Clustering Of Membrane Proteins Research Articles

Symmetry and Stability of Membrane Protein Lattices

Experimental surveys of the protein content in cell membranes suggest that membrane proteins exhibit great diversity in their oligomeric state and transmembrane shape. Furthermore, membrane proteins are often observed to form clusters, which can be composed of thousands of proteins. Recent breakthroughs in high-resolution imaging techniques have shown that the arrangement of proteins in membrane protein clusters is often far from random, with spatial ordering of proteins into regular two-dimensional lattices and, depending on the protein oligomeric state and transmembrane shape, distinct orientational ordering of neighboring membrane proteins. These observations have led, on the one hand, to the proposal that the regular arrangement of membrane proteins may play a role in the biological function of cell membranes. On the other hand, the question arises as to what are the physical mechanisms responsible for the self-assembly, symmetry, and stability of membrane protein lattices. Based on experimental observations and physical models, membrane-mediated interactions between proteins, which result from protein-induced lipid bilayer deformations, have been advanced as a general mechanism for long-ranged interactions between membrane proteins. Here we develop a computational framework for the calculation of membrane-mediated interactions in the large and complicated membrane protein lattices observed in experiments. We find that, depending on the specific shape and oligomeric state of the protein under consideration, membrane-mediated interactions can be attractive or repulsive, several kBT in strength, and depend crucially on the spatial and orientational symmetry in membrane protein lattices. Combining our approach with the theory of regular lattices of polygons, we carry out a systematic survey of the connection between the shape of membrane proteins, and the symmetry and stability of membrane protein lattices. Our results suggest direct experimental tests of how the self-assembly and biological function of membrane protein lattices is influenced by membrane-mediated interactions.

Oligomeric States and Cooperative Gating in Clusters of Mechanosensitive Membrane Proteins

The mechanosensitive channel of large conductance (MscL) provides a paradigm for mechanosensation and mechanotransduction. It also provides a model system for how bilayer material properties regulate membrane protein function. The basic phenomenology of MscL gating as a function of membrane tension, and the dependence of MscL gating on bilayer material properties, can be understood by considering the difference in membrane deformation energy between open and closed states of MscL. However, even basic issues such as the physiologically relevant oligomeric states of MscL remain a matter of intense debate. Is MscL a tetramer or a pentamer under physiological conditions, or do both structures occur in cell membranes? Moreover, MscL proteins have recently been observed to form clusters, and the gating behavior of MscL in clusters was found to be different from the gating behavior of single MscL. What are the physical mechanisms behind the clustering of MscL, and what is the biological significance of MscL clusters? The complexity of the observed MscL structures and the large size of MscL clusters have so far prohibited a theoretical analysis of the relation between the oligomeric states of MscL and the properties of MscL clusters. Here we develop a mathematical approach capable of predicting the coupling between MscL shape and function. Combining this approach with the theory of regular lattices of polygons, we study the interplay between the oligomeric state of MscL, membrane-mediated interactions between MscL proteins, the structure of MscL clusters, and cooperative gating of MscL. We predict that the architecture of MscL clusters may provide a signature of MscL shape, with distinct molecular structures of MscL yielding distinct spatial and orientational organization patterns. Our work establishes a bridge between the oligomeric state of MscL and the self-assembly of MscL into clusters.

The interplay between the clustering of transmembrane proteins and coupling of anchored membrane proteins

Clustering of membrane proteins plays an important role in many cellular activities such as protein sorting and signal transduction. In this study, we used dissipative particle dynamics simulation method to investigate the clustering of anchored membrane proteins (AMPs) in the presence of transmembrane proteins (TMPs). First, our simulation results show that clustering of AMPs and that of TMPs are in fact interdependent, and depending on their hydrophobic length, both protein mixing and protein demixing are observed. Especially, the protein demixing occurs only when the hydrophobic mismatch of TMPs is negative while that of AMPs is positive. Second, our simulation results indicate that the clustering of TMPs also modulates the coupling of the clustering of AMPs in both leaflets. On the one hand, the coupling between AMPs in different leaflets will be strongly restrained if TMPs form protein mixing with AMPs in one leaflet and protein demixing with AMPs in the other leaflet. On the other hand, the coupling between AMPs can be enhanced or mediated by TMPs when TMPs mix with AMPs in both leaflets. Our results may have some implications on our understanding of how different types of membrane proteins cluster and provide a possible explanation of how TMPs participate in signal transduction across cellular membranes.

Polka-Dotted Vesicles: Lipid Bilayer Dynamics and Cross-Linking Effects

We have investigated the effects of cross-linking perturbations on lipid phase-domain coalescence. Our model system explores cross-linking in the fluid-disordered phase of two-phase vesicles. Here, we quantify the vesicle population shift from the expected predominance of two-domain, two-phase configuration to a multidomain vesicle majority. We have found that the increase in multidomain vesicles is a distinct outcome from the cross-linking of biotinylated lipids and avidin. Analysis of our cross-linking data suggests that avidin forms clusters on the surface of the fluid-disordered domains, resulting in a large immobile fraction and restricted diffusion. In cellular membranes, receptor concentrations are similar to our experimental model, and we expect similar cluster formations, leading to nonideal mixing and lateral heterogeneity. We have induced and quantified a global response by cross-linking only a small percentage of lipids in our system, similar to receptor-ligand interactions on the cell membrane. Common activities, such as ligand-receptor coupling, contribute to lateral heterogeneity and membrane protein clustering, adding to cell membrane complexity. Fundamental studies into subtle shifts such as cross-linking events, which induce global cellular response, are pertinent to understanding membrane activities and effects of external stimuli.

A novel cellular stress response characterised by a rapid reorganisation of membranes of the endoplasmic reticulum

Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER.

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125–1000μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2′,7′ dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.

Bond Flexibility and Low Valence Promote Finite Clusters of Self-Aggregating Particles

Systems of complex particles such as proteins or colloidal particles have a widely observed tendency to form nonconnected nanometer-size clusters at steady state, but the underlying mechanisms remain poorly understood. We report here a numerical study on the self-aggregation of low-valence particles with flexible bonds (i.e., free bond orientations) in two dimensions and predict the formation of a stable cluster phase for average valences ranging from 2 to 3.6. For the intermediate case of trivalent particles, we show that a cluster phase is present over a wide range of concentrations and interaction energies. The clusters are polydisperse in size, have a fractal dimension of 1.5, and tend to fully saturate their bonds at high interaction energies. The number of unformed bonds scales linearly with the number of particles in a cluster, which implies the absence of phase transition in the explored region of interaction energies and concentrations. We discuss possible implications of our model for membrane protein clustering.

Mathematical simulation of membrane protein clustering for efficient signal transduction.

Initiation and propagation of cell signaling depend on productive interactions among signaling proteins at the plasma membrane. These diffusion-limited interactions can be influenced by features of the membrane that introduce barriers, such as cytoskeletal corrals, or microdomains that transiently confine both transmembrane receptors and membrane-tethered peripheral proteins. Membrane topographical features can lead to clustering of receptors and other membrane components, even under very dynamic conditions. This review considers the experimental and mathematical evidence that protein clustering impacts cell signaling in complex ways. Simulation approaches used to consider these stochastic processes are discussed.

The Amyloid Precursor Protein Forms Plasmalemmal Clusters via Its Pathogenic Amyloid-β Domain

The amyloid precursor protein (APP) is a large, ubiquitous integral membrane protein with a small amyloid-β (Aβ) domain. In the human brain, endosomal processing of APP produces neurotoxic Aβ-peptides, which are involved in Alzheimer's disease. Here, we show that the Aβ sequence exerts a physiological function when still present in the unprocessed APP molecule. From the extracellular site, Aβ concentrates APP molecules into plasmalemmal membrane protein clusters. Moreover, Aβ stabilization of clusters is a prerequisite for their targeting to endocytic clathrin structures. Therefore, we conclude that the Aβ domain directly mediates a central step in APP trafficking, driving its own conversion into neurotoxic peptides.

Do membrane proteins cluster without binding between molecules?

Clustering is a basic event for the initiation of immune cell responses, and simulation analyses of clustering of membrane proteins have been performed. It was claimed that a cluster is formed by the self-assembly induced by protein dimerization with a high binding speed (Woolf and Linderman, Biophys. Chem. 104, 217-227, 2003). We examined the cluster formation with Monte Carlo simulation using two algorithms. The first was that simulation processes were divided into two substeps. All proteins were subjected to movement in the first substep, followed by reaction in the second substep. The second algorithm was that proteins were first selected to react and proteins which did not react were subjected to movement. The self-assembly induced by dimerization was simulated only with the second algorithm. In this algorithm, monomers dissociated from dimers do not move because these monomers are not selected for movement, and a large proportion of such monomers are selected to form dimers in the next step. The self-assembly was again simulated with the first algorithm containing the conditions that monomers dissociated from dimers did not move in the next movement substep. This algorithm seems to be far removed from natural conditions. Thus, it is inferred that the self-assembly induced by dimerization is unlikely in situ, and that some interaction between proteins is required for cluster formation. In contrast to algorithms in previous simulations, our results suggest that it is more appropriate that proteins move to the same direction for a while and reflect when the collision occurs.

Shape as a determinant of membrane protein cluster formation

Cluster formation of membrane proteins is a crucial event in many vital cellular processes. Here, we present a thorough examination of the oligomerisation ability of membrane proteins with different geometries. By means of mesoscopic membrane simulations we show that lipid-mediated interactions between proteins depend both on the shape of the hydrophobic domain of the proteins and on their hydrophobic mismatch. Based on that, we find that protein interactions can be either attractive or repulsive, depending on the characteristic bilayer perturbations induced by the proteins. The influence of these perturbations is quantified via the associated potential of mean force. Such geometry-dependent interactions are likely to fine-tune protein oligomerization events during cellular processes, for example signal transduction or protein sorting.

Membrane Protein Cluster Titration in Early Lymphocyte Signaling

T cells are known to be sensitive to extremely small numbers of agonist peptides. Early T cell signaling originates in and is sustained by T Cell Receptor (TCR) microclusters. Recent reports indicate that T cell triggering thresholds are determined by the number of agonist peptide in individual TCR microclusters, a conclusion supported by the observation that 2D peptide-MHC kinetics are characterized by unexpectedly fast on-rates. Yet, questions remain regarding the interplay, if any, between signaling microclusters associated with the plasma membrane. We have studied the relationship between peptide number and protein cluster formation in early T cell signaling by direct, simultaneous observation of fluorescently labeled agonist peptides and the early T cell signaling molecule Zap70 genetically fused to EGFP in primary murine T cells. Our experiments replace the antigen-presenting cell with a supported lipid bilayer (SLB) chemically functionalized with peptide-MHC and ICAM-1. This approach allows us to titrate the density of triggering agonist peptide, while also providing a flat interface suitable for high-resolution TIRF imaging. By integrating micro-patterned chromium supports onto a cover glass we can also segregate the SLB into an array of spatially distinct micron-scale reaction centers. Using this method we measure antigen input vs. TCR triggering output in living primary T cells. These results have important implications concerning the role of cooperativity between adjacent TCR clusters in tuning the sensitivity of T cell triggering by exogenous agonist peptides.

Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid–protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14–47μm), protein clusters exhibited a narrow size distribution (2.6–4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid–protein interactions on lateral membrane architecture.

Protein-coat dynamics and cluster phases in intracellular trafficking

Clustering of membrane proteins is a hallmark of biological membranes’ lateralorganization and crucial to their function. However, the physical properties of these proteinaggregates remain poorly understood. Ensembles of coat proteins, the example consideredhere, are necessary for intracellular transport in eukaryotic cells. Assembly and disassemblyrates for coat proteins involved in intracellular vesicular trafficking must be carefullycontrolled: their assembly deforms the membrane patch and drives vesicle formation, yetthe protein coat must rapidly disassemble after vesiculation. Motivated by recentexperimental findings for protein-coat dynamics, we study a dynamical Ising-type model forcoat assembly and disassembly, and demonstrate how simple dynamical rulesgenerate a robust, steady-state distribution of protein clusters (corresponding tointermediate budded shapes) and how cluster sizes are controlled by the kinetics. Weinterpret the results in terms of both vesiculation and the coupling to cargo proteins.

Using Hierarchical Clustering and Dendrograms to Quantify the Clustering of Membrane Proteins

Cell biologists have developed methods to label membrane proteins with gold nanoparticles and then extract spatial point patterns of the gold particles from transmission electron microscopy images using image processing software. Previously, the resulting patterns were analyzed using the Hopkins statistic, which distinguishes nonclustered from modestly and highly clustered distributions, but is not designed to quantify the number or sizes of the clusters. Clusters were defined by the partitional clustering approach which required the choice of a distance. Two points from a pattern were put in the same cluster if they were closer than this distance. In this study, we present a new methodology based on hierarchical clustering to quantify clustering. An intrinsic distance is computed, which is the distance that produces the maximum number of clusters in the biological data, eliminating the need to choose a distance. To quantify the extent of clustering, we compare the clustering distance between the experimental data being analyzed with that from simulated random data. Results are then expressed as a dimensionless number, the clustering ratio that facilitates the comparison of clustering between experiments. Replacing the chosen cluster distance by the intrinsic clustering distance emphasizes densely packed clusters that are likely more important to downstream signaling events.We test our new clustering analysis approach against electron microscopy images from an experiment in which mast cells were exposed for 1 or 2 minutes to increasing concentrations of antigen that crosslink IgE bound to its high affinity receptor, FcϵRI, then fixed and the FcϵRI β subunit labeled with 5nm gold particles. The clustering ratio analysis confirms the increase in clustering with increasing antigen dose predicted from visual analysis and from the Hopkins statistic. Access to a robust and sensitive tool to both observe and quantify clustering is a key step toward understanding the detailed fine scale structure of the membrane, and ultimately to determining the role of spatial organization in the regulation of transmembrane signaling.

Regulation of Zebrafish Hatching by Tetraspanin cd63

Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen

Anaplasma platys infects peripheral blood platelets and causes infectious cyclic thrombocytopenia in canines. The genes, proteins, and antigens of A. platys are largely unknown, and an antigen for serodiagnosis of A. platys has not yet been identified. In this study, we cloned the A. platys major outer membrane protein cluster, including the P44/Msp2 expression locus (p44ES/msp2ES) and outer membrane protein (OMP), using DNA isolated from the blood of four naturally infected dogs from Venezuela and Taiwan, Republic of China. A. platys p44ES is located within a 4-kb genomic region downstream from a putative transcriptional regulator, tr1, and a homolog of the Anaplasma phagocytophilum, identified here as A. platys omp-1X. The predicted molecular masses of the four mature A. platys P44ES proteins ranged from 43.3 to 43.5 kDa. Comparative analyses of the deduced amino acid sequences of Tr1, OMP-1X, and P44/Msp2 proteins from A. platys with those from A. phagocytophilum showed sequence identities of 86.4% for Tr1, 45.9% to 46.3% for OMP-1X, and 55.0% to 56.9% for P44/Msp2. Comparison between A. platys and Anaplasma marginale proteins showed sequence identities of 73.1% for Tr1/Tr, 39.8% for OMP-1X/OMP1, and 41.5% to 42.1% for P44/Msp2. A synthetic OMP-1X peptide was shown to react with A. platys-positive sera but not with A. platys-negative sera or A. phagocytophilum-positive sera. Together, determination of the genomic locus of A. platys outer membrane proteins not only contributes to the fundamental understanding of this enigmatic pathogen but also helps in developing A. platys-specific PCR and serodiagnosis.

Ca2+induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.

Molecular Mechanisms of T Cell Signal Transduction Revealed by Super-Resolution Fluorescence Microscopy

We employ single molecule imaging techniques to understand how activation of the T cell receptor on the cell surface leads to an intracellular signaling response. To function in an immune response, T cells become activates when a highly specific pathogen-derived peptide binds to the T cell receptor (TCR). However, the concomitant signaling events are not specific to the TCR raising the question how T cells recognize specific signals for activation. The organization of signaling proteins in time and space may establish hierarchies and, ultimately, control signaling outcomes that determine cell function in health and disease.By manipulating lipid packing densities (Rentero et al PLoS One, 2008) and quantify membrane order microscopically (Gaus et al, J Cell Biol, 2005), our previous data revealed that membrane lipids and proteins co-operate to form stable membrane domains and protein clusters that are necessary for full T cell activation. To understand the underlying principles of the organization of signaling proteins in the T cell membrane, we established super-resolution microscopy approaches based on photo-activation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). These are a single molecule imaging technique that allows us to quantify the number of proteins participating in signaling clusters, the number of clusters and the ratio of proteins within clusters (Owen et al. J Biophotonics, 2010). In other words, we are able to quantify signaling efficiency and thus determine how lipids influence the organization and regulation of signaling process.

Analysis of Her2/neu membrane protein clusters in different types of breast cancer cells using localization microscopy

The Her2/neu tyrosine kinase receptor is a member of the epidermal growth factor family. It plays an important role in tumour genesis of certain types of breast cancer and its overexpression correlates with distinct diagnostic and therapeutic decisions. Nevertheless, it is still under intense investigation to improve diagnostic outcome and therapy control. In this content, we applied spectral precision distance/position determination microscopy, a technique based on the general principles of localization microscopy in order to study tumour typical conformational changes of receptor clusters on cell membranes. We examined two different mamma carcinoma cell lines as well as cells of a breast biopsy of a healthy donor. The Her2/neu receptor sites were labelled by immunofluorescence using conventional fluorescent dyes (Alexa conjugated antibodies). The characterization of the Her2/neu distribution on plasma membrane sections of 176 different cells yielded a total amount of 20 637 clusters with a mean diameter of 67 nm. Statistical analysis on the single molecule level revealed differences in clustering of Her2/neu between all three different cell lines. We also showed that using spectral precision distance/position determination microscopy, a dual colour reconstruction of the 3D spatial arrangement of Her2/neu and Her3 is possible. This indicates that spectral precision distance/position determination microscopy could be used as an enhanced tool offering additional information of Her2/neu receptor status.