David Holowka

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below ≈25°C. At 37°C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcεRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.

COMPARTMENTALIZED ACTIVATION OF THE HIGH AFFINITY IMMUNOGLOBULIN E RECEPTOR WITHIN MEMBRANE DOMAINS

The earliest known step in the activation of the high affinity IgE receptor, FcεRI, is the tyrosine phosphorylation of its β and γ subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of FcεRI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of β and γ occurs in intact cells only for FcεRI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of FcεRI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in FcεRI activation. Participation of membrane domains accounts for previously unexplained aspects of FcεRI-mediated signaling and may be relevant to signaling by other multichain immune receptors.

Critical Fluctuations in Plasma Membrane Vesicles

We demonstrate critical behavior in giant plasma membrane vesicles (GPMVs) that are isolated directly from living cells. GPMVs contain two liquid phases at low temperatures and one liquid phase at high temperatures and exhibit transition temperatures in the range of 15 to 25 degrees C. In the two-phase region, line tensions linearly approach zero as temperature is increased to the transition. In the one-phase region, micrometer-scale composition fluctuations occur and become increasingly large and long-lived as temperature is decreased to the transition. These results indicate proximity to a critical point and are quantitatively consistent with established theory. Our observations of robust critical fluctuations suggest that the compositions of mammalian plasma membranes are tuned to reside near a miscibility critical point and that heterogeneity corresponding to < 50 nm-sized compositional fluctuations are present in GPMV membranes at physiological temperatures. Our results provide new insights for plasma membrane heterogeneity that may be related to functional lipid raft domains in live cells.

Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells.

Tumor progression involves the ability of cancer cells to communicate with each other and with neighboring normal cells in their microenvironment. Microvesicles (MV) derived from human cancer cells have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Here we show that MV may play another important role in oncogenesis. In particular, we demonstrate that MV shed by two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (tTG). We further demonstrate that tTG is not sufficient to transform fibroblasts but rather that it must collaborate with another protein to mediate the transforming actions of the cancer cell-derived MV. Proteomic analyses of the MV derived from MDAMB231 and U87 cells indicated that both these vesicle preparations contained the tTG-binding partner and cross-inking substrate fibronectin (FN). Moreover, we found that tTG cross-links FN in MV from cancer cells and that the ensuing MV-mediated transfers of cross-linked FN and tTG to recipient fibroblasts function cooperatively to activate mitogenic signaling activities and to induce their transformation. These findings highlight a role for MV in the induction of cellular transformation and identify tTG and FN as essential participants in this process.

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.

Membrane organization in immunoglobulin E receptor signaling

The structure and dynamics of the plasma membrane are proposed to be critical for the initial steps of signal transduction by the high-affinity immunoglobulin E receptor. Recent experimental advances indicate that interactions between the high-affinity immunoglobulin E receptor and the tyrosine kinase Lyn with cholesterol- and sphingolipid-rich regions within the plasma membrane are important for receptor function. This accumulating evidence points to spatio-temporal control of immunoglobulin E receptor signaling by the organization of the plasma membrane; an attractive hypothesis is that ligand-dependent receptor aggregation causes the segregation of Lyn-containing ordered regions of the plasma membrane from disordered regions.

Molecular Clustering of STIM1 with Orai1/CRACM1 at the Plasma Membrane Depends Dynamically on Depletion of Ca2+ Stores and on Electrostatic Interactions

Activation of store operated Ca(2+) entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), localized to the plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion of ER Ca(2+) stores in RBL mast cells causes a redistribution of STIM1, labeled with monomeric red fluorescent protein (mRFP), to micrometer-scale ER-plasma membrane junctions that contain Orai1/CRACM1, labeled with monomeric Aequorea coerulescens green fluorescent protein (AcGFP). Using fluorescence resonance energy transfer (FRET), we determine that this visualized coredistribution is accompanied by nanoscale interaction of STIM1-mRFP and AcGFP-Orai1/CRACM1. We find that antigen stimulation of immunoglobulin E receptors causes much less Orai1/CRACM1 and STIM1 association, but strong interaction is observed under conditions that prevent refilling of ER stores. Stimulated association monitored by FRET is inhibited by sphingosine derivatives in parallel with inhibition of Ca(2+) influx. Similar structural and functional effects are caused by mutation of acidic residues in the cytoplasmic segment of Orai1/CRACM1, suggesting a role for electrostatic interactions via these residues in the coupling of Orai1/CRACM1 to STIM1. Our results reveal dynamic molecular interactions between STIM1 and Orai1/CRACM1 that depend quantitatively on electrostatic interactions and on the extent of store depletion.

Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcɛRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcɛRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcɛRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcɛRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcɛRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein–protein interactions in intact, living cells.

Structural Aspects of the Association of FcεRI with Detergent-resistant Membranes

We recently showed that aggregation of the high affinity IgE receptor on mast cells, FcεRI, causes this immunoreceptor to associate rapidly with specialized regions of the plasma membrane, where it is phosphorylated by the tyrosine kinase Lyn. In this study, we further characterize the detergent sensitivity of this association on rat basophilic leukemia-2H3 mast cells, and we compare the capacity of structural variants of FcεRI and other receptors to undergo this association. We show that this interaction is not mediated by the β subunit of the receptor or the cytoplasmic tail of the γ subunit, both of which are involved in signaling. Using chimeric receptor constructs, we found that the extracellular segment of the FcεRI α subunit was not sufficient to mediate this association, implicating FcεRI α and/or γ transmembrane segments. To determine the specificity of this interaction, we compared the association of several other receptors. Interleukin-1 type I receptors on Chinese hamster ovary cells and α4 integrins on rat basophilic leukemia cells showed little or no association with isolated membrane domains, both before and after aggregation on the cells. In contrast, interleukin-2 receptor α (Tac) on Chinese hamster ovary cells exhibited aggregation-dependent membrane domain association similar to FcεRI. These results provide insights into the structural basis and selectivity of lipid-mediated interactions between certain transmembrane receptors and detergent-resistant membranes.

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.