that the algorithm can deduce the diffusion coefficient for an arbitrary bleaching geometrys.The authors gratefully acknowledge a grant from the US-Israel Binational Science Foundation, #2009345.

Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo , which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4 + T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat.

Dietary supplementation with polyunsaturated fatty acids (PUFAs), especially those of the n-3 class, has immunosuppressive effects on both innate and adaptive immunity through various mechanisms. In this review, we focus on the PUFA modulation of membrane architecture and its consequent effects on both T cell responses and antigen presentation. We first use data from in vitro and in vivo experiments to make the case that the immunosuppressive effects of PUFAs begin with membrane incorporation and modulation of lipid-protein lateral organization. This in turn inhibits downstream signaling mediated by T cell receptors and suppresses T cell activation and proliferation. Next, we review evidence for PUFA-mediated alteration of major histocompatibility complex class I and II surface expression and antigen presentation. We propose that PUFAs influence the expression of major histocompatibility complex by altering its conformation, orientation, lateral organization, and trafficking, with consequences for recognition by effector T cells. Finally, we present data from model membrane studies to explain the physical principles that make PUFA acyl chains unique in modifying membrane lateral organization and protein function. An important concept to emerge from these studies is that PUFA acyl chains and cholesterol molecules are sterically incompatible. By applying this concept to the T cell activation and signaling model, mechanisms emerge by which PUFAs can modulate membrane lipid-protein lateral organization. Our data-based models show that membrane modification of both effectors and targets is an important, often overlooked, mechanism of immunomodulation by PUFAs.

The assembly of class I MHC molecules and their export from the endoplasmic reticulum (ER) is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with yellow fluorescent protein-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ER/Golgi intermediate compartment, and second, by retrieving class I molecules that have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, because we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31.

T cell recognition of peptide-MHC is highly specific and is sensitive to very low levels of agonist peptide; however, it is unclear how this effect is achieved or regulated. In this study we show that clustering class I MHC molecules on the cell surface of B lymphoblasts enhances their recognition by mouse and human T cells. We increased clustering of MHC I molecules by two methods, cholesterol depletion and direct cross-linking of a dimerizable MHC construct. Imaging showed that both treatments increased the size and intensity of MHC clusters on the cell surface. Enlarged clusters correlated with enhanced lysis and T cell effector function. Enhancements were peptide-specific and greatest at low concentrations of peptide. Clustering MHC class I enhanced recognition of both strong and weak agonists but not null peptide. Our results indicate that the lateral organization of MHC class I on the cell surface can modulate the sensitivity of T cell recognition of agonist peptide.

B7-1 and B7-2 are homologous costimulatory ligands expressed on the surface of antigen presenting cells (APCs). Binding of these molecules to the T cell costimulatory receptors, CD28 and CTLA-4, is essential for the activation and regulation of T cell immunity. Despite strong structural similarities, B7-1 and B7-2 exhibit different biochemical features, and their binding to the costimulatory receptors results in distinct T cell functional outcomes. Using photobleaching based fluorescence resonance energy transfer (FRET), our previous studies have demonstrated that B7-1 and B7-2 have different cell surface oligomeric states. While B7-1 is present as a dimer, B7-2 exists as a monomer on the cell surface suggesting that the unique cell surface oligomeric states of the costimulatory ligands may play a key role in the regulation of T cell responses. Moreover, signaling via B7-1 and B7-2 in dendritic cells has been reported to be dependent on their simultaneous expression, raising the possibility that their direct interaction or their involvement in synergistic signaling pathways may play a role in the function of antigen presenting cells. We discuss physiological relevance of distinct oligomeric states of B7-1 and B7-2 and address whether these molecules can associate with one another on the cell surface to form hetero-oligomers. Our findings suggest that B7-1 and B7-2 do not form hetero-oligomers, underscoring the biological relevance of dimeric and monomeric state of B7-1 and B7-2, respectively.

Our present picture of cell membranes as lipid bilayers is the legacy of a century's study that concentrated on the lipids and proteins of cell-surface membranes. Recent work is changing the picture and is turning the snapshot into a video.

Tapasin retains empty or suboptimally loaded MHC class I molecules in the endoplasmic reticulum (ER). However, the molecular mechanism of this process and how tapasin itself is retained in the ER are unknown. These questions were addressed by tagging tapasin with the cyan fluorescent protein or yellow fluorescent protein (YFP) and probing the distribution and mobility of the tagged proteins. YFP-tapasin molecules were functional and could be isolated in association with TAP, as reported for native tapasin. YFP-tapasin was excluded from ER exit sites even after accumulation of secretory cargo due to disrupted anterograde traffic. Almost all tapasin molecules were clustered, and these clusters diffused freely in the ER. Tapasin oligomers appear to be retained by the failure of the export machinery to recognize them as cargo.

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.

Thirty years of study of the major histocompatibility complex (MHC) have resulted in a satisfying picture of this complex in two species, mice and humans. Drawn by immunologists, it shows us a multigene complex whose main function is recognition in immune responses. The picture is only sketched in many places, but it fills its canvas. It is vivid and aesthetically pleasing and in many places details have been added to the bare outlines. But this is not the only picture extant of MHC function.

Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb3, on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb3 in lipid rafts with another glycosphingolipid GM1 on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.

Near-field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far-field microscopes. Near-field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near-field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.

Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.

Activation of phosphatidylinositol 3′-kinase (PI 3′-K) after ligation of CD3 protects Th2 cells from CD95-mediated apoptosis. Here we show that protection is achieved by inhibition of the formation of CD95 aggregates and consequent activation of caspase-8. Inhibition of aggregate formation is mediated by changes in the actin cytoskeleton, which in turn inhibit lateral diffusion of CD95, reducing its diffusion coefficient, D, 10-fold. After cytochalasin D treatment of stimulated cells, the lateral diffusion of CD95 increases to the value measured on unstimulated cells, and CD95 molecules aggregate to process caspase-8 and mediate apoptosis. Regulation of functional receptor formation by modulating lateral diffusion is a novel mechanism for controlling receptor activity.

While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.

Patches (lateral heterogeneities) of cell surface membrane proteins and lipids have been imaged by a number of different microscopy techniques. This patchiness has been taken as evidence for the organization of membranes into domains whose composition differs from the average for the entire membrane. However, the mechanism and specificity of patch formation are not understood. Here we show how vesicle traffic to and from a cell surface membrane can create patches of molecules of the size observed experimentally. Our computer model takes into account lateral diffusion, barriers to lateral diffusion, and vesicle traffic to and from the plasma membrane. Neither barriers nor vesicle traffic alone create and maintain patches. Only the combination of these produces a dynamic but persistent patchiness of membrane proteins and lipids.

We test here a previously proposed hypothesis about lateral heterogeneity of cell membranes, a model predicting that heterogeneity is maintained by a combination of delivery and intake of molecules with barriers to lateral free diffusion. To test the validity of the model, we observed green fluorescent protein tagged major histocompatibility complex class I patches on the plasma membrane of mouse fibroblasts, using total internal reflection fluorescence microscopy in real time. The dynamic characterization revealed the life course of these patches comprises delivery of molecules at a short instant, followed by a slow, exponential decay, corresponding to diffusion of the molecules over dynamic barriers to free lateral diffusion. The characteristic lifetime of the patches, extracted from the measurements, is approximately 30 s, in excellent agreement with the predictions of the model.

Near-field scanning optical microscopy (NSOM) uses the near-field interaction of light from a sharp fiber-optic probe with a sample of interest to image surfaces at a resolution beyond the diffraction limit of conventional optics. We used NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblasts, either dried or in buffer. A patchy distribution of a fluorescent lipid analog suggestive of lipid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluorescence photobleaching recovery measurements. Patches of fluorescent lipid analog were not spatially correlated with patches of transmembrane proteins, HLA class I molecules labeled with fluorescent antibody; the patchiness of the HLA class I molecules was on a smaller scale and was not localized to the same regions of membrane as the lipid analog. Sizes of HLA patches were deduced from a two-dimensional spatial autocorrelation analysis of NSOM images that resolved patches with radii of approximately 70 and approximately 600 nm on fixed, dried cells labeled with IgG and 300-600 nm on cells labeled with Fab and imaged in buffer. The large-size patches were also resolved by far-field microscopy. Both the spatial autocorrelation analysis and estimates from fluorescence intensity indicate that the small patches seen on fixed, dried cells contain approximately 25-125 HLA-I molecules each.

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, β2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.

We used binding of a fluorescent adduct of beta2-microglobulin, fluorescein beta2m, to probe the stability of class I HLA molecules on the surface of human cells. The weight of the literature suggests that this ligand binds to heavy chains that have lost beta2m and possibly peptide as well. Hence Fl-beta2m reports on the stability of the class I HLA trimer. A small fraction of HLA molecules, approximately 5%, binds Fl-beta2m on both resting and activated T cells. A larger fraction of all HLA molecules binds Fl-beta2m in FO-1 cells, beta2m-deficient cells, transfected with various B2m genes. HLA molecules of FO-1 cells are more stable when expressed with human beta2m, than when expressed with mouse beta2m. The non-covalent association of HLA heavy chains, beta2m and peptide implies that eventually every molecule of HLA trimer ought to dissociate and bind Fl-beta2m. In fact, the extent of exchange is limited by the lifetime of a given molecule at the cell surface. beta2m exchange decreases as cell concentration increases, suggesting that some density-dependent process acts to enhance degradation or denaturation of beta2m-free HLA heavy chains.

Activation of T cells through the TCR is mediated by the TCR-CD3 signaling complex. Cross linking of this complex with Abs directed against CD3 leads to potent activation of T cells. However, such activation is not Ag-specific. We exploited the observation that the TCR-CD3 complex is clustered on T cells that have been activated by Ag by using anti-CD3 nanoparticles to selectively activate Ag-experienced mouse T cells. We find that constraining anti-CD3 on the surface of a nanoparticle markedly and selectively enhances proliferation and cytokine production of Ag-experienced T cells but does not activate naive T cells. This effect was recapitulated in heterogeneous cultures containing mixtures of Ag-specific CD4+ or CD8+ T cells and bystander T cells. Furthermore, in vivo anti-CD3–coated nanoparticles increased the expansion of Ag-specific T cells following vaccination. Overall, these findings indicate that anti-CD3–coated nanoparticles could be use to enhance the efficacy of vaccines and immunotherapy. The results also suggest constraining a ligand on the surface of a nanoparticle might as general strategy for selectively targeting clustered receptors.

Near-field scanning optical and atomic force microscopy were used to image structural details of supported lipid monolayers and bilayers. Compressed DPPC monolayers exhibited dense web-like structures whereas DPPE monolayers were more uniform with elongated inclusion domains of fluorescent probes. When a second lipid monolayer was self-assembled on a supported monolayer [Kalb et al., Biochim. Biophys. Acta 1103 (1992) 307], the structure of the resulting supported bilayer strongly depended on the coupling monolayer. On coupling monolayers of DPPC, the bilayers exhibited domains with finger-like extensions. Much more uniform bilayers were obtained when the coupling layer was DPPE. Towards the goal of developing a biosensor for the detection of viruses in biological fluids, bilayers were constructed with 10 mol% of the ganglioside G D1a in the outer monolayer. Specific binding of influenza viruses to G D1a receptors was monitored by total internal reflection fluorescence and atomic force microscopy.

Quenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor association/clustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water-soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein-protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, self-association of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of fibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fractional density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Forster-type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra- or intermolecular ruler in a 0.5–2.5 nm distance range. This approach is simpler (measurements only on donor side) and faster than many other experimental techniques in screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging. © 1995 Wiley-Liss, Inc.

The lateral mobility of cell membrane glycoproteins is often restricted by dynamic barriers. These barriers have been detected by measurements of fluorescence photobleaching and recovery (FPR) and barrier-free path (BFP). To define the location and properties of the barriers, we compared the lateral mobility, measured by FPR and BFP, of wild-type class I major histocompatibility complex (MHC) membrane glycoproteins with the lateral mobility of mutant class I MHC glycoproteins truncated in their cytoplasmic domains. Mutants with 0 or 4 residues in the cytoplasmic domain were as mobile as lipid-anchored class I MHC molecules, molecules whose lateral mobility is relatively unrestricted by barriers. In contrast, mobility of class I MHC molecules with 7-residue cytoplasmic domains was as restricted as mobility of class I molecules with full-length, 31-residue cytoplasmic domains. Though some of the difference between the mobilities of mutants with 4- or 0-residue domains and the other class I molecules may be due to differences in the net charge of the cytoplasmic domain, FPR measurements of the mobility of molecules with 7-residue domains show that length of the cytoplasmic domain has an important influence on the lateral mobility. Model calculations suggest that the barriers to lateral mobility are 2-3 nm below the membrane bilayer.

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

SUMMARY Cell plasma membranes appear to be composed of domains, patches whose composition and function differ from the average for an entire membrane surface. Proteins and lipids may be segregated into domains by different mechanisms. Some of these mechanisms are discussed, followed by a summary of the evidence for membrane domains obtained in my laboratory. This evidence is largely based on measurements of the lateral diffusion of membrane proteins and lipids. Recent new approaches to the interpretation of lateral diffusion measurements, consideration of so-called fractal or long time-tails promise to give new insights into the stability and lifetime of membrane domains.

Class I major histocompatibility complex molecules have been shown to be physically associated with insulin receptor molecules on the surfaces of a number of different cell types. The class I human leukocyte antigen (human MHC) phenotype of B lymphoblasts correlates with the affinity of IR for insulin. This correlation could be the result of association of some but not other HLA molecules with IR, or could reflect differences in the way in which association with an HLA molecule affects the function of IR. To distinguish between these possibilities, this study isolated complexes of four different class I HLA molecules with IR. Expression of some of these molecules correlates with high-affinity IR, whereas expression of others correlates with 5- to 10-fold lower affinity IR. This study found that chemical cross-linking was necessary to stabilize HLA-IR complexes, and all four of the class I HLA molecules studied, HLA-A1, HLA-A2, HLA-B5, and HLA-B8, can form complexes with IR.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are sorted to the apical surface of many epithelial cell types. To better understand the mechanism for apical segregation of these proteins, we analyzed the lateral mobility and molecular associations of a model GPI-anchored protein, herpes simplex virus gD1 fused to human decay accelerating factor (gD1-DAF) (Lisanti, M. P., I. W. Caras, M. A. Davitz, and E. Rodriguez-Boulan. 1989. J. Cell Biol. 109:2145-2156) shortly after arrival and after long-term residence at the surface of confluent, polarized MDCK cells. FRAP measurements of lateral diffusion showed that the mobile fraction of newly arrived gD1-DAF molecules was much less than the mobile fraction of long-term resident molecules (40 vs. 80-90%). Fluorescence resonance energy transfer measurements showed that the newly arrived molecules were clustered, while resident molecules were not. Newly delivered gD1-DAF molecules were clustered but not immobilized in mutant, Concanavalin A-resistant MDCK cells that failed to sort gD1-DAF. Our results indicate that GPI-anchored proteins in MDCK cells are clustered before delivery to the surface. However, clustering alone does not target molecules for apical delivery. The immobilization observed when gD1-DAF is correctly sorted suggests that the clusters must associate some component of the cell's cytoplasm.

Recent findings have indicated the presence of micrometre-scale protein-based domains in the membranes of several cell types. What are the implications of this organization for membrane function? Here, Michael Edidin describes the formation of protein-based domains, and discusses their possible effects on protein interactions within the bilayer.

Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)