Cholesterol-dependent Domains Research Articles

Cholesterol affects voltage-gated proton channels indirectly.

A number of membrane proteins, including ion channels, interact directly with cholesterol within the plasma membrane. This interaction may affect physiologically relevant properties of the protein. Often, protein-sterol interaction is facilitated by a cholesterol recognition amino acid consensus sequence (CRAC) domain with (L/V)-X1-5-(Y)-X1-5-(K/R) or the inverse sequence CARC (K/R)-X1-5-(Y/F)-X1-5-(L/V) (Fantini & Barrantes, 2013, Front. Physiol. 4:31). The human proton channel hHV1 contains a putative CARC sequence KNNYAAMV (amino-acid residues 131-138) within the extrafacial part of its S2 transmembrane domain. We explored effects of cholesterol depletion from or addition to the membrane. Modulation of cholesterol levels using methyl-β-cyclodextrin (MBCD) dramatically altered current amplitude in inside-out membrane patches. Mutations to the CARC domain did not alter the effects of cholesterol, indicating that cholesterol binding to the CARC domain is not responsible. We propose that hHV1 molecules preferentially associate with cholesterol-dependent domains within the membrane. Cholesterol might affect proton currents if varying cholesterol changes the distribution of hHV1 molecules in the membrane between the cholesterol-dependent lipid domains and the rest of the membrane. A previous study reported that suction increased proton currents in excised patches and this was interpreted as evidence for mechanosensitivity of the HV1 protein (Pathak et al, 2016, J. Gen. Physiol. 148:405). We have confirmed that suction applied to the pipette increases proton currents. This observation is consistent with our hypothesis since membrane tension will undoubtedly affect the association of individual Hv1 molecules with lipid domains without the need for bona fide channel mechanosensitivity. Increase in membrane tension would result in the increased proton currents due to the dispersal of condensed lipid domains in a similar way to the cholesterol depletion. This study provides the first indication that HV1 associates with membrane lipid domains, sometimes called lipid rafts.

The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.

Ligand-Induced Growth of CD36-Fyn Clusters Induces Signaling

Nanoclustering is emerging as a key organizational principle of membrane proteins. Using superresolution imaging, we investigated the molecular organization of the clustering-responsive receptor CD36 and its downstream effector Fyn in response to thrombospondin-1 (TSP-1), an anti-angiogenic ligand that promotes endothelial cells apoptosis. At steady state, CD36 receptors pre-exist in nanoclusters (diameter of about 100 nm). Fyn is already present in these clusters, and its N-terminal membrane targeting domain is sufficient for this association. Even if already associated, we found that Fyn only becomes activated activated upon TSP-1 binding through an enhancement of CD36-Fyn clustering, forming larger and denser clusters. These enhancements are supported by the actin cytoskeleton and the presence of plasma membrane cholesterol as their perturbation abolishes signaling. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the reorganization of the receptor-effector pair CD36-Fyn that enables signaling during TSP-1 stimulation.

Plasma Membrane Organization Promotes CD36 Signal Transduction in Endothelial Cells

CD36 is a multiligand scavenger receptor that ligates Thrombospondin-1 (TSP-1) at the surface of endothelial cells and induces their apoptosis. Recent evidences have shown that clustering of CD36 is necessary for signal transduction in macrophages and is regulated by the architecture of the cortical actin cytoskeleton apposed to the plasmalemma. Here, we investigated the role of the cortical actin cytoskeleton and plasma membrane nanodomains in the control of CD36 activation in endothelial cells. Stimulation with multivalent ligands (TSP-1 and anti-CD36 IgM) resulted in the downstream phosphorylation of the Src Family kinase, Fyn. Disruption of the actin cytoskeleton or removal of cholesterol blocked this activation. To gain molecular details on the rearrangement of the receptors during TSP-1 binding, we conducted superresolution approaches (based on PhotoActivated Localization Microscopy or PALM) and quantitative spatial distribution analysis. Endothelial cell lines stably expressing CD36-PAmCherry were generated for that purpose. At steady state, CD36 receptors pre-exist in small clusters (average diameter of 100 nm), in which Fyn was also present. Upon TSP-1 binding, CD36 clusters increased in size (average diameter of 140 nm) and also became denser. The average distance between CD36 molecules in these clusters was in the range of 8 nm compare to 11 nm in the control condition. F-actin depolymerization or cholesterol depletion reduced the capacity of the ligand to induce formation of larger clusters resulting in a decreased recruitment and activation of Fyn. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the organization of CD36 receptors and Fyn before and during TSP-1 stimulation.

Role of Glycolipid-Binding Bacterial Toxins in the Assembly and Function of Membrane Domains

Cell membranes are characterized by the presence of small regions of specialized protein and lipid composition termed membrane domains. Cholesterol-dependent domains serve as important regulators of several membrane trafficking pathways, including the process of clathrin-independent endocytosis. Some of the best studied examples of proteins that are internalized into cells via clathrin independent endocytic pathways include bacterial toxins in the AB5 family such as Shiga toxin and cholera toxin. Recently, the intrinsic ability of cholera toxin and related glycolipid-binding toxins to initiate domain assembly has been postulated to trigger toxin internalization by generating a novel class of endocytic intermediates. Although the properties of domains induced by toxin binding have been well characterized in model systems, the nature of these structures in intact cells is still unclear, and their exact role in toxin uptake in cells remains to be fully elucidated. We have therefore focused on understanding the properties of toxin-induced domains in living cells, using the membrane binding subunit of cholera toxin (CTxB) as a model. To do so, we are utilizing a variety of methods to study domains induced by CTxB-binding to cell membranes, including FRAP and FCS-based measurements of protein dynamics, as well as live cell imaging and cell biological approaches. These studies are providing new insights into how CTxB-induced domains are assembled, how their structure and dynamics are regulated, the pathways they utilize to enter cells, and the role of both the glycolipid receptor of CTxB and toxin structure itself in these events.

Calcium-Dependent Lateral Organization in Phosphatidylinositol 4,5-Bisphosphate (PIP2)- and Cholesterol-Containing Monolayers

Biological membrane function, in part, depends upon the local regulation of lipid composition. The spatial heterogeneity of membrane lipids has been extensively explored in the context of cholesterol and phospholipid acyl-chain-dependent domain formation, but the effects of lipid head groups and soluble factors in lateral lipid organization are less clear. In this contribution, the effects of divalent calcium ions on domain formation in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2), a polyanionic, multifunctional lipid of the cytosolic leaflet of the plasma bilayer, are reported. In binary monolayers of PIP2 mixed with zwitterionic lipids, calcium induced a rapid, PIP2-dependent surface pressure drop, with the concomitant formation of laterally segregated, PIP2-rich domains. The effect was dependent upon head-group multivalency, because lowered pH suppressed the surface-pressure effect and domain formation. In accordance with previous observations, inclusion of cholesterol in lipid mixtures induced coexistence of two liquid phases. Phase separation strongly segregated PIP2 to the cholesterol-poor phase, suggesting a role for cholesterol-dependent lipid demixing in regulating PIP2 localization and local concentration. Similar to binary mixtures, subphase calcium induced contraction of ternary cholesterol-containing monolayers; however, in these mixtures, calcium induced an unexpected, PIP2- and multivalency-dependent decrease in the miscibility phase transition surface pressure, resulting in rapid dissolution of the domains. This result emphasizes the likely critical role of subphase factors and lipid head-group specificity in the formation and stability of cholesterol-dependent domains in cellular plasma membranes.

Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

Breaking Up Isn’t So Hard to Do

Lipid rafts are among the best-studied, yet least understood features of cell membranes. Most researchers agree that lipid rafts are a type of membrane domain that contains mixtures of cholesterol and either phospholipids or sphingolipids with high melting temperatures (1). Such lipid mixtures exhibit both high order and rapid lateral motion of their components and are therefore referred to as liquid-ordered phases (Lo) to contrast them with lipids in liquid-crystalline/liquid-disordered (Ld) states. The hypothesis that cell membranes contain a mixture of Lo and Ld domains has spawned intense study of the phase behavior of lipids that support Lo/Ld phase separation in artificial bilayers (2). One of the most striking features of the lipid domains observed in artificial membranes is their ability to grow to spectacularly large sizes. However, study after study has failed to detect large-scale domains in living cells. Even when scrutinized using highly sophisticated imaging approaches, most cholesterol-dependent domains appear to be, at most, on the order of 5–10 nanometers in size under steady-state conditions. What makes cell membranes so recalcitrant to forming micron-scale lipid domains? On page 3113 of this issue, Yethiraj and Weisshaar use an in silico approach to test the hypothesis that the presence of obstacles precludes the formation of large lipid domains (3). They postulate that integral membrane proteins attached to the cytoskeleton act as immobile obstacles. Using an Ising model of a binary lipid mixture whose phase behavior is tuned to match that of a canonical lipid raft-forming mixture (1:1:1 palmitoyl-sphingomyelin, dioleoyl phosphatidylcholine, and cholesterol), they show that the effect of the inclusion of obstacles is to depress the temperature at which phase separation occurs. The model predicts that for this lipid mixture, fluid-fluid phase separation normally occurs at 37°C. However, in the presence of obstacles, domains are broken up into nanoscale clusters, reducing the tendency of the lipids to phase separate. Remarkably, as little as 10% coverage of the bilayer by obstacles is sufficient to cause such behavior. The findings of Yethiraj and Weisshaar (3) thus support a role for cytoskeletally bound proteins as negative regulators of lipid domain formation. This model predicts that in the absence of the cytoskeleton, domain formation should be enhanced, in excellent agreement with recent experimental studies showing that Lo-like/Ld-like domain separation can occur in plasma membrane blebs devoid of the cytoskeleton (4). The model further raises the interesting possibility that the mean spacing of obstacles in cell membranes can influence the size of lipid domains. In addition to demonstrating a role of proteins in determining the properties of Lo/Ld domains, the results of this study provide a tantalizing clue as to how the distribution of proteins with respect to domains may be controlled. To date, most experimental studies have focused on partitioning into the liquid-ordered versus liquid-disordered phase. In such assays, transmembrane proteins and peptides tend to show a strong preference for partitioning in Ld domains, whereas GPI-anchored proteins prefer an Lo-like phase. However, the existence of a class of proteins that act as “two-dimensional surfactants,” helping to stabilize the interface of Lo and Ld domains, has also been postulated by a number of workers in the field. Consistent with this possibility, Yethiraj and Weisshaar (3) find that in their simulations, obstacles tend to fall at interdomain boundaries, and speculate that this is a function of their energetic neutrality in the model. It will be of interest to determine how the attractive and repulsive interactions between proteins and lipids further shape the size and shape of lipid domains using the types of modeling approaches described here in future work. Is the presence of obstacles the only reason lipid rafts are not readily observed in vivo? A number of alternative models remain to be tested. For example, cell membrane lipid composition may not support fluid-fluid phase immiscibility (2). In addition, triggering events may be required to coalesce and stabilize small rafts. Indeed, this notion has become so widespread that it was incorporated into a recently proposed formal definition of rafts: “Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions” (1). The size of lipid domains in cells is also likely to be controlled by features of cell membranes not easily mimicked in artificial membranes such as membrane turnover, the high concentrations of membrane proteins in cells, and the complex composition of cellular membranes (2,4). There is also building evidence that some types of microdomains, such as nanoclusters enriched in the small GTPase Ras or GPI-anchored proteins, are actively regulated to maintain a constant fraction of clustered to monomeric molecules over a wide range of expression levels. Clearly, with the development of new modeling and experimental systems to probe membrane domain structure, lipid rafts cannot remain an elusive entity for much longer.

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.

Integrin-mediated adhesion regulates membrane order

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.

Binding of Cross-Linked Glycosylphosphatidylinositol-Anchored Proteins to Discrete Actin-Associated Sites and Cholesterol-Dependent Domains

The mechanism by which cross-linked glycosylphosphatidylinositol (GPI)-anchored proteins are immobilized has been a mystery because both the binding to a transmembrane protein and attachment to a rigid cytoskeleton are needed. Using laser tweezers surface scanning resistance (SSR) technology, we obtained physical evidence for cross-linked GPI-anchored protein, Qa-2, binding to a transmembrane protein and for diffusion to discrete cytoskeleton attachment sites. At low levels of cross-linking of Qa-2 molecules, the resistance to lateral movement was that expected of monomeric lipid-anchored proteins, and no specific binding to cytoskeleton-attached structures was observed. When aggregates of the GPI-anchored protein, Qa-2, were scanned across plasma membranes, the background resistance was much higher than expected for a GPI-anchored protein alone and submicron domains of even higher resistance were observed (designated as elastic or non-elastic barriers) at a density of 82 (61 elastic and 21 small non-elastic barriers) per 100 μm 2. Elastic barriers involved weak but specific bonds to the actin cytoskeleton (broken by forces of 2 or 4 pN and were removed by cytochalasin D). Small non-elastic barriers (50–100 nm) depended upon membrane cholesterol and were closely correlated with caveolae density. Thus, cross-linked GPI-anchored proteins can diffuse through the membrane in complex with a transmembrane protein and bind weakly to discrete cytoskeleton attachment sites either associated with flexible actin networks or sphingolipid-cholesterol rich microdomains in live cell membranes. Our SSR measurements provide the first description of the physical characteristics of the interactions between rafts and stable membrane structures.