Specific Membrane Domains Research Articles

Balance between Exocytosis and Endocytosis Determines the Efficacy of Sterol-Targeting Antibiotics

Antifungals targeting membrane ergosterol are longstanding, yet indispensable drugs in clinical use. However, the mechanisms by which the cellular membrane domains recognized by these antibiotics are generated remain largely unknown. Here, we demonstrate that the balance between endocytosis and exocytosis in membrane trafficking is a critical factor in the action of sterol-targeting antibiotics. When fission yeast cells were treated with manumycin A, cellular binding and the action of the antifungals filipin, amphotericin B, and theonellamides, all of which are ergosterol-binders, were abolished. Additionally, manumycin A treatment attenuated Cdc42 activity and inhibited exocytosis, while endocytosis was only moderately suppressed. Similar defects in membrane trafficking could be reproduced by heat shock and genetic perturbation,which also abolished the action of the antibiotics. We propose that exocytosis and endocytosis respectively supply and internalize the specific plasma membrane domains recognized by sterol-targeting antibiotics.

Eisosomes and membrane compartments in the ascomycetes: A view from Aspergillus nidulans.

Eisosomes are punctate structures located in the cytoplasmic side of the cell membrane of ascomycetes. In Saccharomyces cerevisiae they coincide topologically with and are necessary for the organisation of specific membrane domains. The eisosomal proteins are universally and quite strictly conserved in the sub-phylum, however this evolutionary conservation is in apparent contradiction with an elusive functional significance. The comparative analysis of the eisosomes of S. cerevisiae and Aspergillus nidulans reveal striking differences in the assembly and developmental fate of these structures between these two model organisms.

Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos.

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis.

Specialized membrane domains of plasmodesmata, plant intercellular nanopores.

Plasmodesmata (PD) are plant-specific membrane-lined channels that connect neighboring cells across the cell wall and are indispensable for intercellular communication, development and defense against pathogens. They consist of concentric membrane tubules of the plasma membrane (PM) on the outside and endoplasmic reticulum (ER) on the inside. The biophysical properties and molecular composition of both membranes are most likely distinct from the respective bulk membranes with which they are continuous. This specialization of PD membranes is expected to guarantee not only the compartmentalization of PD-related function but also to accommodate the requirement for highly curved

Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.

Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins? In this paper, we show that disruption of SM homeostasis at the trans-Golgi network (TGN) by treatment of HeLa cells with d-ceramide-C6, which was converted together with phosphatidylcholine to short-chain SM and diacylglycerol by SM synthase, led to the segregation of Golgi-resident proteins from each other. We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other. Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

Evidences of endocytosis via caveolae following blood–brain barrier breakdown by Phoneutria nigriventer spider venom

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid

Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way. Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization. We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids. This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.

Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis.

Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. Here, we show that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. We found that an increase in PI(4,5)P2 levels caused premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/PI(3,4,5)P3 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape.

Ammonia transport in the kidney by Rhesus glycoproteins.

Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4(+) with a new model in which specific and regulated transport of both NH3 and NH4(+) across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.

Refurbishing the plasmodesmal chamber: a role for lipid bodies?

Lipid bodies (LBs) are universal constituents of both animal and plant cells. They are produced by specialized membrane domains at the tubular endoplasmic reticulum (ER), and consist of a core of neutral lipids and a surrounding monolayer of phospholipid with embedded amphipathic proteins. Although originally regarded as simple depots for lipids, they have recently emerged as organelles that interact with other cellular constituents, exchanging lipids, proteins and signaling molecules, and shuttling them between various intracellular destinations, including the plasmamembrane (PM). Recent data showed that in plants LBs can deliver a subset of 1,3-β-glucanases to the plasmodesmal (PD) channel. We hypothesize that this may represent a more general mechanism, which complements the delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the PD exterior via the secretory pathway. We propose that LBs may contribute to the maintenance of the PD chamber and the delivery of regulatory molecules as well as proteins destined for transport to adjacent cells. In addition, we speculate that LBs deliver their cargo through interaction with membrane domains in the cytofacial side of the PM.

Phosphoinositides Coated Beads Binding Assay

The PIs coated beads assay or “PIP-Beads” developed by Echelon Biosciences (Salt Lake City, USA) is a quick assay to recognize which PIs are able to bind to a given protein domain, in a quantitative way. It is much faster and cheaper than liposomes and more reproducible than PIP-strip assays. The “PIP-Beads” assay is a biochemical assay that basically involves an incubation of a purified protein or protein domain with the appropriate PI-coated set of beads. After washing, drying and resuspending the samples, they can be easily analyzed by SDS-PAGE separation. Phosphoinositides (PIs) have been characterized as important determinants of cell membrane domains, such as the apical and basolateral domains in epithelial polarized cells (Martin-Belmonte and Mostov, 2007), controlling membrane trafficking (Szentpetery et al., 2010) or determining the presynaptic or postsynaptic terminal in neurons, among other functions (Di Paolo and De Camilli, 2006). These phosphoinositides enriched membranes bring the proteomic machinery together, confers to these membrane their different identities and functions. This protein-PIs interaction in many cases involves direct binding of specific protein membrane domains with certain PIs. Some of these domains are characterized such as PH domains from phospholipase-C- or synaptotagmin-like C2 domains (Galvez-Santisteban et al., 2012), while some of them are not. To determine which PI is binding to a given protein domain, it is important to have a quick and efficient assay. The liposome binding assays are very good to establish the kinetic properties of binding, but they are expensive and permit only to test a few PIs per experiment. On the other hand, PIP-strip (phosphatidil-inositol-phosphate) based analysis is easy and fast, however the PIs are presented in a flat surface and the reproducibility is sometimes limited.

Update of the 1972 Singer-Nicolson Fluid-Mosaic Model of Membrane Structure.

The Fluid-Mosaic Membrane Model of cell membrane structure was based on thermodynamic principals and the available data on component lateral mobility within the membrane plane [Singer SJ, Nicolson GL. The Fluid Mosaic Model of the structure of cell membranes. Science 1972; 175: 720-731]. After more than forty years the model remains relevant for describing the basic nano-scale structures of a variety of biological membranes. More recent information, however, has shown the importance of specialized membrane domains, such as lipid rafts and protein complexes, in describing the macrostructure and dynamics of biological membranes. In addition, membrane-associated cytoskeletal structures and extracellular matrix also play roles in limiting the mobility and range of motion of membrane components and add new layers of complexity and hierarchy to the original model. An updated Fluid-Mosaic Membrane Model is described, where more emphasis has been placed on the mosaic nature of cellular membranes where protein and lipid components are more crowded and limited in their movements in the membrane plane by lipid-lipid, protein-protein and lipid-protein interactions as well as cell-matrix, cell-cell and cytoskeletal interactions. These interactions are important in restraining membrane components and maintaining the unique mosaic organization of cell membranes into functional, dynamic domains.

Lateral organization of biological membranes

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a 'lipophobic effect.'

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Phosphorylation‐dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

Arabidopsis Tetraspanins Are Confined to Discrete Expression Domains and Cell Types in Reproductive Tissues and Form Homo- and Heterodimers When Expressed in Yeast

Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms. In animals, they are known to act as central organizers of membrane complexes and thought to facilitate diverse biological processes, such as cell proliferation, movement, adhesion, and fusion. The genome of Arabidopsis (Arabidopsis thaliana) encodes 17 members of the tetraspanin family; however, little is known about their functions in plant development. Here, we analyzed their phylogeny, protein topology, and domain structure and surveyed their expression and localization patterns in reproductive tissues. We show that, despite their low sequence identity with metazoan tetraspanins, plant tetraspanins display the typical structural topology and most signature features of tetraspanins in other multicellular organisms. Arabidopsis tetraspanins are expressed in diverse tissue domains or cell types in reproductive tissues, and some accumulate at the highest levels in response to pollination in the transmitting tract and stigma, male and female gametophytes and gametes. Arabidopsis tetraspanins are preferentially targeted to the plasma membrane, and they variously associate with specialized membrane domains, in a polarized fashion, to intercellular contacts or plasmodesmata. A membrane-based yeast (Saccharomyces cerevisiae) two-hybrid system established that tetraspanins can physically interact, forming homo- and heterodimer complexes. These results, together with a likely genetic redundancy, suggest that, similar to their metazoan counterparts, plant tetraspanins might be involved in facilitating intercellular communication, whose functions might be determined by the composition of tetraspanin complexes and their binding partners at the cell surface of specific cell types.

The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca(2+) channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca(2+) channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.

G-protein signaling, lipid rafts and the possible sites of action for the antidepressant effects of n-3 polyunsaturated fatty acids.

Dietary fish oil, a source of polyunsaturated fatty acids (n-3 PUFA), has become increasingly popular for antidepressant therapy, in part because about half of patients treated with conventional antidepressants either fail to remit or discontinue therapy due to side effects. The inception of n-3 PUFA as a putative depression therapeutic may have stemmed from reports suggesting that dietary n-3 PUFA deficiency is linked to both altered membrane PUFA content as well as clinical depression. Several studies have examined n-3 PUFA treatment in depression, either singly or in combination with conventional antidepressant drugs. While results have been encouraging, fish oil treatment remains controversial. At least some of the reason for this is the lack of a defined site of action for n-3 PUFA that would be consistent with an antidepressant effect. This review will address this issue. While it is possible, even likely, that n-3 PUFA have multiple sites of action, this chapter will focus on sites at which n-3 PUFA modify G protein signaling and how those sites relate to both depression and antidepressant action. Much of the focus herein will be on specialized membrane domains (lipid rafts) and the effects that agents modifying those rafts have on elements of G protein signaling cascades. The relevance of specific alterations of G protein signaling for both depression and antidepressant action will be discussed, as will the ability for n-3 PUFA to act either as an antidepressant or in concert with conventional antidepressants.

What does S‐palmitoylation do to membrane proteins?

S-palmitoylation is post-translational modification, which consists in the addition of a C16 acyl chain to cytosolic cysteines and which is unique amongst lipid modifications in that it is reversible. It can thus, like phosphorylation or ubiquitination, act as a switch. While palmitoylation of soluble proteins allows them to interact with membranes, the consequences of palmitoylation for transmembrane proteins are more enigmatic. We briefly review the current knowledge regarding the enzymes responsible for palmitate addition and removal. We then describe various observed consequences of membrane protein palmitoylation. We propose that the direct effects of palmitoylation on transmembrane proteins, however, might be limited to four non-mutually exclusive mechanistic consequences: alterations in the conformation of transmembrane domains, association with specific membrane domains, controlled interactions with other proteins and controlled interplay with other post-translational modifications.