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Open accessJournal ArticleDOI: 10.1073/PNAS.2025343118

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.

02 Mar 2021-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 118, Iss: 9
Abstract: Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Forster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

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Topics: Phosphatidylinositol 4,5-bisphosphate (54%), Phospholipase C (52%), Diacylglycerol kinase (52%) ... read more
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6 results found



Open accessJournal ArticleDOI: 10.1242/JCS.259365
Alex G. Batrouni1, Nirmalya Bag1, Henry T. Phan1, Barbara Baird1  +1 moreInstitutions (1)
Abstract: PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B "lipoforms" affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein-protein and protein-lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway.

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Topics: Palmitoylation (64%), Transmembrane protein (52%)

Open accessJournal ArticleDOI: 10.3390/IJMS222111727
Abstract: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Forster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

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Topics: Cytoskeleton organization (58%), Cytoskeleton (54%), Pleckstrin homology domain (53%) ... read more

Open accessJournal ArticleDOI: 10.3390/BIOLOGY10111119
30 Oct 2021-Biology
Abstract: TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity—from very low to very high—independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

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Topics: Patch clamp (57%), Membrane potential (56%), Calcium signaling (55%) ... read more

Open accessPosted ContentDOI: 10.1101/2021.06.14.447954
Alex G. Batrouni1, Nirmalya Bag1, Henry T. Phan1, Barbara Baird1  +1 moreInstitutions (1)
14 Jun 2021-bioRxiv
Abstract: PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B “lipoforms” affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein–protein and protein–lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway. SUMMARY STATEMENT Different palmitoylation patterns on a lipid kinase adaptor protein control partitioning of the kinase between two spatiotemporally and functionally distinct complexes within the plasma membrane.

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References
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69 results found


Journal ArticleDOI: 10.1038/42408
05 Jun 1997-Nature
Abstract: A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.

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Topics: Fluid mosaic model (64%), Lipid raft (62%), Liquid ordered phase (62%) ... read more

9,050 Citations


Journal ArticleDOI: 10.1126/SCIENCE.1068539
03 May 2002-Science
Abstract: Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

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Topics: Peripheral membrane protein (67%), Lipid raft (64%), Membrane fluidity (64%) ... read more

2,092 Citations


Open accessJournal ArticleDOI: 10.1172/JCI16390
Kai Simons1, Robert Ehehalt1Institutions (1)
Abstract: Lipid rafts are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms. Rafts are receiving increasing attention as devices that regulate membrane function in eukaryotic cells. In this Perspective, we briefly summarize the structure and regulation of lipid rafts before turning to their evident medical importance. Here, we will give some examples of how rafts contribute to our understanding of the pathogenesis of different diseases. For more information on rafts, the interested reader is referred to recent reviews (1, 2). Composition of lipid rafts Lipid rafts have changed our view of membrane organization. Rafts are small platforms, composed of sphingolipids and cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. These assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase (3). Thus, the presence of liquid-ordered microdomains in cells transforms the classical membrane fluid mosaic model of Singer and Nicholson into a more complex system, where proteins and lipid rafts diffuse laterally within a two-dimensional liquid. Membrane proteins are assigned to three categories: those that are mainly found in the rafts, those that are present in the liquid-disordered phase, and those that represent an intermediate state, moving in and out of rafts. Constitutive raft residents include glycophosphatidylinositol-anchored (GPI-anchored) proteins; doubly acylated proteins, such as tyrosine kinases of the Src family, Gα subunits of heterotrimeric G proteins, and endothelial nitric oxide synthase (eNOS); cholesterol-linked and palmitate-anchored proteins like Hedgehog (see Jeong and McMahon, this Perspective series, ref. 4); and transmembrane proteins, particularly palmitoylated proteins such as influenza virus hemagglutinin and β-secretase (BACE) (1). Some membrane proteins are regulated raft residents and have a weak affinity for rafts in the unliganded state. After binding to a ligand, they undergo a conformational change and/or become oligomerized. When proteins oligomerize, they increase their raft affinity (5). A peripheral membrane protein, such as a nonreceptor tyrosine kinase, can be reversibly palmitoylated and can lose its raft association after depalmitoylation (6). By these means, the partitioning of proteins in and out of rafts can be tightly regulated.

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Topics: Lipid raft (70%), Fluid mosaic model (66%), Membrane lipids (64%) ... read more

1,015 Citations


Open accessJournal ArticleDOI: 10.1038/NRM.2017.16
Abstract: Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

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Topics: Membrane raft (64%), Lipid raft (55%)

966 Citations


Open accessJournal ArticleDOI: 10.1016/J.BBAMEM.2007.03.026
Raphael Zidovetzki1, Irena Levitan2Institutions (2)
Abstract: The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.

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Topics: Biological membrane (57%), Membrane (57%), Cell membrane (54%) ... read more

861 Citations