Membrane Compartments Related to Signaling and Trafficking in Immune Cells
Stephanie Hammond, David Holowka and Barbara Baird, Cornell University, Ithaca, New York
Cells of the immune system detect and respond to the presence of foreign antigens by signaling through an array of receptors, which include the multichain immune recognition receptor (MIRR) family. Stimulated changes in membrane architecture, which includes protein and lipid segregation within membranes as well as membrane trafficking, regulate these cellular responses. We provide a brief overview of MIRR signaling, which focuses on signaling through the IgE receptor in mast cells, and we highlight specific examples in which membrane compartmentalization plays a role in this signaling pathway. We summarize biochemical methods used to isolate and characterize membrane subdomains. Finally, we discuss cross-correlation microscopy methods, application of antigen-patterned surfaces, and fluorescence-based trafficking assays to study dynamics of stimulated proteins interactions and membrane trafficking.
Membrane compartmentalization is required for rapid and efficient signaling responses in immune cells, such as mast cells, T cells, and B cells. Changes in the organization of membrane proteins and lipids are necessary for a spatially regulated response, and trafficking of intracellular compartments contribute to this spatial reorganization. Mast cells that express the high-affinity receptor for IgE, FceRI, are a well-characterized model system for investigating the role of membrane domains known as lipid rafts in the signaling pathway. However, changes in membrane compartmentalization necessary for signaling are not limited to plasma membrane lipid rafts. Subdomains of intracellular organelles, specifically the endoplasmic reticulum and the endosomal system, function to regulate membrane interactions and directional trafficking in this system. In this article, we discuss molecular biology tools that are being applied to the study of protein trafficking and membrane compartmentalization. We outline standard methods to isolate membrane subdomains, which include isolation of detergent-resistant membranes, cholesterol depletion, and subcellular fractionation. We then conclude this article by discussing spectrofluorometric methods used to study protein colocalization and membrane trafficking in living cells.
Signaling Through Antigen-Binding Receptors
Immune cells express a variety of receptors to detect foreign antigens and respond in a selective manner to clear pathogens and infected cells from the body. Both soluble and membrane-bound antigens are recognized by the multichain immune recognition receptor (MIRR) family of antigen-binding receptors. This family includes three well-characterized members: the high-affinity receptor for IgE (FcεRI) expressed on mast cells and basophils, the T cell receptor (TCR), and the B cell receptor (BCR). These receptors consist of transmembrane ligand-binding subunits that are noncovalently associated with signaling subunits that contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains (Fig. 1a). The binding of specific ligands cross-links these receptors and initiates signaling.
The FcεRI consists of the IgE-binding α subunit, which associates with the signal-transducing γ-homodimer and the signal-amplifying β subunit, and it serves as an archetype for studying immunoreceptor signaling. The TCR consists of an antigen-binding αβ heterodimer, the signal-transducing CD3 (δεγ) complex, and the ξ-chain homodimer. TCRs recognize short peptide sequences bound to major histocompatability complex (MHC) proteins on the surface of antigen-presenting-cells. The BCR consists of an antigen-binding membrane immunoglobulin subunit that associates with the signal-transducing Ig-α/Ig-β complex. B-cell signaling is enhanced through interactions with the B cell coreceptor complex that contributes additional phosphorylation sites.
Mast cell signaling through FcεRI and lipid rafts
Mast cells are the primary cellular mediators of allergic reactions and also function in innate immunity and defense against helminths (1). These tissue-resident cells reside in strategic locations throughout the body, most prominently in the mucosal membranes of the respiratory and digestive tracts. The rat basophilic leukemia (RBL-2H3) cell line, which is derived from mucosal mast cells, is often used as a model system to study FcεRI signal transduction. These cells abundantly express FcεRI (~200,000 receptors per cell) at the plasma membrane, where they bind allergen-specific IgE with high affinity (KD ~ 10-10 M) (2), sensitizing the cell and priming it to respond rapidly during subsequent exposure. Mast cells contain numerous secretory granules that store preformed allergic mediators, which include histamine, and stimulated release of these contents is responsible for the immediate symptoms of allergic reactions.
Signaling through FcεRI is initiated by binding of multivalent antigen by receptor-bound IgE and consequent aggregation of individual receptor complexes. FcεRI clustering promotes stable association with specialized regions of the plasma membrane, which are termed lipid rafts (3). Lipid rafts are microdomains of ordered lipids that are enriched in cholesterol and phospholipids with saturated acyl chains, including sphingolipids, and they coexist with regions of more fluid, disordered lipids (4). Several types of measurements indicate that as much as 40% of membrane lipids exist in ordered regions, although most transmembrane proteins prefer disordered regions (3). In unstimulated cells, lipids rafts seem to be small, 20-100 nm, and highly dynamic structures (5, 6). Coalescence and stabilization of lipid rafts by antigen-mediated cross-linking of IgE-FcεRI is believed to initiate this signaling cascade. This step is a ligand-sensing event that depends on antigen valency and receptor affinity for the antigen; receptor oligomerization increases receptor residency time in lipid rafts and allows for signal initiation (4).
Lipid rafts are one mechanism by which cells can segregate plasma membrane components and compartmentalize signaling events. Lyn, which is a Src family kinase, is dually acylated at its N-terminus by myristoylation and palmitoylation that target Lyn to the plasma membrane and confer its association with lipid rafts. Monomeric receptors are located largely outside of lipid rafts and are effectively segregated from the activating kinase in unstimulated cells. After receptor cross-linking and association with lipid rafts, Lyn phosphorylates FcεRI ITAMs (Fig. 1b). The lipid raft environment enhances signaling by protecting Lyn from inactivation through the exclusion of transmembrane phosphatases (7). Phosphorylation of the P and y subunits is the first biochemically detectable step in FcεRI signaling. The actin cytoskeleton plays a negative regulatory role in mast cell signaling (8), in part by limiting the functional interactions of FcεRI and Lyn through segregation of Lyn from cross-linked FcεRI complexes (9). The lipid composition of ordered microdomains in the plasma membrane is also regulated by the actin cytoskeleton (3).
In the primary signaling pathway, Syk kinase is recruited to the phosphorylated γ subunit via its tandem Src homology 2 (SH2) domains (10). Once activated through phosphorylation and conformational changes, Syk phosphorylates several downstream targets, which include the linker for the activation of T cells (LAT), an adaptor protein with multiple ITAMs that, when phosphorylated, serve as binding sites for SH2 domain-containing proteins (11). LAT forms complexes with several signaling proteins downstream of Syk including phospholipase C γ (PLCγ). Activated PLCγ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to produce soluble inositol-1,4,5-trisphosphate (IP3) and membrane-bound diacyl-glycerol (DAG) (10). These products lead to calcium mobilization and protein kinase C activation, which are both required for mast cell degranulation. Additionally, scaffolding proteins recruit several guanine nucleotide exchange factors (GEFs), which in turn bind to low-molecular-weight GTPases, leading to activation of the MAPK signaling cascade and cytokine production, as well as cell morphological changes.
A complementary pathway activated during FcεRI stimulation is initiated by the Src family kinase Fyn, as revealed in bone-marrow-derived mast cells. Fyn mediates phosphatidylinositol 3-kinase (PI-3-K) activation that phosphorylates PIP2 to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (10). Increasing plasma membrane PIP3 levels recruit pleckstrin homology (PH) domain-containing proteins, such as the Tec family kinase Btk, PLCγ, and GEFs that regulate changes in the actin cytoskeleton through the Rho family of GTPases. PI-3-K activation is necessary for the maintenance, but not the initiation, of calcium signaling required for degranulation and may influence cytokine production because of its ability to affect intracellular calcium concentrations (10). Studies in RBL mast cells with trivalent ligands of defined lengths are consistent with a PI-3-K pathway that operates in parallel to the Lyn-Syk pathway (12).
Figure 1. Signal transduction pathways from antigen receptors. (a) The MIRR family of receptors consists of the ligand-binding units associated with signal transducing units that contain ITAM sequences on transmembrane receptor subunits. (b) In unstimulated mast cells, monomeric IgE receptors largely reside outside of lipid rafts. After cross-linking by a multivalent antigen, IgE receptors cluster and stably associate with lipid rafts where Lyn phosphorylates the receptors and initiates a signaling cascade that culminates in the release of allergic mediators. Molecules are not shown to scale.
Elevation of intracellular calcium levels
Cytoplasmic calcium concentrations in unstimulated cells are ~100 nM but can rapidly increase to ≥1 μM during stimulation. In lymphocytes, this increase is a biphasic process. The initial increase is caused by IP3 binding to the IP3 receptor (IP3R) in the endoplasmic reticulum (ER) and rapid release from intracellular stores. The second, sustained phase results from extracellular calcium influx through calcium-release activated calcium (CRAC) channels in a process termed store-operated calcium entry (SOCE) and also through other calcium channels including the transient receptor potential channels and plasma membrane IP3RS (13). SOCE is the main mechanism by which cytoplasmic calcium levels are increased in lymphocytes (14). Rapid increases in intracellular calcium are necessary to trigger secretory vesicle fusion and extracellular secretion, whereas sustained calcium elevation is required to activate gene transcription and cytokine production.
ER heterogeneity and organization of ER subdomains in relation to calcium signaling received increased attention recently after several groups independently identified an ER calcium sensor protein, STIM1, and a component of the CRAC channel, Orai1/CRACM1, using genetic approaches (15). After depletion of ER calcium, STIM1 oligomerizes and accumulates in ER subregions near the plasma membrane. Orai1 in the plasma membrane coclusters with STIM1 in the ER, and the simultaneous accumulation of both proteins allows for activation of CRAC channels (16). Calcium levels are returned to basal levels after stimulation through the actions of sarco/endoplasmic reticulum Ca2+-ATPase channels in the ER, which refill the calcium store, and plasma membrane calcium pumps and exchangers, which export calcium into the extracellular environment.
Degranulation and stimulated membrane trafficking
Mast cell stimulation results in spatially regulated trafficking of several intracellular compartments that include secretory lysosomes, which release allergic mediators during degranulation, and recycling endosomes, a heterogeneous perinuclear compartment of mildly acidic membranes (17). Stimulation increases the outward trafficking of a lipid-raft component of recycling endosomes, as monitored by cholera toxin subunit B (CTxB) binding to GM1 (18), which are targeted toward sites of cross-linked IgE receptors (19), whereas secretory lysosome fusion occurs at sites distinct from receptor-signaling complexes (19). Furthermore, secretory lysosomal fusion is differentially regulated from cytokine trafficking; the latter may occur through recycling endosomal trafficking (20) as recently demonstrated in macrophages (21).
Members of the Rab and Arf GTPase families organize membrane compartmentalization within the endosomal system that is important for regulating transport through these organelles. Several Rab proteins localize to distinct subdomains on early and recycling endosomes and contribute to the sorting and trafficking functions of these compartments (22). These proteins also regulate the outward trafficking of lipid rafts from recycling endosomes (23).
T cell signaling through the TCR
T cell stimulation begins with binding of the antigenic peptide- MHC complex on antigen-presenting-cells to the TCR, and the Src family kinase Lck initiates a phosphorylation cascade (24). The Syk family kinase Zap-70 is recruited to phosphorylated TCR ITAMs, where it is phosphorylated by Lck. Activated Zap-70 then phosphorylates the adaptor protein LAT, which coordinates the recruitment and activation several downstream targets including PLCγ, PI-3-K, and GEFs that activate low molecular weight GTPases. These proteins couple receptor activation to calcium mobilization and changes in the actin cytoskeleton. Sustained calcium signaling is required for the formation of the immunological synapse at the interface between the T cell and the antigen-presenting-cell (14). In this structure, the TCR is localized to a central supremolecular activation cluster and is surrounded by a peripheral ring of adhesion molecules (25). The immunological synapse may facilitate sustained signaling necessary to induce cytokine production and T cell proliferation (14).
Initial signaling begins in small microclusters that form quickly after T cell stimulation and precedes formation of the immunological synapse (26). Coalescence of small structures into a larger signaling complex is dependent on stimulated actin polymerization and requires signaling through additional costimulatory molecules, which lead to actin cytoskeleton-regulated reorganization of lipid rafts (26). TCR signaling induces a transient dephosphorylation of the ezrin, radixin, and moesin (ERM) protein family, which temporarily uncouples lipid rafts from the actin cytoskeleton and allows for their coalescence into larger signaling structures (27). Trafficking of raft-enriched intracellular compartments increases the raft content of the plasma membrane and may contribute to targeting of rafts to the immunological synapse (26).
Concurrent with polarization of actin polymerization, the microtubule organizing center and secretory granules, which contain cytolytic agents in cytotoxic T cells and cytokines in helper T cells, are also polarized toward the antigen-presenting-cell (27). Helper T cells also spatially regulate cytokine secretion, releasing IL-2 and INF-γ into the immunological synapse but releasing TNF multidirectionally (28). Targeting granule release to the target cell produces locally high concentrations while minimizing unwanted effects on surrounding cells, whereas multi-directional release promotes recruitment of additional immune cells.
B cell signaling through the BCR
The BCR mediates antigen uptake for processing and presentation of the antigenic peptide-MHC class II complex to T cells, which promotes B cell differentiation into antibody-producing plasma cells and memory cells. Antigen binding to the BCR initiates a signaling cascade that involves Src, Syk, and Tec kinases that results in phosphorylation of the adaptor protein BLINK (B cell linker protein), which couples BCR stimulation to calcium mobilization similarly to LAT (29). The role lipid rafts play in BCR signaling is still under investigation as some BCR signaling can occur outside of rafts (30), and the outcome of BCR raft association changes during B cell development (31). BCR stimulation also results in transient dephosphorylation of ERM proteins and rapid depolymerization of the actin cytoskeleton, which facilitates coalescence of lipid rafts into stable signaling domains (32). In this system, the actin cytoskeleton may segregate lipid rafts in unstimulated cells and, after a strong stimulus, extensive actin depolymerization leads to sustained BCR signaling (32).
BCR signaling coordinates receptor internalization and reorganization of the endomembrane system essential for B cell antigen presentation. Lipid raft association triggers endocyto- sis of the antigen-BCR complex and delivery to MHC class
II-containing intracellular compartments for peptide loading onto MHC class II molecules (30), which is necessary for antigen presentation to helper T cells. Consequent interactions with helper T cells sustain B cell activation, which promotes immunoglobulin class switching and antibody production. The strength of the activation signaling (32) and the B cell microenvironment determines the ultimate outcome of B cell activation.
Techniques to Study Membrane Compartmentalization
Changes in protein and membrane interactions are most effectively studied with integrated biological and physical techniques. Recent advances in molecular biology enable mutation, overexpression, or depletion of a specific protein to assess its function within the signaling pathway. Biophysical methods to isolate functional membrane domains are used to assess stimulated changes in protein and lipid content. Fluorescence techniques are used to visualize and quantify protein interactions and directional membrane trafficking in live cells under physiological conditions.
Molecular biology methods
A protein’s role in a signaling pathway can be assessed by introducing mutations that disrupt interactions, localization, or activity or by silencing gene expression. The dynamics of engineered proteins can be monitored via selective placement of fluorescent tags within the protein sequence.
DNA engineering has become a standard laboratory procedure by which engineered proteins are used to study the role of protein localization and function within signaling pathways. Site-directed mutagenesis, in which only one or a few specific amino acids are altered in a protein, is a valuable tool for elucidating the structural properties of proteins that mediate protein dynamics. This approach was used, for example, to examine the role of MIRR transmembrane domains in mediating lipid raft association in mast cells (33) and B cells (34). The addition or deletion of a lipid modification site within the target protein can also be used as a tool to either enhance or decrease membrane or lipid raft association. A membrane targeted form of the C-terminal Src kinase (Csk), mCsk, was generated via the addition of a myristoylation site and was found to suppress basal Lyn activity in mast cells by phosphorylating the C-terminus of Lyn (35). Site-directed mutation of the myristoylation and palmitoylation sites in Lyn demonstrated that both modifications are required for membrane targeting and that palmityolation enhances lipid raft association (36).
Differential epitope tagging
One way to distinguish the genetically engineered protein from the cellular endogenous protein is to tag the engineered protein with either a fluorescent label or a nonfluorescent epitope tag. Fusion proteins tagged with green fluorescent protein (GFP) or any flavor of the many spectral variants of fluorescent proteins (FPs) (37) can be used to visualize the spatio-temporal distribution of several different proteins simultaneously within a single cell. Protein dynamics and interactions can be monitored using real-time fluorescence microscopy and quantitative microscopy techniques described below. Protein localization is often mediated by modular protein domains, such as PH domain binding to lipid species within the target membrane or SH2 and SH3 domains that bind to specific protein sequences of the target protein, which results in protein redistribution. The fusion of FPs to these modular domains generates fluorescent probes that can be used to monitor changes in lipid composition (38) and protein phosphorylation (39).
Alternatively, epitope tagging of proteins, in which a small epitope is inserted into the target protein sequence, is used to visualize protein localization by immunofluorescence microscopy of fixed and permeabilized cells. A variety of well-characterized epitope tags are available (40), and commercially available antibodies are used to detect the epitope tagged protein. These tags are relatively small compared with the full-length protein and can be inserted within or at the end of the protein sequence. Selective placement of the epitope tag, either on a cytoplasmic/intracellular or a luminal/extracellular portion of the protein, can be used to monitor trafficking of the protein from an internal pool to the plasma membrane. In live cells, the luminal epitope tag will be available for binding only if a membrane fusion event has occurred, which exposes this tag to the extracellular environment. This strategy has been used to monitor GLUT4 trafficking in adipocytes using an HA-GLUT4-GFP construct (41). FPs sometimes perturb the structure and/or interactions of labeled proteins, and small epitope tags may be necessary alternatives in this situation for protein localization studies.
Protein overexpression and knock down
Two standard methods used to study protein function are transfection of the protein of interest or knock down of the target protein. Transfected proteins may compete with endogenous proteins for substrates or interaction partners, and the expression level of the transfected protein can affect the measured response. For example, overexpression of mCsk reduces basal Lyn activity in mast cells (35), and this system has been used to examine the role of Src family members in mast cell signaling (42).
Alternatively, researchers can assess protein function with targeted degradation of the endogenous protein. RNA interference (RNAi), in which short interfering double-stranded RNA sequences (siRNA) target the degradation of specific mRNA sequences that lead to knock down of the target protein, has been developed within the last decade (43). Protein knock down using siRNA is transient, in which maximum silencing occurs 24-72 hours after transfection. For example, the regulation of endocytic and exocytic pathways in mast cells by the synaptotagmin family of proteins has been examined using both protein overexpression and knock-down strategies (44).
Stable protein knock down can be achieved with short hairpin RNAs (shRNA), which are processed inside the cell to generate active siRNA structures (43). Viral-mediated RNAi can reduce endogenous mRNA levels by ~75-90%, and cotransfection with multiple shRNA sequences can be used to produce double knockdowns (45). These double knockdowns can be used to generate cell lines or transgenic animals with stably silenced gene expression (43). Another approach to produce stably silenced cell lines is to encode antisense cDNA within a plasmid vector, and this method can reduce the levels of endogenous protein by ~90% (46). Either transient or stable knock down provides an environment for directly assessing the tagged-protein function without the endogenous protein background.
Biophysical and biochemical methods
Two common methods to study the role of lipid rafts in signaling pathways are detergent insolubility followed by isolation on a density gradient and cholesterol depletion. Other methodologies exist for isolating plasma membrane samples and intracellular organelles for additional characterization (47).
Detergent-resistant membranes (DRMs) were initially defined in terms of their resistance to solubilization by nonionic detergents, such as Triton X-100 (Pierce, Rockland, IL), at low temperatures, followed by floatation at low densities in equilibrium sucrose gradients (Fig. 2a) (3, 48). Lipids that are tightly packed in ordered regions of membrane are less susceptible to solubilization by small amounts of nonionic detergents than more disordered lipid regions (49). DRMs are also sensitive to the level of cholesterol, which is an abundant lipid in mammalian plasma membranes that comprises ~30-40 molar percent of total plasma membrane lipids (49). Cyclodextrins, which rapidly remove cholesterol from cellular membranes, are used to extract cholesterol and perturb raft association and, thereby, MIRR signaling (50).
DRMs do not correspond directly to pre-existing lipid rafts in cell membranes. Because detergents can cause reorganization of membrane lipids, isolated DRMs do not strictly measure lipid and protein associations that occur prior to detergent treatment (5). However, protein association with DRMs provides a correlative indication of association with lipid rafts in cell membranes (48, 51). Cholesterol plays multiple roles in cells, and cholesterol depletion can alter the lateral mobility of both raft and non-raft proteins as well as affecting association of the actin cytoskeleton with the plasma membrane (3, 6, 24). Although detergent insolubility and cholesterol depletion have limitations, they remain useful tools to investigate the role of membrane microdomains in signaling pathways (48).
Subcellular fractionation separates organelles based on their biophysical and biochemical properties. Several methods exist for cellular disruption, which include homogenization, sonication, and nitrogen cavitation (47). Homogenization shears a sample by forcing suspended cells through a narrow space and can result in membrane fragments of various size caused by variability in shear forces. When smaller cells, such as lymphocytes, are homogenized, the nuclei may be fragmented along with the plasma membrane. Sonication shears samples by using high-frequency sound waves to disrupt membranes, which nonselectively disrupts intracellular membranes as well as the plasma membrane and can damage membranes because of production of local heating. Nitrogen cavitation ruptures suspended cells, which are first equilibrated with high pressures of an inert gas, during rapid return to atmospheric pressure. This method fragments the ER and plasma membrane into uniform vesicles under inert conditions, which minimizes sample damage. After disruption, the nuclei and large cellular debris (nuclear pellet) are separated from the other organelles and cytosolic components (postnuclear supernatant) by a low-speed centrifugation step. Each of these components can be purified for analysis.
Centrifugation in a dense medium is a common method for separating organelles and membranes from the postnuclear supernatant (Fig. 2b). Differential centrifugation separates organelles based on size and weight; larger, heavier membranes pellet at slower speeds than smaller, lighter membranes. Successive centrifugation at faster speeds fractionates the sample into cellular components. Equilibrium, or isopycnic, centrifugation separates organelles based on their buoyant density independent of their size and shape. Cellular components are separated on a discontinuous gradient, and fractions float in the medium density that matches their own buoyant density. Sucrose is often used to create discontinuous gradients, but the relative separation of organelles and their distribution within the gradient will vary depending on the gradient medium and the method of sample loading.
Figure 2. Density gradient centrifugation to isolate membrane domains. (a) To isolate DRMs, cells are lysed with cold, nonionic detergent, and the resulting lysate is loaded on the bottom of a discontinuous sucrose gradient. The DRM fractions float, whereas the soluble fraction remains at the bottom of the gradient. (b) To isolate plasma membrane or other intracellular organelles, cells are lysed by means of homogenization, sonication, or nitrogen cavitation, and the postnuclear supernatant is loaded onto a discontinuous gradient. Vesicles derived from different organelles will float at different densities in the gradient, which allows for selective recovery of membrane populations.
Fluorescence spectroscopic methods
Temporal and spatial changes of fluorescently tagged proteins can be monitored in live cells under physiological conditions using a variety of spectroscopic methods that allow for the quantification of dynamic protein and membrane trafficking responses. Increasing capabilities to monitor protein translocation, protein-protein interactions, lipid turnover, and membrane trafficking will provide a more complete picture of the large-scale reorganization in cells that occurs during antigen receptor stimulation.
Direct visualization of fluorescently tagged proteins continues to be a powerful method for examining subcellular localization and dynamics within a single cell. Confocal microscopy, in which only a thin, optical section (~1 μm) of the sample is imaged, is commonly used to study protein localization in both fixed and living samples. The development of advanced imaging techniques at the micron- and submicron-scale resolution have been essential to monitoring small domains of the plasma membrane and intracellular organelles. A more-detailed discussion of advanced imaging techniques such as fluorescence recovery after photobleaching, single particle tracking, and fluorescence correlation spectroscopy used to study protein dynamics is provided elsewhere (52). Additionally, ultra high-resolution techniques, such as photoactivated localization microscopy and stimulated emission depletion microscopy are being developed to investigate plasma membrane dynamics (53, 54, 55).
Protein colocalization is qualitatively assessed by overlaying individual confocal images and looking for overlapping protein distributions, which is an approximate measurement of protein colocalization on the scale of optical resolution (~300 nm). Alternatively, a structure can be manually identified, and the fluorescence intensity profiles of two proteins along the trace can be correlated. This approach has been used to quantify receptor-protein interactions in mast cells (39, 56).
Several image correlation techniques, termed image correlation microscopy or image correlation spectroscopy (ICS), have been developed to analyze the density, diffusion, velocity, and interactions of fluorescently labeled membrane proteins. Spatial ICS measures the number and density of protein aggregates as well as changes in aggregation state, whereas temporal ICS measures diffusion coefficients and flow rates (57, 58). A recent extension of these techniques, which is termed spatio-temporal image correlation spectroscopy, can be used to monitor interactions between two fluorescently labeled proteins and also enables vector mapping of directed protein movement (58) even when a large percentage of the protein population is immobile (59). Raster image correlation spectroscopy can provide diffusion rates for both fast moving cytosolic species and slow moving membrane species (60).
Another recently developed cross-correlation microscopy methodology analyzes protein redistribution that occurs at the plasma membrane by imaging equatorial sections of the cell (61). This automated methodology allows for rapid analysis of a time-lapse image series in which a plasma membrane trace, defined with a fluorescent probe, is generated for each time point and, therefore, it can accommodate small changes in cell morphology (Fig. 3a). The stimulated recruitment of cytosolic proteins to the membrane (Fig. 3b), and their interactions with aggregated receptors (Fig. 3c) are quantified as a function of time. This methodology was used to analyze early signaling events in FcεRI signaling (61). Because events that occur at the plasma membrane are selectively analyzed, stimulated membrane trafficking events to and from the plasma membrane can also be studied with this approach.
Figure 3. Cross-correlation microscopy to study protein dynamics at the plasma membrane. (a) Methodology for analyzing protein dynamics at the plasma membrane. Cells are transiently transfected with the fluorescently labeled protein of interest. The antigen receptor and plasma membrane are also labeled with fluorescent markers. Plasma membrane masks generated for each time point are used to quantify protein dynamics at the plasma membrane over the time course. Representative traces of two cells that express GFP-PLCγ-(SH2)2 showing (b) recruitment to the plasma membrane and (c) interactions with clustered IgE receptors after stimulation as described in Reference 61.
Mast cells, in addition to responding to soluble antigen, can also polarize responses to the cell-pathogen interface when stimulated by helminths or large pathogens. Patterned silicon surfaces, in which the antigen is deposited on a silicon chip in micron-sized features (Fig. 4a), are used to study protein trafficking and membrane reorganization events that occur at the cell-stimulus interface (Fig. 4b). These surfaces have been used to investigate protein recruitment to aggregated FcεRI complexes in mast cells and demonstrate differential redistribution of inner-leaflet and outer-leaflet markers (62). The differential trafficking of recycling endosomal membranes, which are trafficked to sites of aggregated receptors, and histamine-containing secretory granules, polarized to the interface but not toward aggregated receptors, was examined using antigen-patterned surfaces (19).
Antigen-patterned surfaces have also been used to study the formation and regulation of the immunological synapse in T cells (63). Patterned surfaces with micron-sized corrals have been used to investigate the dynamics of immunological synapse formation by preventing central clustering of TCR ligands (64). Patterns that prevented TCR microclusters from reaching the central synapse enhanced T cell signaling, which suggests that spatial translocation of TCR microclusters to the central synapse is a method to downregulate TCR signaling (64). The role of peripheral TCR microclusters in T cell signaling can be further investigated with new technologies to produce sub-micron scaled patterns (63).
Figure 4. Antigen-patterned surfaces to study cellular activation. (a) A lipid mixture that contains antigen is patterned onto silicon surfaces in micron-sized features (62). (b) Mast cells that were labeled with Alexa488-IgE (top) show clustering over the antigen clusters (bottom).
Spectroscopic methods to study membrane trafficking
The organelles of the endocytic pathway are characterized by progressively more acidic structures as endocytosed material moves through early endosomes, late endosomes, and sent to either acidic lysosomes for degradation or to recycling endosomes for return to the plasma membrane. Fluorescent labels that are sensitive to pH, such as pH-sensitive GFP derivatives (pHluorins) or fluorescein isothiocyanate (FITC), can be used to monitor changes in the trafficking along the endocytic and exocytic pathways (65). A “targeted fluorescence” strategy to characterize pH gradients along the endocytic pathway takes advantage of pH-sensitive changes in FITC fluorescence (66).
Fluorophore pH-sensitivity can be used to quantify the trafficking of internal membranes to and fusion with the plasma membrane. FITC fluorescence is quenched in endocytic organelles, but fluorescence increases after exposure of the FITC-tagged protein to more pH-neutral environments. A trafficking assay has been developed to characterize the stimulated outward trafficking of recycling endosomes labeled with either FITC-CTxB or FITC-anti-transferrin receptor in mast cells (18). This assay can be used to screen for inhibitors of recycling endosomal trafficking and to elucidate the mechanisms that regulate trafficking of raft, CTxB-labeled endosomes, versus trafficking of nonraft, transferrin receptor-containing endosomes.
The redistribution of plasma membrane lipid rafts from small, dynamic entities to stable signaling complexes represents one example of stimulated membrane reorganization central to MIRR signaling. However, changes in membrane compartmentalization necessary for sustained immune cell signaling are not restricted to the plasma membrane; the membrane organization of intracellular organelles such as the ER and vesicles along the endocytic and exocytic pathways contribute to the functionality of these organelles and also to changes that occur at the plasma membrane. Biochemical methods to characterize membrane subdomains, such as detergent resistance, cholesterol depletion, and subcellular fractionation, used in combination with imaging techniques to study protein and membrane dynamics in living cells, are essential to understanding the role and regulation of membrane compartmentalization in immune cell signaling and trafficking.
Our research described in this review was supported by the Nanobiotechnology Center (NSF: ECS9876771) and by NIH grants: R01-AI22449, R01-AI18306, and T32-GM08210.
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Cell Membranes, Dynamics of
Imaging Techniques for Proteins
Lipid Domains, Chemistry of