*Institut Curie, Centre de Recherche, F-75248 Paris, France†CNRS, UMR 168, PhysicoChimie Curie, F-75248 Paris, France‡CNRS, UMR 144, Subcellular Structure and Cellular Dynamics, F-75248 Paris, France§Université Pierre et Marie Curie, F-75252 Paris, France
AbstractThe understanding of lipid membranes and their organization has undergone signifi-cant development with better techniques and therefore more resolved experiments. Many new factors and organizing principles have been discovered, and interplay between these factors is expected to result in rich functional behaviours. The major factors regulating the lateral membrane heterogeneity, apart from the well-studied phase separation, are cytoskeleton pinning, clustering of lipids and curvature. These factors are effective means to create membrane domains that provide rich biological functionality. We review the recent advances and concepts of membrane heterogeneity organization by curvature, cytoskeleton and clustering proteins.
IntroductionCellular membranes are highly regulated barriers that separate the cytoplasm of the cell from the external world as well as the intra-organelle environment from the cytoplasm. They are not
simple inert barriers, and they can constantly remodel and reorganize due to physical mecha-nisms such as diffusion and demixing, or owing to their mechanical properties combined with their ability to interact with a rich plethora of proteins. Moreover, cell membranes are non-equilibrium systems where the components constantly exchange with the surrounding medium due to membrane trafficking. The membranes undergo continuous shape transforma-tions, reorganization of membrane structures (domains) and are under constant regulation of the lipid contents. The view of the plasma membrane has been constantly refined since the Singer–Nicolson fluid mosaic model [1]. Many more details have emerged regarding organiza-tion of the membrane or non-equilibrium processes regulating the dynamics. The presence of dynamical domains in cell membranes is now recognized, but how they form and evolve is still a matter of debate [2]. Studies on lipid mixtures in vitro have demonstrated that lipid mem-branes can phase separate, depending on the composition and temperature. These studies gen-erally correspond to equilibrium conditions, not representative of biological ones. Nevertheless, in vivo, many parameters can contribute to local demixing.
Membrane shapes are very diverse in a cell. The plasma membrane is macroscopically flat but microscopically shows dynamic generation of high curvatures owing to actions of clathrin, caveolin or other tubulating proteins with other exquisite dynamics owing to fluctuations and interaction with other components of the cell. The smooth endoplasmic reticulum or the mito-chondria are made of highly curved (30–100 nm in diameter) cylindrical structures. Golgi bodies represent complex dynamic structures of flat stacked membranes (cisternae), locally connected by tubules. Endosomes are small spherical organelles of 40–1000 nm diameter. Nevertheless, all of these structures are highly dynamic because membrane components are constantly exchanged between the compartments and with the plasma membrane by budding and fusion of small vesicles or tubular structures with curvatures higher than 0.01 nm−1 [3]. Lipids also display preferences for curvature dependent on their molecular packing in the bilayer. In the present chapter, we will address how membrane subdomains can be driven by membrane curvature and vice versa.
Electron micrographs of cytoplasmic surface of plasma membrane reveal that the mem-brane is extensively associated with a meshwork of filamentous actin and are locally deformed by membrane buds consisting of clathrin-coated pits and caveolae [4]. Plasma membrane con-tains many types of transmembrane proteins and proteins associated with it. It is conspicuous that interaction with cytoskeletal elements, bound- and trans-membrane proteins can modify and affect the organization of membranes [2]. We will discuss the latest developments with respect to membrane cytoskeletons and proteins influencing membrane phase behaviour.
Thus we will review in the present chapter the contribution to membrane nanodomain formation and regulation at the plasma membrane, and in intracellular membrane, by two important factors: (1) membrane shape (curvature) and (2) interacting proteins and cytoskeleton.
Phase separation and nanodomainsLipid demixing in membranesFrom a physical point of view, phases in lipid membranes are characterized by their molecular order. Membranes show three general types of lipid organization: the Ld (liquid disordered), the Lo (liquid ordered) and the Lβ (solid gel) phases. Heterogeneities in cell plasma membrane
are thought to be related to the Lo–Ld phase coexistence. Such phase coexistence and demixing have been observed in vitro in membranes composed of two lipids and cholesterol [5]. Phase separation occurs when the entropy of mixing (increase in disorder of multi-component sys-tem transitioning from a fully demixed state to an ideally mixed state [6]) is overcome by the interactions between components favouring demixing. In vivo, the existence of microdomains in cell membranes was first hypothesized by Simons and van Meer [7] to explain lipid sorting in polarized cells; the lateral organization by phase coexistence was next formulated in the concept of ‘lipid rafts’ by Simons and Ikonen [8]. Rafts result from the local dynamic enrich-ment of sphingolipids and cholesterol, allowing a better packing owing to their molecular geometry and accompanied by a preferential partitioning of certain proteins, in particular GPI (glycosylphosphatidylinositol)-anchored proteins. At compositions close to miscibility transi-tion points, phase separating lipid mixtures exhibit critical fluctuations in vitro [9]. Monte Carlo simulation studies show that in quasi-abrupt phase transition (first-order transition) regimes, transient short-lived fluid or gel domains are observed that are thermodynamically unstable [10]. The sizes of these short-lived nanodomains are below optical resolution. Interestingly, GPMVs (giant plasma membrane vesicles) isolated from cells show critical fluc-tuations [11]. This led to the proposal that the physical basis for plasma membrane heteroge-neity is near-critical fluctuations [11]. Whether the cellular membrane composition is tuned to be at criticality or quasi-abrupt transition in living organisms needs further experimental con-firmation. Nevertheless, an important consequence of being poised to demixing is that any external factor contributing to sort lipids or proteins can easily counterbalance the entropy of mixing, which facilitates heterogeneity. This can be achieved either by segregating some lipids or some proteins upon membrane bending and thus minimizing bending energy, or by immo-bilizing membrane components upon binding of proteins or cytoskeletal polymers that possess multiple binding sites [2].
Partitioning of phosphoinositidesAn important interplay to be considered with respect to cellular functions is the partitioning of phosphoinositides. PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) is a key phospholipid that is involved in a wide range of cellular functions [12]. PI(4,5)P2 also interacts with mole-cules regulating the organization of the actin cytoskeleton and proteins containing PH (pleck-strin homology) domains [13]. In a cellular context, the local distribution of phosphoinositides is regulated by specialized enzymes that can create local increased concentration of specific phosphoinositides. The local transient enrichment of phosphoinositides may also be defined as a membrane nanodomain, albeit kinetically far from equilibrium, and can control and regulate cellular processes [14]. Phosphoinositides are generally localized in Ld domains, to which the phosphoinositide nanodomains may also localize. The functional implications of such non-equilibrium nanodomains are discussed below.
Curvature and membrane nanodomainsVery early, after the hypothesis of ‘rafts’ in cell membranes, it was proposed that the existence of highly curved geometry could also account for the redistribution of lipids. Indeed, it was shown that different fluorescent DiI derivatives, varying solely in their alkyl chains (saturated vs. unsaturated) were located in different zones of endosomes with distinct curvatures (the
unsaturated in highly curved tubules) and followed different trafficking pathways [15]. This supported the hypothesis that different domains existed in plasma membranes and suggested that lipids could be differentially sorted, depending on their capacity to form membranes that can easily be curved [16]. Different in vitro experiments and theoretical models have tested this interesting hypothesis [17]. They demonstrate that for lipid membranes, curvature-induced lipid sorting is in general negligible and dominated by entropy of mixing. Only for membranes poised to demixing where entropy of mixing vanishes, unsaturated lipids (pre-dominantly forming Ld phases) can be enriched in curved areas, whereas saturated lipids or sphingolipids (predominantly in Lo phases) are depleted [18]. Nevertheless, as mentioned in the introduction, plasma membranes are thought to be near critical, and this effect might be all the more significant because cell membranes contain proteins and interact with underlying cytoskeleton. Proteins have a much larger size than lipids and entropy of mixing cost upon sorting is reduced compared with lipids. Different proteins have been found enriched or depleted in tubular structures in vitro, independently of the surrounding lipid composition [19,20]. This shows that membrane shape can directly influence protein distribution and that complex feedback, even shape instabilities could be expected since membrane curvature is in general generated by protein binding to membranes. For proteins that interact preferentially with lipids found in Lo phases, such as Shiga toxin or cholera toxin that bind to glycosphin-golipids, it was demonstrated that: (1) these toxins can not only induce clustering of lipids and nanodomains (see the subsection ‘Binding of proteins clustering lipids’ below), but also gener-ate the formation of their own tubular trafficking intermediates; and (2) these tubular struc-tures are enriched in sphingolipids [21], showing that protein–lipid interactions are essential in nanoclustering in membranes (Figure 1). These different in vitro experiments and modelling provided a useful basis for demonstrating and evaluating the contribution of membrane shape to the local enrichment of lipids and proteins. Specific binding of individual sphingolipid spe-cies to a defined signature within the transmembrane domain has also been described [22], where similar protein-mediated enrichment may take place. More complex effects can be expected when membrane curvature influences the recruitment or the activity of enzymes act-ing on lipids or proteins, such as phosphatases, kinases and GAPs (GTPase-activating pro-teins). Thus, nanodomain formation might result from a rich coupling between membrane shape, membrane organization and biochemistry.
Cytoskeleton, membrane proteins and membrane nanodomainsPinning of cytoskeleton to cell membranesThe cytoskeletal elements may interact with the membrane directly or indirectly. Indirect interactions may occur through cytoskeletal elements linked to transmembrane proteins or to other proteins, which interact with the inner leaflet of the plasma membrane or organelle membrane (for a review, see Kusumi et al. [23]). The molecular details of such interactions have been described in detail elsewhere [13]. If we consider the case where the cytoskeleton interacts preferentially with one type of domain (Ld or Lo) of a membrane showing phase coex-istence, the lateral distribution of these domains is then directly related to the cytoskeletal organization. In the case where cytoskeleton interaction is mediated by proteins (transmembrane
or cytosolic) with a preferential partition into a specific domain type [e.g. the ERM (ezrin, radixin and moesin) family of proteins/talin/WAVE (WASP verprolin homologous)/WASP (Wiskott–Aldrich syndrome protein)] [24], cytoskeleton meshwork organization and lipid domain distribution are intimately related; however, the details of their preferred phases in physiological conditions are yet to be illustrated. A similar nucleating effect or pinning of a specific domain is also expected with transmembrane proteins at gap junctions, synaptic junc-tions or focal adhesions containing proteins such as cadherins or integrins, which pin the Lo phase (Figure 2). Pinning of a specific phase by the preference of pinning molecules has been qualitatively shown in vitro [25]. Experiments using Monte Carlo simulations have shown that cytoskeletal pinning inhibits large-scale phase separation (note that phase separation as such is
Figure 1. Selective lipid sorting by proteins(A) In the absence of any protein, unsaturated lipids (Ld) are enriched in tubes, which minimize bending energy. (B) Proteins preferring negative curvature, such as Shiga toxin, are depleted from the highly positively curved tube, and are preferentially found on much less curved membrane. (C) If Shiga toxin is on the other side of the bilayer such as to induce/recognize negative curvature, the protein is enriched in the tubes. (D) Binding of Shiga toxin to a membrane with a composition close to phase coexistence can induce domain formation. Experimental evidence suggests that Shiga toxin binds and induces Lo domains, even though it may bind otherwise Ld-enriched unsaturated Gb3. (E) Enrichment of Shiga toxin in a tube drags along Lo-preferring sphingolipids and cholesterol, overcoming the increase in bending stiffness due to sphingolipids in highly curved geometry. Grey circles with dotted outline show bound lipid molecules.
not inhibited, but coalescence to form a large single domain is inhibited) and the domain size is determined by the characteristic compartment size defined by the actin meshwork [26], which could explain the small size of domains in vivo. A recent in vitro study cap-tures this effect on a reconstituted system on supported lipid bilayers [27]. Presence of cytoskeleton can preserve the micro-heterogeneities and fluctuations over a wider range of temperature, resulting in condensation of phases along a filament even for a near-criti-cal mixture [26]. This might reconcile the fact that GPMVs show critical fluctuations at 22°C, but interactions with cytoskeleton can preserve the heterogeneities even at a higher temperature of 37°C.
PI(4,5)P2 is an important regulator of actin cytoskeleton. Local concentrations of phosph-oinositides are actively regulated by kinases and phosphatases, depending on biological stimu-lus. Therefore, it can also be envisaged that the underlying actin meshwork organization can also be actively remodelled by motors [28] or triggered through these enzymes and may recip-rocally regulate organization of domains. It has been shown that ordered phase is formed by actin attachment on the membrane [29]. This view is supported by in vitro experiments show-ing that interaction with actin cytoskeleton binding to an originally homogeneous membrane through PI(4,5)P2 and N-WASP (neuronal WASP) nucleates domains, as expected when a pol-ymer binds to a membrane and reduces entropy of mixing. Furthermore, this interaction also leads to a positive shift in miscibility transition temperature [30], demonstrating that cytoskel-etal interactions in fact maintain membrane heterogeneities at temperatures higher than the phase transition temperatures for a lipid mixture.
Membrane heterogeneity induction by membrane–cytoskeleton interaction also has con-sequences on lateral diffusion of membrane components, and eventually on their functionality. Measurements from single-molecule trajectories of μ-opioid receptor and unsaturated phos-pholipid at high time resolution have revealed a confined hop diffusion leading to a picket-fence
Figure 2. Organizers of membrane heterogeneity: pinning of phases and reciprocal partitioning of protein into a preferred phase are expected to occurPinning can occur through the underlying actin cytoskeleton or through interaction of transmembrane proteins exemplified by cell–cell junctions and extracellular matrix. External ligands for glycosphingolipids cross-link and organize lipids, affecting their partitioning. Recruitment of phosphoinositide enzymes upon activation of receptor systems results in local enrichment of phosphoinositides (PI; magenta) and result in functionally active phosphoinositide nanodomains. Black, cholesterol; yellow, orange and grey, unsaturated lipids; purple, saturated lipids. CTx, cholera toxin; STx, Shiga toxin.
model of the cytoskeletal interaction with the membrane [31]. However, one should be careful about curvature effects and interpretation of single-molecule trajectory analysis [32]. Monte Carlo studies combining phase separation and cytoskeletal pinning show that subdiffusion is enhanced and eventually leads to hop diffusion of lipids [10]. With the complexity of cytoskel-eton interacting with membranes showing phase coexistence, diffusion of components (lipids and membranes) is expected to show very rich behaviour. In a hypothetical case where Ld is condensed along the filaments, lipids and proteins enriched in Ld phase may show quasi one-dimensional diffusion, which may have effects on reaction rates [33] (Figure 3). Such directional observation of proteins embedded in the plasma membrane along cytoskeletal net-works have been observed using single-particle tracking and FCS (fluorescence correlation
Figure 3. Interaction of cytoskeletal and phosphoinositide binding proteins with the plasma membrane(A) Cytoskeletal pinning of domains. (i) Phosphoinositide modification results in generation of phosphoinositide-enriched domains that recruit various actin nucleators and recruit actin to the membrane. (ii) Stable actin meshwork with pinning of elements to a preferred phase (in this Figure, Ld) results in condensation of the phase (Ld) along the filament. The condensed phase is expected to show two distinct diffusion behaviours exemplified by the dashed line path predominantly in the Ld phase (along the filaments) and the solid line path predominantly in the Lo phase. (iii) The actin meshwork is also subject to dynamic rearrangement by active cross-linking myosins, thereby rearranging the lipid phases. (B) Generation of a functional transient phosphoinositide-rich nanodomain. Phosphoinositide-modifying enzymes (striped large magenta circle) result in a local enriched concentration of phosphoinositide (small magenta circles); this recruits phosphoinositide-binding proteins (grey triangles), which in turn cluster phosphoinositide. A positive feedback loop results in growth and stabilization of a phosphoinositide-rich nanodomain. This does not necessarily result in partitioning to Lo phase, but results in a domain enriched in phosphoinositide.
spectroscopy) [27,34]. All of these studies suggest that even a simple equilibrium model of cytoskeletal interaction with the membrane results in very rich effects.
Binding of proteins clustering lipidsBinding of molecular cross-linkers such as Shiga toxin, cholera toxin or the pentameric capsid protein VP1 of the SV40 virus to glycosphingolipids on the extracellular side reorganizes the lipids and changes partitioning of glycosphingolipids, leading eventually to the endocytosis of tubular carriers self-induced by these molecules. It has been shown to induce phase separation in vitro. Interestingly, all of these three cross-linkers have similar geometry (pentagonal), bind a few glycosphingolipids simultaneously and thus reduce entropy of mixing [35]. Recently, it was also shown that galectin-3, belonging to the lectin family of carbohydrate-binding pro-teins, can cluster and form CLICs (clathrin-independent carriers) [36].
Even more complex effects occur with phosphoinositides. Phosphoinositides constitute only 1% of the lipids of the cytosolic leaflet, but are highly regulated by enzymes and have wide implications in many biochemical pathways. Electrostatic interactions and specific interactions with membrane protein PH domains can affect their lateral distribution upon interaction with proteins. In contrast with most lipids, polyphosphoinositides are highly charged and are there-fore subject to clustering by basic-residue-bearing molecules at the cytoplasm–membrane interface. In vitro studies have shown that PI(4,5)P2-binding peptides cluster PI(4,5)P2 on the membrane and show decreased diffusion coefficients [37]. Local PIP synthesis combined with the positive feedback established by binding of proteins leading to PIP clustering may provide functionally relevant localized stable nanodomains (see Chapter 12 [38]) (Figure 3B). Phosphoinositide-rich nanodomains have been described in vivo [39].
Generation of specific nanodomains in a spatio-temporally controlled manner regulates recruitment of proteins and other downstream processes [14]. Interestingly, scaffolding by BAR domain proteins has been shown to generate diffusion barriers along tubular structures and to induce stable PI(4,5)P2 nanodomains and phase boundaries [40]. This mechanochemi-cal feedback also recruits phosphatases at the neck, while the stable BAR domain proteins pro-tect the PI(4,5)P2 from phosphatases. This can have strong effects on membrane fission owing to generation of phase boundaries and increased line tension. A recent example is in the pro-cess of clathrin-mediated endocytosis, where formation of PI(3,4)P2 by class II PI(3)K C2α (phosphoinositide 3-kinase C2α) at the neck of clathrin-coated buds occurs at a pre-scission point. This regulates recruitment of SNX9, a BAR protein at endocytic intermediates at the plasma membrane [41].
Conclusion and future goalsMembrane compartmentalization and presence of dynamic heterogeneities are now well estab-lished. Cells exploit a plethora of physical and biochemical mechanisms to modulate lipid and protein localization spatially and temporally on membranes. With steep development in high-resolution imaging, single-molecule techniques and reconstitution methods, many of the mechanisms – curvature, lipid clustering, cytoskeletal pinning, effects on diffusion and phos-phoinositide clustering – have been put to the test. The consensus is that although substantial progress has been made in explaining many phenomena, the nuances are still to be explored.
Membrane-dependent complex phenomena such as self-organization have not yet been well explored [42]. Depending on the nature of the cell, specialized mechanisms may be employed for specific consequences. The diversity of membrane composition in different cells and their effects are slowly being recognized with lipidomics. One of the much needed improvements is in lipid labelling. Fluorescent tagging can lead to a change in the properties of the molecule in a bilayer, and reliable fluorescent lipids remain a challenge. Another area that has been chal-lenging to investigate is the interleaflet coupling. Although in vitro studies indicate very tight coupling between the two leaflets, with a highly asymmetric membrane and asymmetry in the biochemical processes, the nature of interleaflet coupling and its consequences are unknown. Interdisciplinary integration, chiefly involving imaging techniques, chemical synthesis, recon-stitution and biochemical tools have a lot to offer in terms of providing accurate details to gen-eral mechanisms that govern the lipid membrane.
SummaryCellular membranes function in synergy with proteins. Transmembrane pro-teins, cytoskeletal proteins and peripheral membrane-binding proteins all affect membrane heterogeneity.Membranes are shaped by proteins, which feeds back by redistributing pro-teins and lipids heterogeneously.Cytoskeletal pinning contributes to organization of the heterogeneity and affects diffusion of components.Phosphoinositides are very low in concentration, but are highly regulated to bring about a variety of functions. Phosphoinositide domains result from the interplay between protein-binding-induced clustering and the action of spe-cific enzymes. They have essential functional roles.
S.A. acknowledges a Labex CelTisPHyBio postdoctoral fellowship. We apologize to all of the authors whose work we could not cite owing to space limitations.
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