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Alternative models explaining the biliary lipid secretion at the canalicular membrane of hepatocytes exist: successive lipid extraction by preformed bile salt micelles, or budding of membrane fragments with formation of mixed micelles. To test the feasibility of the latter mechanism, we developed a mathematical model that describes the formation of lipid microdomains in the canalicular membrane. Bile salt monomers intercalate into the external hemileaflet of the canalicular membrane, to form a rim to liquid disordered domain patches that then pinch off to form nanometer-scale mixed micelles. Model simulations perfectly recapitulate the measured dependence of bile salt-dependent biliary lipid extraction rates upon modulation of the membrane cholesterol (lack or overexpression of the cholesterol transporter Abcg5-Abcg8) and phosphatidylcholine (lack of Mdr2, also known as Abcb4) content. The model reveals a strong dependence of the biliary secretion rate on the protein density of the membrane. Taken together, the proposed model is consistent with crucial experimental findings in the field and provides a consistent explanation of the central molecular processes involved in bile formation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F 2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF 2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P < 0.01). In experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger, and residence times were reduced for receptors in fish oil-treated cells (P < 0.05). In experiment 3, single particle tracking was used to determine the effects of PGF 2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil-treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF 2α ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015)., (© 2016 International Society for Neurochemistry.)

Lipid modifications of mammalian proteins are widespread, modifying thousands of targets involved in all aspects of cellular physiology cellular physiology. Broadly, lipidations serve to increase protein hydrophobicity and association with cellular membranes. Often, these modifications are absolutely essential for protein stability and localization, and serve critical roles in dynamic regulation of protein function. A number of lipidated proteins are associated with diseases, including parasite infections, neurological diseases, diabetes, and cancer, suggesting that lipid modifications represent potentially attractive targets for pharmacological intervention. This review briefly describes the various types of posttranslational protein lipid modifications, proteins modified by them, and the enzymatic machinery associated with these. We then discuss several case studies demonstrating successful development of lipidation inhibitors of potential (and more rarely, realized) clinical value. Although this field remains in its infancy, we believe these examples demonstrate the potential utility of targeting protein lipidation as a viable strategy for inhibiting the function of pathogenic proteins.

The APOE genotype is the major risk factor for Alzheimer's disease (AD); however, it remains unclarified how the ε4 allele accelerates whereas the ε2 allele suppresses AD development, compared with the more common ε3 allele. On the basis of the previous finding that the assembly of the amyloid-β protein (Aβ) into fibrils in the brain, an early and invariable pathological feature of AD, depends on the lipid environment, we determined the levels of synaptic membrane lipids in aged individuals of different APOE genotypes. In the comparison between amyloid-free ε2/ε3 and ε3/ε3 brains, the presence of the ε2 allele significantly decreased the level of cholesterol. Alternatively, in the comparison among ε3/ε3 brains, the presence of AD pathology substantially decreased the levels of cholesterol. This study suggests that the ε2 allele suppresses the initiation of AD development by lowering the cholesterol levels in synaptic membranes., (Copyright © 2014 Wiley Periodicals, Inc.)

One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome’s proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.

Cold acclimation results in changes of the plasma membrane (PM) composition. The PM is considered to contain specific lipid/protein-enriched microdomains which can be extracted as detergent-resistant plasma membrane (DRM). Previous studies in animal cells have demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be targeted to microdomains and/or the apoplast. However, the functional significance of GPI-APs during cold acclimation in plants is not yet fully understood. In this study, we aimed to investigate the responsiveness of GPI-APs to cold acclimation treatment in Arabidopsis. We isolated the PM, DRM, and apoplast fractions separately and, in addition, GPI-AP-enriched fractions were prepared from the PM preparation. Label-free quantitative shotgun proteomics identified a number of GPI-APs (163 proteins). Among them, some GPI-APs such as fasciclin-like arabinogalactan proteins and glycerophosphoryldiester phosphodiesterase-like proteins predominantly increased in PM- and GPI-AP-enriched fractions while the changes of GPI-APs in the DRM and apoplast fractions during cold acclimation were considerably different from those of other fractions. These proteins are thought to be associated with cell wall structure and properties. Therefore, this study demonstrated that each GPI-AP responded to cold acclimation in a different manner, suggesting that these changes during cold acclimation are involved in rearrangement of the extracellular matrix including the cell wall towards acquisition of freezing tolerance. [ABSTRACT FROM AUTHOR]

Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax. [ABSTRACT FROM AUTHOR]

Photosynthesis is the key bioenergetic process taking place in the chloroplast. The components of the photosynthetic machinery are embedded in a highly dynamic matrix, the thylakoid membrane. This membrane has the capacity to adapt during developmental transitions and under stress conditions. The galactolipids are the major polar lipid components of the thylakoid membrane conferring bilayer properties, while neutral thylakoid lipids such as the prenyllipids and carotenoids contribute to essential functions such as electron transport and photoprotection. Despite a large number of studies, the intriguing processes of thylakoid membrane biogenesis and dynamics remain unsolved. Plastoglobules, thylakoid-associated lipid droplets, appear to actively participate in thylakoid function from biogenesis to senescence. Recruitment of specific proteins enables the plastoglobules to act in metabolite synthesis, repair and disposal under changing environmental conditions and developmental stages. In this review, we describe plastoglobules as thylakoid membrane microdomains and discuss their involvement in lipid remodeling during stress and in the conversion from one plastid type to another. This article is part of a Special Issue entitled: Chloroplast Biogenesis. [ABSTRACT FROM AUTHOR]

Membranes are an important signalling platform in plants. The plasma membrane is the point where information about the external environment must be converted into intracellular signals, while endomembranes are important sites of protein trafficking, organization, compartmentalization, and intracellular signalling. This requires co-ordinating the spatial distribution of proteins, their activation state, and their interacting partners. This regulation frequently occurs through post-translational modification of proteins. Proteins that associate with the cell membrane do so through transmembrane domains, protein-protein interactions, lipid binding motifs/domains or use the post-translational addition of lipid groups as prosthetic membrane anchors. S-acylation is one such lipid modification capable of anchoring proteins to the membrane. Our current knowledge of S-acylation function in plants is fairly limited compared with other post-translational modifications and S-acylation in other organisms. However, it is becoming increasingly clear that S-acylation can act as more than just a simple membrane anchor: it can also act as a regulatory mechanism in signalling pathways in plants. S-acylation is, therefore, an ideal mechanism for regulating protein function at membranes. This review discusses our current knowledge of S-acylated proteins in plants, the interaction of different lipid modifications, and the general effects of S-acylation on cellular function. [ABSTRACT FROM AUTHOR]

Carbohydrates on the glycoproteins and glycosphingolipids expressed on the cell surface membrane play crucial roles in the determination of cell fates by being involved in the fine tuning of cell signalling as reaction molecules in the front line to various extrinsic stimulants. In glycoproteins, modification of proteins is performed by substitution of sugar chains to one or multiple sites of individual proteins, leading to quantitative and qualitative changes of receptor functions in the cell membrane. As for glycosphingolipids, majority of them consist of two moieties, i.e. carbohydrates and ceramides, and are localized in the microdomains such as lipid rafts or detergent-resistant microdomains. They generate and/or modulate cell signals to determine the cell fates by interacting with various carbohydrate-recognizing proteins. Modes of glycosylation and mechanisms by which glycosylation is involved in the regulation of cell signals are now hot subjects in glycobiology. [ABSTRACT FROM AUTHOR]

Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin-enriched microdomains (TEMs) are a unique type of protein-based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV-1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV-1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft-associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions. [ABSTRACT FROM AUTHOR]

Ca2+-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca2+-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca2+ sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca2+ sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca2+ sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca2+-triggered membrane fusion. [ABSTRACT FROM AUTHOR]

Specialized lipid microdomains in the cell plasma membrane, referred to as ’lipid rafts’, are enriched in sphingolipids and cholesterol and have drawn considerable interest as platforms for the recruitment of signaling molecules. Despite numerous biochemical and cellular studies, debate persists on the size, lifetime and even the existence of lipid rafts, emphasizing the need for reliable lipid probes to study in situ membrane lipid organization. In this review, we summarize our recent data on living cells using two specific probes of raft components: lysenin, a sphingomyelin- binding protein and the fluorescein ester of poly(ethyleneglycol)cholesteryl ether that labels cholesterol-rich domains. Sphingomyelin-rich domains that are spatially and functionally distinct from the GM1 ganglioside-rich domains were found at the plasma membrane of Jurkat T cells. In addition, the dynamics of cholesterol-rich domains could be monitored at the cell surface as well as inside the cells. [ABSTRACT FROM AUTHOR]

Abstract: Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with ≥0.25% Triton X-100 at 4 °C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 °C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue. [Copyright &y& Elsevier]

One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome's proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting. [ABSTRACT FROM AUTHOR]