Analyzing Single Giant Unilamellar Vesicles With a Slotline-Based RF Nanometer Sensor.

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 radio-frequency (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 ~1.76×107 V/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 ~2 GHz, ~2.5 GHz, and ~2.8 GHz with an initial |S21 | min of ~-100 dB. Corresponding GUV dielectric properties are obtained. A one-dimensional scanning of single GUV is also demonstrated.

Trafficking of MHC molecules to the cell surface creates dynamic protein patches.

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).

A high fat diet containing saturated but not unsaturated fatty acids enhances T cell receptor clustering on the nanoscale.

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.

Universal Approach to FRAP Analysis of Arbitrary Bleaching Patterns.

The original approach to calculating diffusion coefficients of a fluorescent probe from Fluorescence Recovery After Photobleaching (FRAP) measurements assumes bleaching with a circular laser beam of a Gaussian intensity profile. This method was used without imaging the bleached cell. An empirical equation for calculating diffusion coefficients from a rectangular bleaching geometry, created in a confocal image, was later published, however a single method allowing the calculation of diffusion coefficients for arbitrary geometry does not exist. Our simulation approach allows computation of diffusion coefficients regardless of bleaching geometry used in the FRAP experiment. It accepts a multiple-frame TIFF file, representing the experiment as input, and simulates the (pure) diffusion of the fluorescent probes (2D random walk) starting with the first post-bleach frame of the actual data. It then fits the simulated data to the real data and extracts the diffusion coefficient. We validate our approach using a well characterized diffusing molecule (DiIC18) against well-established analytical procedures. We show that the algorithm is able to calculate the absolute value of diffusion coefficients for arbitrary bleaching geometries, including exaggeratedly large ones. It is provided freely as an ImageJ plugin, and should facilitate quantitative FRAP measurements for users equipped with standard fluorescence microscopy setups.

Light and life in Baltimore--and beyond.

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.

Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity.

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.

Nanoscale artificial antigen presenting cells for T cell 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.

Selective activation of antigen-experienced T cells by anti-CD3 constrained on nanoparticles.

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.

Modulation of MHC binding by lateral association of TCR and coreceptor.

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 K(b)-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand L(d)-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.

Lifetime of major histocompatibility complex class-I membrane clusters is controlled by 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.

Quantum dot fluorescence characterizes the nanoscale organization of T cell receptors for antigen.

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.

Class I MHC molecules as probes of membrane patchiness: from biophysical measurements to modulation of immune responses.

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.

Staphylococcal enterotoxin A induces small clusters of HLA-DR1 on B cells.

The superantigen SEA causes non-specific hyperactivation of T and B cells at low concentrations. Studies of mutants or soluble proteins suggest SEA is bivalent for its ligand, MHC class II. However, the interaction between these molecules on intact cells is unknown. On primary mouse B cells expressing the MHC class II allele HLA-DR1, measurements of Förster Resonance Energy Transfer between HLA-DR1 molecules on SEA-treated cells indicated specific clustering, not observed in untreated or monovalent superantigen treated cells. Tomographic visualization and electron microscopy of immunogold-labeled SEA-treated B cells revealed small clusters of surface HLA-DR1 (< or = 4 gold labels). These results present direct visual evidence of SEA-mediated clustering of MHC class II molecules on treated antigen presenting cells, and provide a new structural approach to addressing problems of this nature.

Interaction of Bap31 and MHC class I molecules and their traffic out of the endoplasmic reticulum.

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.

Cutting edge: phosphatidylinositol 4,5-bisphosphate concentration at the APC side of the immunological synapse is required for effector T cell function.

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)P(2)) at the APC side of the immunological synapse. In both human and mouse cells, a PI(4,5)P(2)-specific fluorescent reporter, PH-GFP (where PH is pleckstrin homology), detected an Ag-dependent enrichment of PI(4,5)P(2) at the synapse between Ag-specific T cells and APC. When PIP(4,5)P(2) 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.

In vivo test of the vertical phase separation hypothesis: the display of major histocompatibility complex (MHC) class I molecules on membranes of B cells from mice fed high-fat diets.

The membrane vertical phase separation hypothesis predicts that a decrease in plasma membrane acyl chain order will increase major histocompatibility complex (MHC) class I surface expression. The hypothesis is based on modification of plasma membrane acyl chain order in cell culture and has not been tested in vivo. In the present study, we isolated splenic B cells from C57/BL6 mice fed either a normal diet or high-fat diets enriched in SFA or MUFA and assayed for changes in plasma membrane acyl chain order and MHC class I surface expression. Plasma membranes of B cells from MUFA-fed mice had significantly decreased acyl chain order and increased headgroup order. The decrease in acyl chain order correlated with a significant increase in the acyl chain unsaturation of B cells from the MUFA-fed mice. MHC class I surface levels on B cells were not affected by the MUFA-rich diet. This study suggests that the membrane vertical phase separation hypothesis may have limited application in a physiologically relevant setting.

Polyunsaturated fatty acids and membrane organization: elucidating mechanisms to balance immunotherapy and susceptibility to infection.

Polyunsaturated fatty acids (PUFAs), notably of the n-3 series, have immunosuppressive effects which make these molecules candidates for treating inflammatory symptoms associated with cardiovascular disease, obesity, arthritis, and asthma. However, immunosuppression by PUFAs could increase susceptibility to bacterial and viral infection. A detailed molecular picture is required in order to understand the balance between the benefits and risks of utilizing PUFAs as adjuvant immunosuppressants. Here we review evidence that incorporation of PUFAs into membrane lipids of antigen presenting cells (APCs) downregulates APC function. We propose that PUFAs modulate antigen presentation by altering the organization of lipid and protein molecules of the plasma membrane and endomembranes; this alters recognition and responses by T cells. The foundation of our hypothesis is built on data from artificial bilayer experiments which provide the physical principles by which PUFA acyl chains affect membrane architecture. This review also reconciles conflicting results in the literature by discussing the advantages and disadvantages of differing methods of PUFA treatment of cells. We suggest that membrane modulation of immune cells may be an important and overlooked mechanism of immunomodulation. In addition, we propose that mechanistic studies with defined experimental protocols will speed the translation of laboratory studies on PUFAs to the clinic.

Plasma membrane lipid diffusion and composition of sea urchin egg membranes vary with ocean temperature.

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.

Tapasin increases efficiency of MHC I assembly in the endoplasmic reticulum but does not affect MHC I stability at the cell surface.

Cell surface expression of MHC I molecules depends on the chaperone tapasin; how tapasin functions is not fully understood. We created three fluorescent tapasin constructs: wild-type tapasin, soluble tapasin, which does not interact with TAP, and N300 tapasin, which does not interact with MHC I. In contrast to earlier reports, all three constructs localize to the endoplasmic reticulum (ER), though soluble tapasin is more mobile than wild type and N300. Soluble tapasin does not increase MHC I surface levels to the same extent as wild type, which suggests that proximity to TAP is necessary for full tapasin function. N300 acts as a dominant-negative perhaps by blocking wild-type tapasin access to TAP. None of the constructs affects MHC I stability at the cell surface, although stability of ER resident MHC I is decreased in tapasin-negative cells. We propose that tapasin acts primarily to increase efficiency of assembly of MHC I within the ER.