Search

A myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Herein, we quantified the diffusion properties of a panel of structurally distinct lipid-anchored and transmembrane (TM) probes in RBL mast cells by multiplexed Imaging Fluorescence Correlation Spectroscopy. We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences as small as 10% in diffusion coefficients, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid-anchored probes can be explained by their dynamic partitioning into ordered proteo-lipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion by functional protein modules in both lipid-anchored and TM probes reflect additional complexity in steady-state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represent the dynamic resting steady-state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the major enzyme responsible for generating phosphatidylinositol (4)-phosphate [PI(4)P] at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-cysteine motif in EFR3B (Cys5, Cys7 and Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B ‘lipoforms’ affects both interactions between EFR3B and TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] resynthesis following phospholipase C signaling, and EFR3B partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code involved in controlling protein–protein and protein–lipid interactions that affect a plasma membrane-resident lipid biosynthetic pathway.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FceRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FceRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FceRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FceRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FceRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FceRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.

PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B “lipoforms” affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein–protein and protein–lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway.SUMMARY STATEMENTDifferent palmitoylation patterns on a lipid kinase adaptor protein control partitioning of the kinase between two spatiotemporally and functionally distinct complexes within the plasma membrane.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a non-functional threshold is achieved in the stimulated state, but not in the resting state, and current evidence supports the hypothesis that this relies on disruption by Ag-crosslinking of a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to supra-threshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia (RBL) mast cells. Our high-precision diffusion measurements by Imaging Fluorescence Correlation Spectroscopy (ImFCS) on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains and concomitant sharpening of segregation from liquid-disordered (Ld)-like regions. With other structural variants we determine that lipid-based interactions are essential for access by Lyn leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions by steric repulsion of TM segments and preference for Ld-like regions. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.SIGNIFICANCE STATEMENTLipid organization of the plasma membrane is known to be important for facilitating protein interactions in transmembrane signaling. However, the orchestration of these interactions in live cells has been elusive. We employed ImFCS to systemically investigate the interplay of lipids and proteins during signaling in mast cells, initiated as phosphorylation of Ag-crosslinked IgE-FcεRI by lipid-anchored Lyn kinase. We find lipid-based interactions are first required for protein-based phosphorylation of the clustered FcεRI within Lo-like nanodomains. Transmembrane phosphatases must be excluded from these regions, and we find this is mediated by their preference for Ld-like regions and by steric exclusion from the clustered FcεRI proteins. ImFCS provides quantitative characterization of the functional link between features of plasma membrane organization and transmembrane signaling.

Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1-6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930-34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1-6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR.

Significance Membrane phase behavior in cells permits transient concentration of specific proteins and lipids into dynamic nanoscopic domains. Here, we tested the existence and role of such phase behavior in endoplasmic reticulum (ER) membranes. Employing hypotonic cell swelling, we created large intracellular vesicles (LICVs) from internal organelles. ER LICVs maintained stable interorganelle contacts, with known protein tethers concentrated at the contact sites. Cooled ER LICVs underwent reversible phase separation into microscopically visible domains with different lipid order and membrane fluidity. The phase-separated domains specified sites of contact between the ER and different organelles., The endoplasmic reticulum (ER) is the site of synthesis of secretory and membrane proteins and contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner. How the ER spatially segregates its numerous and diverse functions, including positioning nanoscopic contact sites with other organelles, is unclear. We demonstrate that hypotonic swelling of cells converts the ER and other membrane-bound organelles into micrometer-scale large intracellular vesicles (LICVs) that retain luminal protein content and maintain contact sites with each other through localized organelle tethers. Upon cooling, ER-derived LICVs phase-partition into microscopic domains having different lipid-ordering characteristics, which is reversible upon warming. Ordered ER lipid domains mark contact sites with ER and mitochondria, lipid droplets, endosomes, or plasma membrane, whereas disordered ER lipid domains mark contact sites with lysosomes or peroxisomes. Tethering proteins concentrate at ER–organelle contact sites, allowing time-dependent behavior of lipids and proteins to be studied at these sites. These findings demonstrate that LICVs provide a useful model system for studying the phase behavior and interactive properties of organelles in intact cells.

Surface-patterned ligands reveal spatially targeted signaling events mediated by FcεRI on mast cells. Distinctively clustered IgE-FcεRI recruits signaling proteins, including Syk, LAT, and activated PLCγ1. β1- and β3-integrins also colocalize at patterned sites, involving actin polymerization and stimulated trafficking from recycling endosomes., We examined the spatial targeting of early and downstream signaling mediated by the immunoglobulin E (IgE) receptor (FcεRI) in RBL mast cells using surface-patterned 2,4-dinitrophenyl (DNP) ligands. Micron-sized features of DNP are presented as densely immobilized conjugates of bovine serum albumin (DNP-BSA) or mobile in a supported lipid bilayer (DNP-SLB). Although soluble anti-DNP IgE binds uniformly across features for both pattern types, IgE bound to FcεRI on cells shows distinctive distributions: uniform for DNP-SLB and edge concentrated for DNP-BSA. These distributions of IgE-FcεRI propagate to the spatial recruitment of early signaling proteins, including spleen tyrosine kinase (Syk), linker for activation of T-cells (LAT), and activated phospholipase C gamma 1 (PLCγ1), which all localize with engaged receptors. We found stimulated polymerization of F-actin is not required for Syk recruitment but is progressively involved in the recruitment of LAT and PLCγ1. We further found β1- and β3-integrins colocalize with IgE-FcεRI at patterned ligand surfaces as cells spread. This recruitment corresponds to directed exocytosis of recycling endosomes (REs) containing these integrins and their fibronectin ligand. Together our results show targeting of signaling components, including integrins, to regions of clustered IgE-FcεRI in processes that depend on stimulated actin polymerization and outward trafficking of REs.

We characterized phenotypes in RBL-2H3 mast cells transfected with human alpha synuclein (a-syn) using stimulated exocytosis of recycling endosomes as a proxy for similar activities of synaptic vesicles in neurons. We found that low expression of a-syn inhibits stimulated exocytosis and that higher expression causes slight enhancement. NMR measurements of membrane interactions correlate with these functional effects: they are eliminated differentially by mutants that perturb helical structure in the helix 1 (A30P) or NAC/helix-2 (V70P) regions of membrane-bound a-syn, but not by other PD-associated mutants or C-terminal truncation. We further found that a-syn (but not A30P or V70P mutants) associates weakly with mitochondria, but this association increases markedly under conditions of cellular stress. These results highlight the importance of specific structural features of a-syn in regulating vesicle release, and point to a potential role for a-syn in perturbing mitochondrial function under pathological conditions.

Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high-affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v-SNARE VAMP8, and the lysosomal hydrolase β-hexosaminidase (β-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.

Ca2+ mobilization in response to cross-linking of IgE bound to its high affinity receptor, FcεRI, on mast cells is central to immune allergic responses. Stimulated tyrosine phosphorylation caused by this cross-linking activates store-operated Ca2+ entry that results in sustained Ca2+ oscillations dependent on Rho family GTPases and phosphoinositide synthesis. Coupling of the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1), to the Ca2+-selective channel, Orai1, is regulated by these elements and depends on membrane organization, both at the plasma membrane and at the ER. Mitochondria also contribute to the regulation of Ca2+ mobilization, and we describe recent evidence that the ER membrane protein vesicle-associated membrane protein-associated protein (VAP) plays a significant role in the coupling between ER and mitochondria in this process. In addition to granule exocytosis, Ca2+ mobilization in these cells also contributes to stimulated outward trafficking of recycling endosomes and to antigen-stimulated chemotaxis, and it is pathologically regulated by protozoan parasitic invasion.

The high-affinity receptor for IgE expressed on the surface of mast cells and basophils interacts with antigens, via bound IgE antibody, and triggers secretion of inflammatory mediators that contribute to allergic reactions. To understand how past inputs (memory) influence future inflammatory responses in mast cells, a microfluidic device was used to precisely control exposure of cells to alternating stimulatory and non-stimulatory inputs. We determined that the response to subsequent stimulation depends on the interval of signaling quiescence. For shorter intervals of signaling quiescence, the second response is blunted relative to the first response, whereas longer intervals of quiescence induce an enhanced second response. Through an iterative process of computational modeling and experimental tests, we found that these memory-like phenomena arise from a confluence of rapid, short-lived positive signals driven by the protein tyrosine kinase Syk; slow, long-lived negative signals driven by the lipid phosphatase Ship1; and slower degradation of Ship1 co-factors. This work advances our understanding of mast cell signaling and represents a generalizable approach for investigating the dynamics of signaling systems.

Decreased luminal endoplasmic reticulum (ER) Ca2+ concentration triggers oligomerization and clustering of the ER Ca2+ sensor STIM1 to promote its association with plasma membrane Orai1 Ca2+ channels leading to increased Ca2+ influx. A key step in STIM1 activation is the release of its SOAR domain from an intramolecular clamp formed with the STIM1 first coiled-coil (CC1) region. Using a truncated STIM1(1-343) molecule that captures or releases the isolated SOAR domain depending on luminal ER Ca2+ concentrations, we analyzed the early molecular events that control the intramolecular clamp formed between the CC1 and SOAR domains. We found that STIM1 forms constitutive dimers, and its CC1 domain can bind the SOAR domain of another STIM1 molecule in trans. Artificial oligomerization failed to liberate the SOAR domain or activate STIM1 unless the luminal Ca2+-sensing domains were removed. We propose that the release of SOAR from its CC1 interaction is controlled by changes in the orientation of the two CC1 domains in STIM1 dimers. Ca2+ unbinding in the STIM1 luminal domains initiates the conformational change allowing SOAR domain liberation and clustering, leading to Orai1 channel activation.

Antigen stimulation of mast cells via FcεRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca2+ mobilization for exocytosis of secretory granules during the allergic response. To characterize the role of Rho GTPases in FcεRI signaling, we utilized a mutant RBL cell line, B6A4C1, that is deficient in antigen-stimulated Cdc42 activation important for these processes. Recently the importance of stimulated intracellular oscillations has emerged, and we find that B6A4C1 cells exhibit severely attenuated Ca2+ oscillations in response to antigen, which are restored to wild-type RBL-2H3 levels by expression of constitutively active Cdc42 G12V or by a GEF for Cdc42, DOCK7, but not when the C-terminal di-arginine motif of active Cdc42 is mutated to di-glutamine. We found that antigen-stimulated FcεRI endocytosis, which occurs independently of Ca2+ mobilization, is also defective in B6A4C1 cells, and Cdc42 G12V reconstitutes this response as well. Thus, activation of Cdc42 occurs prior to and is critical for antigen-stimulated pathways leading separately to both Ca2+ mobilization and receptor endocytosis. Accounting for these downstream functional consequences, we show that Cdc42 G12V reconstitutes antigen-stimulated oscillations of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane in mutant B6A4C1 cells, pointing to Cdc42 participation in the regulation of stimulated PIP2 synthesis.

The cell surface receptor for epidermal growth factor (EGFR), a receptor tyrosine kinase, is a key player in normal cell growth and proliferation. Mutations in this receptor often lead to oncological transformation and other pathologies. Because of its representation of the receptor tyrosine kinase family and its important role in health and disease, a broad range of studies have been carried out in many laboratories to investigate the structural basis for transmembrane receptor activation and the resulting assembly of cytosolic signaling components. This review highlights two approaches our laboratory has taken to gain more detailed information about both aspects: Surface patterned ligands to examine recruitment of the signaling machinery, and mutational analysis to examine the regulatory role of EGFR's juxtamembrane segment. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Graphene oxide (GO) has attracted intense interest for use in living systems and environmental applications. GO's compatibility with mammalian cells is sometimes inferred from its low cytotoxicity, but such conclusions ignore non-lethal effects that will influence GO's utility. Here we demonstrate, with rat basophilic leukemia (RBL) cells, profound plasma membrane (PM) ruffling and shedding induced by GO using confocal and live cell fluorescence microscopy, as well as scanning electron microscopy. These membrane structures contain immunoglobulin E receptors, are resistant to detergents, and lack detectable fluorescence labeling of F-actin and fibronectin. The formation of these membrane structures correlates with a loss of contact inhibition between RBL cells. We observe similar cellular responses towards GO for NIH-3T3 fibroblast cells and MDA-MB-231 human breast cancer cells. These findings reveal a previously unreported cellular response towards foreign nanomaterials. Membrane ruffling and shedding raise fundamental questions about how GO interacts with the PM, as well as its potential to modulate cellular mechanosensing for tissue engineering, stem cell differentiation, and other biomedical applications.

Store operated Ca2+ entry through the channel protein Orai1 contributes to mast cell spontaneous motility and antigen-mediated chemotaxis. Migration is a fundamental function of immune cells, and a role for Ca2+ in immune cell migration has been an interest of scientific investigations for many decades. Mast cells are the major effector cells in IgE-mediated immune responses, and cross-linking of IgE-FcɛRI complexes at the mast cell surface by antigen activates a signaling cascade that causes mast cell activation, resulting in Ca2+ mobilization and granule exocytosis. These cells are known to accumulate at sites of inflammation in response to parasite and bacterial infections. Using real-time imaging, we monitored chemotactic migration of RBL and rat BMMCs in response to a gradient of soluble multivalent antigen. Here, we show that Ca2+ influx via Orai1 plays an important role in regulating spontaneous motility and directional migration of mast cells toward antigen via IgER complexes. Inhibition of Ca2+ influx or knockdown of the Ca2+ entry channel protein Orai1 by shRNA causes inhibition of both of these processes. In addition, a mutant Syk− shows impaired spontaneous motility and chemotaxis toward antigen that is rescued by expression of Syk. Our findings identify a novel Ca2+ influx-mediated, Orai1-dependent mechanism for mast cell migration.

Cells respond to their physical environment and to chemical stimuli in terms of collective molecular interactions that are regulated in time and space. Small molecules may engage specific receptors to initiate a transmembrane signal, and the system amplifies this nanoscale interaction to microscale assemblies within the cell and often to longer length scales involving surrounding tissue and ultimately the whole organism. A striking example of signal integration over multiple length scales is the allergic immune response. IgE receptors (FceRI) on mast cells are the gatekeepers of this response, and this system has proven to be a valuable model for investigating receptor-mediated cellular activation. Spanning the range of cellular responses we use super resolution fluorescence localization microscopy to investigate the earliest signaling events and ligands patterned in micron size features together with confocal microscopy to investigate early and later events. We are characterizing distinctive regulatory roles played by the actin cytoskeleton at different stages. We are further investigating spatial redistributions of the cellular integrins that relate to the enhancement of signaling observed for cells spreading on surfaces.

Mast cell stimulation via IgE receptors causes activation of multiple processes, including Ca(2+) mobilization, granule exocytosis, and outward trafficking of recycling endosomes to the plasma membrane. We used fluorescein-conjugated cholera toxin B (FITC-CTxB) to label GM(1) in recycling endsomes and to monitor antigen-stimulated trafficking to the plasma membrane in both fluorimeter and imaging-based assays. We find that the sphingosine derivatives D-sphingosine and N,N'-dimethylsphingosine effectively inhibit this outward trafficking response, whereas a quarternary ammonium derivative, N,N',N″-trimethylsphingosine, does not inhibit. This pattern of inhibition is also found for Ca(2+) mobilization and secretory lysosomal exocytosis, indicating a general effect on Ca(2+)-dependent signaling processes. This inhibition correlates with the capacity of sphingosine derivatives to flip to the inner leaflet of the plasma membrane that is manifested as changes in plasma membrane-associated FITC-CTxB fluorescence and cytoplasmic pH. Using a fluorescently labeled MARCKS effector domain to monitor plasma membrane-associated polyphosphoinositides, we find that these sphingosine derivatives displace the electrostatic binding of this MARCKS effector domain to the plasma membrane in parallel with their capacity to inhibit Ca(2+)-dependent signaling. Our results support roles for plasma membrane polyphosphoinositides in Ca(2+) signaling and stimulated exocytosis, and they illuminate a mechanism by which D-sphingosine regulates signaling responses in mammalian cells.

Ca2+ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca2+ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca2+ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca2+ oscillations throughout the cell that usually follow the initiating Ca2+ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca2+ puffs at the region of cell contact. Initiating Ca2+ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca2+ release from endoplasmic reticulum stores and to extracellular Ca2+, but they do not depend on store-operated Ca2+ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response.

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Signaling in mast cells and basophils is mediated through IgE and its high affinity cell surface receptor, FcϵRI. Crosslinking of the receptors by a cognate multivalent antigen leads to degranulation and release of mediators of the allergic immune response. Using multicolor fluorescence confocal microscopy, we probed the spatio-temporal dynamics of early events in the IgE receptor signal cascade. We monitored the recruitment of GFP-/CFP-labeled signaling proteins by acquiring sequential images with time resolution of 3s during stimulation of RBL-2H3 mast cells with multivalent antigen. A fluorescent tag on the antigen allowed us to visualize the plasma membrane localization of crosslinked receptors, and fluorescent cholera toxin B served as a plasma membrane marker. We developed an automated image analysis scheme to quantify the recruitment of fluorescent intracellular proteins to the plasma membrane and to assess the time-dependent colocalization of these and other membrane-associated proteins with crosslinked receptors as measured by cross-correlation between the plasma membrane distributions of the two fluorophores. This automated method permits analysis of thousands of individual images from multiple experiments for each cross-correlation pair. We systematically applied this analysis to characterize stimulated interactions of IgE receptors with several signaling proteins, including the tyrosine kinases Lyn and Syk, and the adaptor protein LAT. Notably, for Syk-CFP we observed a rapid stimulated translocation to the plasma membrane but very little colocalization with aggregated receptors. Our results demonstrate the utility of this simple, automated method to monitor protein interactions quantitatively during cell signaling.

Advances in microfabrication and nanofabrication are opening new opportunities to investigate complicated questions of cell biology in ways not before possible. In particular, the spatial regulation of cellular processes can be examined by engineering the chemical and physical environment to which the cell responds. Lithographic methods and selective chemical modification schemes can provide biocompatible surfaces that control cellular interactions on the micron and submicron scales on which cells are organized. Combined with fluorescence microscopy and other approaches of cell biology, a widely expanded toolbox is becoming available. This review illustrates the potential of these integrated engineering tools, with an emphasis on patterned surfaces, for investigating fundamental mechanisms of receptor-mediated signaling in cells. We highlight progress made with immune cells and in particular with the IgE receptor system, which has been valuable for developing technology to gain new information about spatial regulation in signaling events.

Novel biophysical approaches combined with modeling and new biochemical data have helped to recharge the lipid raft field and have contributed to the generation of a refined model of plasma membrane organization. In this review, we summarize new information in the context of previous literature to provide new insights into the spatial organization and dynamics of lipids and proteins in the plasma membrane of live cells. Recent findings of large-scale separation of liquid-ordered and liquid-disordered phases in plasma membrane vesicles demonstrate this capacity within the complex milieu of plasma membrane proteins and lipids. Roles for membrane heterogeneity and reorganization in immune cell activation are discussed in light of this new information.

Immune receptors that specifically recognize foreign antigens to activate leukocytes in adaptive immune responses belong to a family of multichain cell surface proteins. All of these contain immunoreceptor tyrosine-based activation motifs in one or more subunits that initiate signaling cascades following stimulated tyrosine phosphorylation by Src-family kinases. As highlighted in this review, lipids participate in this initial activation step, as well as in more downstream signaling steps. We summarize evidence for cholesterol-dependent ordered lipids serving to regulate the store-operated Ca(2+) channel, Orai1, and we describe the sensitivity of Orai1 coupling to the ER Ca(2+) sensor, STIM1, to inhibition by polyunsaturated fatty acids. Phosphoinositides play key roles in regulating STIM1-Orai1 coupling, as well as in the stimulated Ca(2+) oscillations that are a consequence of IgE receptor signaling in mast cells. They also participate in the coupling between the plasma membrane and the actin cytoskeleton, which regulates immune receptor responses in T cells, B cells, and mast cells, both positively and negatively, depending on the cellular context. Recent studies show that other phospholipids with mostly saturated acylation also participate in coupling between receptors and the actin cytoskeleton. Lipid heterogeneity is a central feature of the intimate relationship between the plasma membrane and the actin cytoskeleton. The detailed nature of these interactions and how they are dynamically regulated to initiate and propagate receptor-mediated cell signaling are challenging questions for further investigation. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Our long-term efforts to elucidate receptor-mediated signalling in immune cells, particularly transmembrane signalling initiated by FcɛRI, the receptor for IgE in mast cells, led us unavoidably to contemplate the role of the heterogeneous plasma membrane. Our early investigations with fluorescence microscopy revealed co-redistribution of certain lipids and signalling components with antigen-cross-linked IgE–FcɛRI and pointed to participation of ordered membrane domains in the signalling process. With a focus on this function, we have worked along with others to develop diverse and increasingly sophisticated tools to analyse the complexity of membrane structure that facilitates regulation and targeting of signalling events. The present chapter describes how initial membrane interactions of clustered IgE–FcɛRI lead to downstream cellular responses and how biochemical information integrated with nanoscale resolution spectroscopy and imaging is providing mechanistic insights at the level of molecular complexes.

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, 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-FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM 1 , in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM 1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcepsilonRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcepsilonRI promotes its proximity to active Lyn in a lipid raft environment.

Binding of fluorescein isothiocyanate (FITC)-conjugated cholera toxin B subunit to ganglioside GM1 on RBL-2H3 cells at 37 °C results in labeling of the plasma membrane as well as a pool of perinuclear intracellular membranes identified as the endosomal recycling compartment. Antigen-mediated activation of IgE receptor signaling causes rapid, sustained outward trafficking of these labeled endosomes, that is monitored as an increase in FITC fluorescence due to relief of quenching in the acidic endosomes upon delivery to the plasma membrane. Stimulation of this process depends on the integrity of cholesterol-dependent lipid rafts and occurs in response to Ca2+-mobilizing thapsigargin as well as antigen. Inhibitors of some early signaling enzymes stimulated by FceRI, including Syk tyrosine kinase and phosphoinositide 3-kinase, have little or no effect on this trafficking response. Other signaling pathways, including activation of phospholipase C and Ca2+ influx, do not appear to be necessary for the initiation of the outward trafficking response, but they contribute to maintaining the sustained phase of this process. Consistent with this, antigen-stimulated ruffles are labeled with FITC-cholera toxin B in a Ca2+-dependent manner. Thus, this trafficking response provides a mechanism by which an internal membrane pool can contribute to plasma membrane remodeling during stimulated membrane ruffling, cell motility, and phagocytosis.

Background Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. Results To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. Conclusions Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.

Mast cells play a key role in allergy and inflammation processes as part of the immune response. The activation of mast cells via antigen binding and cross-linking of IgE receptors initiates the onset of dramatic calcium (Ca(2+)) mobilization dynamics that promote the release of mediators of inflammation and allergy. Ca(2+) signaling in mast cells has been studied extensively using a variety of research tools and techniques. In these studies, a large number of proteins have been identified to participate in various stages of these processes. Here we describe single-cell imaging as an important approach for examining Ca(2+) signaling and exocytosis in mast cells. Single-cell imaging tools have advanced significantly over the last 10 years, in part due to improvements in microscope technology and in part due to the development of a new generation of Ca(2+) indicators and genetically encoded Ca(2+) sensors. The single-cell imaging techniques described here provide the spatial and temporal resolution required to decipher the signaling events that are critical for mast cell functions.

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid effectively inhibit of Ca2 + responses in mast cells stimulated by antigen-mediated crosslinking of FceRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca2 + entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca2 + channel protein, AcGFP–Orai1, together with the labeled ER Ca2 + sensor protein, STIM1–mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1–STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1–Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1–Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization. Thisarticle is part of a Special Issue entitled Tools to study lipid functions.

Lipid domains or rafts are currently embraced by immunologists as critical participants in receptor-mediated signaling events occurring at the plasma membrane. This view of membrane heterogeneity and its functional importance is supported by many years of different experimental approaches. We can now refine our investigations, moving beyond the simple models to ask more detailed questions about structural properties and mechanistic interactions. As highlighted for the IgE receptor (Fc ϵ RI), new information about initial engagement with src family kinases, cytoskeletal regulation, and coupling with downstream signaling is beginning to emerge.

Accumulating evidence strongly supports the view that the plasma membrane participates in transmembrane signaling by IgE-receptors (IgE-Fc epsilon RI) through the formation of lipid-based domains, also known as rafts. Ongoing biochemical and biophysical experiments investigate the composition, structure, and dynamics of the corresponding membrane components and how these are related to functional coupling between Fc epsilon RI and Lyn tyrosine kinase to initiate signaling in mast cells.

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcepsilonRI) by the Src family kinase Lyn is the first known biochemical step that occurs during activation of mast cells and basophils after cross-linking of FcepsilonRI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the coupling of Lyn and FcepsilonRI was tested by investigating functional and structural effects of cholesterol depletion on Lyn/FcepsilonRI interactions. We find that cholesterol depletion with methyl-beta-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of FcepsilonRI and other proteins while enhancing more downstream events that lead to stimulated exocytosis. In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated FcepsilonRI and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that membrane structure, maintained by cholesterol, plays a critical role in the initiation of FcepsilonRI signaling.

We recently showed that aggregation of the high affinity IgE receptor on mast cells, FcepsilonRI, 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 FcepsilonRI and other receptors to undergo this association. We show that this interaction is not mediated by the beta subunit of the receptor or the cytoplasmic tail of the gamma subunit, both of which are involved in signaling. Using chimeric receptor constructs, we found that the extracellular segment of the FcepsilonRI alpha subunit was not sufficient to mediate this association, implicating FcepsilonRI alpha and/or gamma 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 alpha4 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 alpha (Tac) on Chinese hamster ovary cells exhibited aggregation-dependent membrane domain association similar to FcepsilonRI. These results provide insights into the structural basis and selectivity of lipid-mediated interactions between certain transmembrane receptors and detergent-resistant membranes.

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a versatile phospholipid that participates in many membrane-associated signaling processes. PI(4,5)P2 production at the plasma membrane (PM) depends on levels of its precursor, phosphatidylinositol 4-phosphate (PI4P), synthesized principally by two intracellular enzymes, PI4-kinases IIIα and IIIb; the former is preferentially inhibited by phenylarsine oxide (PAO). We found that PAO and quercetin, another lipid kinase inhibitor, rapidly inhibit Ca(2+) responses to antigen in IgE-sensitized rat basophilic leukemia mast cells. Quercetin also rapidly inhibits store-operated Ca(2+) influx stimulated by thapsigargin. In addition, quercetin and PAO effectively inhibit antigen-stimulated ruffling and spreading in these cells, and they inhibit endocytosis of crosslinked IgE receptor complexes, evidently by inhibiting pinching off of endocytic vesicles containing the clustered IgE receptors. A minimal model to account for these diverse effects is inhibition of PI(4,5)P2 synthesis by PAO and quercetin. To characterize the direct effects of these agents on PI(4,5)P2 synthesis, we monitored the reappearance of the PI(4,5)P2-specific PH domain PH-phospholipase C δ-EGFP at the PM after Ca(2+) ionophore (A23187)-induced PI(4,5)P2 hydrolysis, followed by Ca(2+) chelation with excess EGTA. Resynthesized PI(4,5)P2 initially appears as micron-sized patches near the PM. Addition of quercetin subsequent to A23187-induced PI(4,5)P2 hydrolysis reduces PI(4,5)P2 resynthesis in PM-associated patches, and PAO reduces PI(4,5)P2 at the PM while enhancing PI(4,5)P2 accumulation at the Golgi complex. Taken together, these results provide evidence that PI4P generated by PI4-kinase IIIα is dynamically coupled to PI(4,5)P2 pools at the PM that are important for downstream signaling processes activated by IgE receptors.

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.

Antigen-mediated crosslinking of IgE bound to its receptor, FcεRI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-FcεRI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nano-scale distribution of IgE-FcεRI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-FcεRI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-FcεRI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of FcεRI phosphorylation and cholesterol dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nano-scale organization of receptor-mediated signaling complexes in the plasma membrane.