Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

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Lipid Rafts As a Membrane-Organizing Principle

▪ Abstract Lipid raft microdomains were conceived as part of a mechanism for the intracellular trafficking of lipids and lipid-anchored proteins. The raft hypothesis is based on the behavior of defined lipid mixtures in liposomes and other model membranes. Experiments in these well-characterized systems led to operational definitions for lipid rafts in cell membranes. These definitions, detergent solubility to define components of rafts, and sensitivity to cholesterol deprivation to define raft functions implicated sphingolipid- and cholesterol-rich lipid rafts in many cell functions. Despite extensive work, the basis for raft formation in cell membranes and the size of rafts and their stability are all uncertain. Recent work converges on very small rafts <10 nm in diameter that may enlarge and stabilize when their constituents are cross-linked.

https://www.physics.iitm.ac.in/~iol/lecture_notes/vani/general-lipid-stuff/lipid-rafts-2003-ann.rev.pdf

The state of lipid rafts: from model membranes to cells.

Ten years ago, we wrote a Review on lipid rafts and signalling in the launch issue of Nature Reviews Molecular Cell Biology. At the time, this field was suffering from ambiguous methodology and imprecise nomenclature. Now, new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking.

Revitalizing membrane rafts: new tools and insights

Lipid rafts bringing order to chaos

The internalization of various cargo proteins and lipids from the mammalian cell surface occurs through the clathrin and lipid-raft endocytic pathways. Protein-lipid and protein-protein interactions control the targeting of signalling molecules and their partners to various specialized membrane compartments in these pathways. This functions to control the activity of signalling cascades and the termination of signalling events, and therefore has a key role in defining how a cell responds to its environment.

/pdf/clathrin-and-non-clathrin-mediated-endocytic-regulation-of-48hm6xvlvc.pdf

Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling

Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent‐insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37°C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of ∼70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR–CD3 complex, Lck and ZAP‐70 kinases, and CD4 co‐receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.

/pdf/tcr-signal-initiation-machinery-is-pre-assembled-and-i97316eqih.pdf

TCR signal initiation machinery is pre‐assembled and activated in a subset of membrane rafts

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTe) system, a recombinant optogenetics-based molecular tool targeting the T Cell Receptor (TCR). The LiTe system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation patterns. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTe system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTe system provides new opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with a high spatiotemporal control.

Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells

Like other cells, the RBC membrane is composed of a lipid bilayer (BL) supported by a cytoskeleton (SC). The BL provides ﬂuidity, area incompressibility and slight resistance to bending. The SC provides in plane shear elasticity. They are connected by protein junction complexes, with possible sliding. Despite this biological reality, the most common modelling strategy is of the capsule type in which the SC and the BL are represented in common. The priority is given to shear and bending elasticity, with area incompressibility only crudely satisﬁed, the BL ﬂuidity sacriﬁced and sliding forbidden. Herein, we show that the modelling strategy of RBC membrane is at least as important as the choice of the stress-free shape, which has been and still is the focus of debate in the community. Four regimes representative of the richness of RBC dynamics in shear ﬂow are considered. We compare the capsule (C) and vesicle (V) models with models that distinguish the BL and the SC. The latter is then represented by a spring network (S) in the coarse-graining spirit or by a capsule model (C) in a continuous medium approach. Both in Tumbling (TU) and Tank-treading (TT) modes a non-negligible inﬂuence on the dynamics is observed. The C-C and V-C models are clearly distinguished. The deviation from the C model of the C-S and V-S models is less. Moreover, the anisotropy artiﬁcially introduced by a coarse spring model and in between for the V-C model. The V model shows a spinning frequency almost half that of the other models.

Rémi Lasserre

Papers

TCR signal initiation machinery is pre‐assembled and activated in a subset of membrane rafts

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells

Is red blood cell a simple capsule?