BBA - Molecular Cell Research (v.1853, #4)

Preface to special issue on nanoscale membrane organisations by Balbino Alarcón; Michael Reth; Wolfgang Schamel (765-766).

The plasma membrane (PM) has to fulfill a wide range of biological functions including selective uptake of substances, signal transduction and modulation of cell polarity and cell shape. To allow efficient regulation of these processes many resident proteins and lipids of the PM are laterally segregated into different functional domains. A particularly striking example of lateral segregation has been described for the budding yeast PM, where integral membrane proteins as well as lipids exhibit very slow translational mobility and form a patchwork of many overlapping micron-sized domains. Here we discuss the molecular and physical mechanisms contributing to the formation of a multi-domain membrane and review our current understanding of yeast PM organization. Many of the fundamental principles underlying membrane self-assembly and organization identified in yeast are expected to equally hold true in other organisms, even for the more transient and elusive organization of the PM in mammalian cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: Plasma membrane; Membrane organization; Yeast;

MHC class II (MHC-II) molecules function by binding peptides derived from either self or foreign proteins and expressing these peptides on the surface of antigen presenting cells (APCs) for recognition by CD4 T cells. MHC-II is known to exist on clusters on the surface of APCs, and a variety of biochemical and functional studies have suggested that these clusters represent lipid raft microdomain-associated MHC-II. This review will summarize data exploring the biosynthesis of raft-associated MHC-II and the role that lipid raft association plays in regulating T cell activation by APCs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: MHC class II; Lipid raft; T lymphocyte; Antigen presenting cell;

Linking form to function: Biophysical aspects of artificial antigen presenting cell design by Karlo Perica; Alyssa K. Kosmides; Jonathan P. Schneck (781-790).
Artificial antigen presenting cells (aAPCs) are engineered platforms for T cell activation and expansion, synthesized by coupling T cell activating proteins to the surface of cell lines or biocompatible particles. They can serve both as model systems to study the basic aspects of T cell signaling and translationally as novel approaches for either active or adoptive immunotherapy. Historically, these reductionist systems have not been designed to mimic the temporally and spatially complex interactions observed during endogenous T cell-APC contact, which include receptor organization at both micro- and nanoscales and dynamic changes in cell and membrane morphologies. Here, we review how particle size and shape, as well as heterogenous distribution of T cell activating proteins on the particle surface, are critical aspects of aAPC design. In doing so, we demonstrate how insights derived from endogenous T cell activation can be applied to optimize aAPC, and in turn how aAPC platforms can be used to better understand endogenous T cell stimulation. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: Immunotherapy; Microparticle; Artificial antigen presenting cell; Microscale interaction; Nanoscale interaction;

Detecting protein association at the T cell plasma membrane by Florian Baumgart; Gerhard J. Schütz (791-801).
At the moment, many models on T cell signaling rely on results obtained via rather indirect methodologies, which makes direct comparison and conclusions to the in vivo situation difficult. Recently, a variety of new imaging methods were developed, which have the potential to directly shed light onto the mysteries of protein association at the T cell membrane. While the new modalities are extremely promising, for a broad readership it may be difficult to judge the results, since technological shortcomings are not always obvious. In this review article, we put key questions on the mechanism of protein interactions in the T cell plasma membrane into relation with techniques that allow to address such questions. We discuss applicability of the techniques, their strengths and weaknesses. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: Protein interaction; T cell plasma membrane; Single molecule microscopy;

Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains by Katharina Beck-García; Esmeralda Beck-García; Sheila Bohler; Carina Zorzin; Erdinc Sezgin; Ilya Levental; Balbino Alarcón; Wolfgang W.A. Schamel (802-809).
In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: TCR; Nanoclustering; Membrane; Lipid; Cholesterol;

Mechanisms of localized activation of the T cell antigen receptor inside clusters by Yair Neve-Oz; Yair Razvag; Julia Sajman; Eilon Sherman (810-821).
The T cell antigen receptor (TCR) has been shown to cluster both before and upon engagement with cognate antigens. However, the effect of TCR clustering on its activation remains poorly understood. Here, we used two-color photo-activated localization microscopy (PALM) to visualize individual molecules of TCR and ZAP-70, as a marker of TCR activation and phosphorylation, at the plasma membrane of uniformly activated T cells. Imaging and second-order statistics revealed that ZAP-70 recruitment and TCR activation localized inside TCR clusters. Live cell PALM imaging showed that the extent of localized TCR activation decreased, yet remained significant, with cell spreading. Using dynamic modeling and Monte-Carlo simulations we evaluated possible mechanisms of localized TCR activation. Our simulations indicate that localized TCR activation is the result of long-range cooperative interactions between activated TCRs, or localized activation by Lck and Fyn. Our results demonstrate the role of molecular clustering in cell signaling and activation, and are relevant to studying a wide range of multi-molecular complexes. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: T cell activation; Immune synapse; T cell receptor; Nanocluster; Microcluster; Super-resolution light microscopy; Photoactivated localization microscopy; Modeling and simulation;

Do mechanical forces contribute to nanoscale membrane organisation in T cells? by Enrico Klotzsch; Johannes Stiegler; Eldad Ben-Ishay; Katharina Gaus (822-829).
Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor–ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.Display Omitted
Keywords: Mechanobiology; T cell receptor; Protein clustering; Signalling; Super-resolution fluorescence microscopy; Traction force microscopy;

The nanoscale organization of the B lymphocyte membrane by Palash Chandra Maity; Jianying Yang; Kathrin Klaesener; Michael Reth (830-840).
The fluid mosaic model of Singer and Nicolson correctly predicted that the plasma membrane (PM) forms a lipid bi-layer containing many integral trans-membrane proteins. This model also suggested that most of these proteins were randomly dispersed and freely diffusing moieties. Initially, this view of a dynamic and rather unorganized membrane was supported by early observations of the cell surfaces using the light microscope. However, recent studies on the PM below the diffraction limit of visible light (~ 250 nm) revealed that, at nanoscale dimensions, membranes are highly organized and compartmentalized structures. Lymphocytes are particularly useful to study this nanoscale membrane organization because they grow as single cells and are not permanently engaged in cell:cell contacts within a tissue that can influence membrane organization. In this review, we describe the methods that can be used to better study the protein:protein interaction and nanoscale organization of lymphocyte membrane proteins, with a focus on the B cell antigen receptor (BCR). Furthermore, we discuss the factors that may generate and maintain these membrane structures.
Keywords: B cell antigen receptor; Nanocluster; Protein islands;

Ras nanoclusters: Versatile lipid-based signaling platforms by Yong Zhou; John F. Hancock (841-849).
Ras proteins assemble into transient nanoclusters on the plasma membrane. Nanoclusters are the sites of Ras effector recruitment and activation and are therefore essential for signal transmission. The dynamics of nanocluster formation and disassembly result in interesting emergent properties including high-fidelity signal transmission. More recently the lipid structure of Ras nanoclusters has been reported and shown to contribute to isoform-specific Ras signaling. In addition specific lipids play critical roles in mediating the formation, stability and dynamics of Ras nanoclusters. In consequence the spatiotemporal organization of these lipids has emerged as important and novel regulators of Ras function. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Keywords: Ras proteins; Nanoclusters; Spatial cross talk; lateral segregation; cholesterol; phosphatidylserine;

On multivalent receptor activity of GM1 in cholesterol containing membranes by Radek Šachl; Mariana Amaro; Gokcan Aydogan; Alena Koukalová; Ilya I. Mikhalyov; Ivan A. Boldyrev; Jana Humpolíčková; Martin Hof (850-857).
Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~ 1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~ 4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.Display Omitted
Keywords: Gangliosides; Cholera toxin; Fluorescence correlation spectroscopy; Energy transfer; Antibunching;

Plasma membrane reorganization: A glycolipid gateway for microbes by Sahaja Aigal; Julie Claudinon; Winfried Römer (858-871).
Ligand–receptor interactions, which represent the core for cell signaling and internalization processes are largely affected by the spatial configuration of host cell receptors. There is a growing piece of evidence that receptors are not homogeneously distributed within the plasma membrane, but are rather pre-clustered in nanodomains, or clusters are formed upon ligand binding. Pathogens have evolved many strategies to evade the host immune system and to ensure their survival by hijacking plasma membrane receptors that are most often associated with lipid rafts. In this review, we discuss the early stage molecular and physiological events that occur following ligand binding to host cell glycolipids. The ability of various biological ligands (e.g. toxins, lectins, viruses or bacteria) that bind to glycolipids to induce their own uptake into mammalian cells by creating negative membrane curvature and membrane invaginations is explored. We highlight recent trends in understanding nanoscale plasma membrane (re-)organization and present the benefits of using synthetic membrane systems. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.Display Omitted
Keywords: Toxin; Virus; Bacterium; Receptor clustering; Membrane invagination; Lipid rafts;