Role Of Membrane Microdomains Research Articles

New Insight Into the Roles of Membrane Microdomains in Physiological Activities of Fungal Cells.

The organization of biological membranes into structurally and functionally distinct lateral microdomains is generally accepted. From bacteria to mammals, laterally compartmentalized membranes seem to be a vital attribute of life. The crucial fraction of our current knowledge about the membrane microdomains has been gained from studies on fungi. In this review we summarize the evidence of the microdomain organization of membranes from fungal cells, with accent on their enormous diversity in composition, temporal dynamics, modes of formation, and recognized engagement in the cell physiology. A special emphasis is laid on the fact that in addition to their other biological functions, membrane microdomains also mediate the communication among different membranes within a eukaryotic cell and coordinate their functions. Involvement of fungal membrane microdomains in stress sensing, regulation of lipid homeostasis, and cell differentiation is discussed more in detail.

Role of membrane microdomains in compartmentation of cAMP signaling.

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.

Statins cause profound effects on gene expression in human cancer cells in vitro: the role of membrane microdomains.

There is increasing evidence that statin treatment can be beneficial in certain cancer patients. To determine if these benefits are a direct result of the cholesterol-lowering effects of statins or a result of secondary, protein transcription effects, the impacts of pravastatin and a cholesterol sequestrating agent methyl-beta-cyclodextrin (MbetaCD) on mRNA expression in the breast cancer cell MDA-MB-231 and the lung carcinoma cell Calu-1 have been compared by microarray techniques. The effects of these agents on cholesterol-rich rafts and caveolae, which have significance in cancer signaling, have also been examined. Both treatments caused a general downregulation of not only signal transduction including cancer pathway proteins, but also apoptosis and chemokine pathways, with statins impacting 35 genes by twofold or greater in MDA-MB-231 and > 300 genes in Calu-1. These manifold dysregulations could also explain the various side effects reportedly caused by statins. MbetaCD produced far fewer statistical events than pravastatin in the breast cancer line but many more in the lung cell line. Pravastatin increased expression of CAV1 but caveolae density decreased and overall raft density was unaffected. MbetaCD also caused an increase in CAV1 expression and reduced the prevalence of both rafts and caveolae. It is proposed that sequestration of cholesterol from the membrane by MbetaCD is not equivalent to blockade of the cholesterol pathway and causes different effects on microdomain-mediated signal transduction dependant on the cell line. The profound effects of statins on mRNA expression can be explained by the failure of caveolin-1 to properly complex with cholesterol in an altered sterol environment, with caveolae acting as the main loci for signaling directed towards those transcription processes unaffected by MbetaCD. Targeted inhibition of the postmevalonate pathway could offer an opportunity to specifically reduce caveolae-based signaling in cancer cells. The observed impact of pravastatin on gene expression may explain the pleiotropic effects of statins when they are used as adjuvants in chemotherapy and suggests impact on gene expression as a possible cause of side effects from statin use.

Receptor-mediated signaling at plasmodesmata

Plasmodesmata (PD) generate continuity between plant cells via the cytoplasm, endoplasmic reticulum (ER) and plasma membrane (PM), allowing movement of different classes of molecules between cells. Proteomic data indicates that the PD PM hosts many receptors and receptor kinases, as well as lipid raft and tetraspanin enriched microdomain associated proteins, suggesting the hypothesis that the PD PM is specialized with respect to both composition and function. PD-located receptor proteins and receptor kinases are responsible for perception of microbe associated molecular patterns at PD and initiate signaling that mediates changes to PD flux. In addition, developmentally relevant receptor kinases have different interactions dependent upon whether located at the PD PM or the cellular PM. The implications of these findings are that receptor-mediated signaling in PD membranes differs from that in the cellular PM and, in light the identification of PD-located proteins associated with membrane microdomains and the role of membrane microdomains in analogous signaling processes in animals, suggests that the PD PM contains specialized signaling platforms.

Relevance of Fatty Acid Covalently Bound to Escherichia coli α-Hemolysin and Membrane Microdomains in the Oligomerization Process

alpha-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell–cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell–cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell–cell contacts contributes to changes in cell behavior.

The roles of membrane microdomains (rafts) in T cell activation.

Detergent-resistant membrane microdomains enriched in sphingolipids, cholesterol and glycosylphosphatidylinositol-anchored proteins play essential roles in T cell receptor (TCR) signaling. These 'membrane rafts' accumulate several cytoplasmic lipid-modified molecules, including Src-family kinases, coreceptors CD4 and CD8 and transmembrane adapters LAT and PAG/Cbp, essential for either initiation or amplification of the signaling process, while most other abundant transmembrane proteins are excluded from these structures. TCRs in various T cell subpopulations may differ in their use of membrane rafts. Membrane rafts also seem to be involved in many other aspects of T cell biology, such as functioning of cytokine and chemokine receptors, adhesion molecules, antigen presentation, establishing cell polarity or interaction with important pathogens. Although the concept of membrane rafts explains several diverse biological phenomena, many basic issues, such as composition, size and heterogeneity, under native conditions, as well as the dynamics of their interactions with TCRs and other immunoreceptors, remain unclear, partially because of technical problems.

Cell adhesion and polarity during immune interactions.

Intercellular interactions are critical for a coordinated function of different cell types involved in the immune response. Here we review the cellular and molecular events occurring during cell-cell immune contacts. Cognate naïve CD4+ T lymphocyte-dendritic cell (DC) and primed T cell-antigen-presenting B lymphocyte interactions are discussed. The engagement of cytotoxic T lymphocytes (CTL) or natural killer cells (NK) with their targets is analyzed and compared to the process of T cell-antigen-presenting cell (APC) conjugate formation. The immunological synapse, a complex cluster of molecules organized at the contact area of cell conjugates, exhibits common features but shows some differences depending on cell types involved. Cellular interactions occur in sequential stages that involve dramatic changes in cell polarity and dynamic redistribution of cell membrane receptors. The role of membrane microdomains, adaptor molecules and the cytoskeleton in the regulation of the molecular reorganization at cell-cell contacts is also discussed.

Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking.

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).