Role of rafts in neurological disorders.

INTRODUCTION: Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders. DEVELOPMENT: We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease. CONCLUSIONS: Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.

The Role of Membrane Lipids in the Formation and Function of Caveolae.

Caveolae are plasma membrane invaginations with a distinct lipid composition. Membrane lipids cooperate with the structural components of caveolae to generate a metastable surface domain. Recent studies have provided insights into the structure of essential caveolar components and how lipids are crucial for the formation, dynamics, and disassembly of caveolae. They also suggest new models for how caveolins, major structural components of caveolae, insert into membranes and interact with lipids.

Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation.

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Cross-linker-Modulated Nanogel Flexibility Correlates with Tunable Targeting to a Sterically Impeded Endothelial Marker.

Deformability of injectable nanocarriers impacts rheological behavior, drug loading, and affinity target adhesion. Here, we present atomic force microscopy (AFM) and spectroscopy measurements of nanocarrier Young's moduli, tune the moduli of deformable nanocarriers with cross-linkers, and demonstrate vascular targeting behavior that correlates with Young's modulus. Homobifunctional cross-linkers were introduced into lysozyme-dextran nanogels (NGs). Single particle-scale AFM measurements determined NG moduli varying from ∼50-150 kPa for unmodified NGs or NGs with a short hydrophilic cross-linker (2,2'-(ethylenedioxy)bis(ethylamine), EOD) to ∼350 kPa for NGs modified with a longer hydrophilic cross-linker (4,9-dioxa-1,12-dodecanediamine, DODD) to ∼10 MPa for NGs modified with a longer hydrophobic cross-linker (1,12-diaminododecane, DAD). Cross-linked NGs were conjugated to antibodies for plasmalemma vesicle associated protein (PLVAP), a caveolar endothelial marker that cannot be accessed by rigid particles larger than ∼100 nm. In previous work, 150 nm NGs effectively targeted PLVAP, where rigid particles of similar diameter did not. EOD-modified NGs targeted PLVAP less effectively than unmodified NGs, but more effectively than DODD or DAD modified NGs, which both yielded low levels of targeting, resembling results previously obtained with polystyrene particles. Cross-linked NGs were also conjugated to antibodies against intracellular adhesion molecule-1 (ICAM-1), an endothelial marker accessible to large rigid particles. Cross-linked NGs and unmodified NGs targeted uniformly to ICAM-1. We thus demonstrate cross-linker modification of NGs, AFM determination of NG mechanical properties varying with cross-linker, and tuning of specific sterically constrained vascular targeting behavior in correlation with cross-linker-modified NG mechanical properties.

A DNA Nanodevice Simultaneously Activating the EGFR and Integrin for Enhancing Cytoskeletal Activity and Cancer Cell Treatment.

Cell-surface receptors (e.g., EGFR and integrin) and their interactions play determining roles in signal transduction and cytoskeletal activation, which affect cell attachment/detachment, invasion, motility, metastasis (intracellular), and cell-cell signaling. For instance, the interactions between the EGFR and integrin (α6ß4) may cause increased mechanical force and shear stress via enhanced cytoskeleton activation. Here, we design a DNA nanodevice (DNA-ND) that can simultaneously target the EGFR and integrin receptors on the caveolae. The piconewton (pN) forces in response to the EGFR-integrin coactivation can be sensed upon the unfolding of the DNA hairpin structure on the side arm of the device via changes of the fluorescence and plasmonic signals. We find that simultaneous activation of EGFR-integrin receptors causes enhanced signal transduction, contractions of the cells, and initiation of the biochemical pathways, thus resulting in a change of the cell division and endocytosis/exocytosis processes that affect the cell proliferation/apoptosis. The DNA-ND further enables us to visualize the cointernalization and degradation of the receptors by lysosomes, providing a novel approach toward bioimaging and mechano-pharmacology.

Caveolins; An Assailant or An Ally of Various Cellular Disorders.

Caveolae have impressive morphological highlights of the cytomembrane of mammalian cells which involve in wide diversity of cellular functions involving signaling pathways and cholesterol hastening. Caveolin proteins possess a 'scaffolding' domain which for caveolin-1 and caveolin-3 appear to act a dominant role in signal regulation through caveolae. Caveolin-1 is treated to be protein in the cytomembrane entrapped with caveolae in endothelial cells and vascular smooth muscle cells which diminish nitric oxide (NO) by fill up the calcium/calmodulin (Ca2+/CaM) confining point of endothelial nitric oxide synthase (eNOS), decrease NO generation produce endothelial dysfunction and atherosclerotic injury development. It is a cholesterol-binding layer protein associated with cell cholesterol transport and also shows cardioprotective action through ischemic preconditioning (IPC) in diabetic and postmenopausal rat heart. Additionally it is ensnared in the procedures of tumorigenesis, prostate disease, and inflammation. The present study in the paper is to explore the structural functionalities of caveolins and their contributory role in CVS disorders and various other diseases.

Caveolae: Structure, Function, and Relationship to Disease.

The plasma membrane of eukaryotic cells is not a simple sheet of lipids and proteins but is differentiated into subdomains with crucial functions. Caveolae, small pits in the plasma membrane, are the most abundant surface subdomains of many mammalian cells. The cellular functions of caveolae have long remained obscure, but a new molecular understanding of caveola formation has led to insights into their workings. Caveolae are formed by the coordinated action of a number of lipid-interacting proteins to produce a microdomain with a specific structure and lipid composition. Caveolae can bud from the plasma membrane to form an endocytic vesicle or can flatten into the membrane to help cells withstand mechanical stress. The role of caveolae as mechanoprotective and signal transduction elements is reviewed in the context of disease conditions associated with caveola dysfunction.

Design of Polymeric Gene Carriers for Effective Intracellular Delivery.

Polymeric carriers have emerged as major non-viral alternatives for gene delivery due to their lower immunogenicity and pathogenicity. However, during intracellular delivery of these carriers, multiple barriers have to be overcome or the efficiency of gene delivery will be impeded. A thorough understanding of these cellular impediments is pivotal to optimizing the efficiency of polymer-based gene delivery. This review delineates the major barriers encountered during intracellular delivery of polyplexes and discusses possible molecular designs to overcome these barriers. Based on a review of the latest strategies to enhance the intracellular delivery process, we provide insights into the further development of polymeric carriers with enhanced efficiency in transfection.

Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

Spelunking for lipids in caveolae.

Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

The caveolae dress code: structure and signaling.

Over the past decade, interest in caveolae biology has peaked. These small bulb-shaped plasma membrane invaginations of 50-80nm diameter present in most cell types have been upgraded from simple membrane structures to a more complex bona fide organelle. However, although caveolae are involved in several essential cellular functions and pathologies, the underlying molecular mechanisms remain poorly defined. Following the identification of caveolins and cavins as the main caveolae constituents, recent studies have brought new insight into their structural organization as a coat. In this review, we discuss how these new data on caveolae can be integrated in the context of their role in signaling and pathophysiology.

Cytosolic co-delivery of miRNA-34a and docetaxel with core-shell nanocarriers via caveolae-mediated pathway for the treatment of metastatic breast cancer.

Co-delivery of microRNAs and chemotherapeutic drugs into tumor cells is an attractive strategy for synergetic breast cancer therapy due to their complementary mechanisms. In this work, a core-shell nanocarrier coated by cationic albumin was developed to simultaneously deliver miRNA-34a and docetaxel (DTX) into breast cancer cells for improved therapeutic effect. The co-delivery nanocarriers showed a spherical morphology with an average particle size of 183.9 nm, and they efficiently protected miRNA-34a from degradation by RNase and serum. Importantly, the nanocarriers entered the cytosol via a caveolae-mediated pathway without entrapment in endosomes/lysosomes, thus improving the utilization of the cargo. In vitro, the co-delivery nanocarriers suppressed the expression of anti-apoptosis gene Bcl-2 at both transcription and protein levels, inhibited tumor cell migration and efficiently induced cell apoptosis and cytotoxicity. In vivo, the co-delivery nanocarriers prolonged the blood circulation of DTX, enhanced tumor accumulation of the cargo and significantly inhibited tumor growth and metastasis in 4T1-tumor bearing mice models. Taken together, the present nanocarrier co-loading with DTX and miRNA-34a is a new nanoplatform for the combination of insoluble drugs and gene/protein drugs and provides a promising strategy for the treatment of metastatic breast cancer.

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs.

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.

Characterization of Caveola-Vesicle Complexes (CVCs) Protein, PHIST/CVC-8195 in Plasmodium vivax.

Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-8195, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-8195 protein in P. vivax. In this study, the recombinant PvPHIST/CVC-8195 N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-8195 N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-8195 N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-8195 which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-8195. These results suggest that the PvPHIST/CVC-8195 is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

Ethanol Enhances TGF-ß Activity by Recruiting TGF-ß Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-ß-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-ß signaling sensor, by ∼ 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-ß receptor (TßR-II) by ∼ 2-3-fold from its intracellular pool, as determined by I(125) -TGF-ß-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TßR-I and TßR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-ß signaling by increasing non-lipid raft microdomain localization of the TGF-ß receptors. Since TGF-ß plays a protective role in ASCVD but can also cause ALD, the TGF-ß enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues.

Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae.

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gαq and stabilizes its activation state resulting in an enhanced Ca(2+) signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gαq association. This loss in Gαq/Cav1 association due to osmotic stress results in a significant reduction of Gαq/phospholipase Cß-mediated Ca(2+) signals. This reduced Ca(2+) response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-ß-cyclodextran. No change in Ca(2+) release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gαq-mediated Ca(2+) signals.