Non-raft Regions Research Articles

How Membrane Phospholipids Containing Long-Chain Polyunsaturated Fatty Acids and Their Oxidation Products Orchestrate Lipid Raft Dynamics to Control Inflammation

BackgroundLong-chain PUFA (LC-PUFA) influence varying aspects of inflammation. One mechanism by which they regulate inflammation is by controlling the size and molecular composition of lipid rafts. Lipid rafts are sphingolipid/cholesterol-enriched plasma membrane microdomains that compartmentalize signaling proteins and thereby control downstream inflammatory gene expression and cytokine production. ObjectivesThis review summarizes developments in our understanding of how LC-PUFA acyl chains of phospholipids, in addition to oxidized derivatives of LC-PUFAs such as oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC), manipulate formation of lipid rafts and thereby inflammation. MethodsWe reviewed the literature, largely from the past 2 decades, on the impact of LC-PUFA acyl chains and oxidized products of LC-PUFAs on lipid raft biophysical organization of myeloid and lymphoid cells. The majority of the studies are based on rodent or cellular experiments with supporting mechanistic studies using biomimetic membranes and molecular dynamic simulations. These studies have focused largely on the LC-PUFA docosahexaenoic acid, with some studies addressing eicosapentaenoic acid. A few studies have investigated the role of oxidized phospholipids on rafts. ResultsThe biophysical literature suggests a model in which n-3 LC-PUFAs, in addition to oxPAPC, localize predominately to nonraft regions and impart a disordering effect in this environment. Rafts become larger because of the ensuing increase in the difference in order between raft and nonrafts. Biochemical studies suggest that some n-3 LC-PUFAs can be found within rafts. This deviation from homeostasis is a potential trigger for controlling aspects of innate and adaptive immunity. ConclusionOverall, select LC-PUFA acyl chains and oxidized acyl chains of phospholipids control lipid raft dynamics and downstream inflammation. Gaps in knowledge remain, particularly on underlying molecular mechanisms by which plasma membrane receptor organization is controlled in response to oxidized LC-PUFA acyl chains of membrane phospholipids. Validation in humans is also an area for future study.

Real-Time FRET-Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons.

Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.

Molecular Organization of a Raft-like Domain in a Polyunsaturated Phospholipid Bilayer: A Supervised Machine Learning Analysis of Molecular Dynamics Simulations

Numerous health benefits are associated with omega-3 polyunsaturated fatty acids (n-3 PUFA) consumed in fish oils. An understanding of the mechanism remains elusive. The plasma membrane as a site of action is the focus in this study. With large-scale all-atom MD simulations run on a model membrane (1050 lipid molecules), we observed the evolution over time (6 μs) of a circular (raft-like) domain composed of N-palmitoylsphingomyelin (PSM) and cholesterol embedded into a surrounding (non-raft) patch composed of polyunsaturated 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) (1:1:1 mol). A supervised machine learning algorithm was developed to characterize the migration of each lipid based on molecular conformation and the local environment. PDPC molecules were seen to infiltrate the ordered raft-like domain in a small amount, while a small concentration of PSM and cholesterol molecules was seen to migrate into the disordered non-raft region. Enclosing the raft-like domain, a narrow (∼2 nm in width) interfacial zone composed of PDPC, PSM, and cholesterol that buffers the substantial difference in order (ΔSCD ≈ 0.12) between raft-like and non-raft environments was seen to form. Our results suggest that n-3 PUFA regulate the architecture of lipid rafts enriched in sphingolipids and cholesterol with a minimal effect on order within their interior in membranes.

Antidepressants Produce Persistent Gα s-Associated Signaling Changes in Lipid Rafts after Drug Withdrawal.

Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of Gα s signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of Gα s protein from lipid rafts to nonraft membrane regions. This results in augmented Gα s signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft-localized cAMP sensor, we show that Gα s signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in Gα s localization observed after antidepressant treatment. Furthermore, we show that suppression of Gα s signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. Gα s localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft Gα s signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane Gα s signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of Gα s out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT: This work explores, for the first time, the effects of antidepressants on Gα s signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment.

Cholesterol Pushes Vitamin E and Polyunsaturated Phospholipids Together

Vitamin E (α-tocopherol) is an essential micronutrient that protects membrane lipids from oxidative damage. Whether this lipid-soluble vitamin preferentially locates in the vicinity of polyunsaturated phospholipids that are most vulnerable to oxidation continues to be debated. We apply MD simulations to address this issue. A key role for cholesterol, ubiquitous in mammalian membranes, is revealed. Binding energies measured for vitamin E in all-atom (AA) umbrella sampling simulations are lower for polyunsaturated phospholipids than less unsaturated phospholipids - they do not indicate preferential interaction with polyunsaturated phospholipids. There is a reduction in the binding energy following the introduction of cholesterol, which we suggest drives vitamin E and polyunsaturated phospholipids together. Support is provided by largescale simulations performed over an extended timescale on vitamin E in mixtures of PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine) with SM (sphingomyelin) and cholesterol that separate into a (raft-like) domain enriched in SM and cholesterol (raft-like) and a (non-raft ) region composed primarily of PDPC (non-raft) but depleted in cholesterol. In coarse-grained (CG) and AA simulations vitamin E is excluded from the raft-like domain and thereby placed in closer proximity to polyunsaturated phospholipids. We surmise that the much greater abundance of cholesterol, which has high affinity for saturated SM, relative to vitamin E is responsible.

The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin.

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.

Amyloid-β Interactions with Lipid Rafts in Biomimetic Systems: A Review of Laboratory Methods.

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.

CAMP Biosensors with Restricted Subcellular Localization Reveal Persistent Signaling Changes Following Antidepressant Withdrawal

Many patients experience a constellation of side effects following termination of antidepressant therapy. Collectively these effects are known as the antidepressant discontinuation syndrome. All currently prescribed antidepressant medications can cause this syndrome, regardless of primary mechanism of action. This suggests that some shared mechanism, independent of these drugs’ known binding targets, may contribute to the discontinuation syndrome.Lipid rafts are membrane microdomains with distinct signaling characteristics and protein expression compared to other membrane regions. Previous work has shown that chronic, but not acute, treatment with traditional antidepressants induces translocation of Gs out of lipid raft microdomains. In contrast, rapid acting antidepressants such as ketamine cause Gs translocation following acute exposure. This promotes Gs signaling in general, likely due to increased coupling to downstream effectors such as adenylyl cyclase.Using a lipid raft restricted cAMP biosensor we show that perturbations of Gs signaling persist for over 24 hr after antidepressant withdrawal in an in vitro model system. These signaling changes are variable between different subcellular microdomains, and between different cell types. In general, signaling changes in lipid rafts are more persistent than changes observed in non‐raft membrane regions, or in the cytoplasm. Effects are compared between c6 glioma, SK‐N‐SH neuronal, and HEK‐293 kidney derived cell lines.We also show that changes in lipid raft cAMP signaling are reflected by persistent reductions in lipid raft Gs expression following drug withdrawal. Protein expression is quantified following membrane isolation and sequential detergent extraction to separate lipid raft from non‐raft membranes.The observed antidepressant effects occur in model systems lacking the primary target for these drugs (e.g. SERT and escitalopram), and as such may represent a novel mechanism by which antidepressants regulate cellular activity. Understanding these residual antidepressant effects may aid in the development of novel antidepressants without an associated discontinuation syndrome, and in the discovery of new strategies to mitigate these persistent effects following drug withdrawal.Support or Funding InformationSupported by: VA Merit BX001149, NIH T32 MH067631 and NIH R01 AT009169

Different regions of synaptic vesicle membrane regulate VAMP2 conformation for the SNARE assembly

Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.

Vitamin E's Affinity for Polyunsaturated Phospholipids Studied by All-Atom MD Simulations

Vitamin E (α-tocopherol) is the principle lipid soluble antioxidant in cell membranes. Its purpose is to protect membrane lipids from oxidative damage. With all-atom (AA) MD simulations we address how affinity to various lipid species optimizes vitamin E's ability to protect polyunsaturated phospholipids that are most vulnerable to oxidation. Umbrella sampling simulations reveal that the binding energy of vitamin E simulations is greater for saturated SM (sphingomyelin) than polyunsaturated PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine). Preferential affinity for polyunsaturated phospholipids is not identified. The binding energy is reduced when cholesterol is added, however, suggesting vitamin E, like polyunsaturated phospholipids, is forced out of (raft-like) regions enriched in cholesterol into (non-raft) regions depleted in cholesterol. With large scale, microsecond-long simulations run on the Anton 2 supercomputer we determine where vitamin E prefers to locate in bilayers assembled with pre-formed PDPC and SM/cholesterol domains. The results of these studies will be presented.

Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by β-2 Adrenoceptors in Human Airway Smooth-Muscle Cells.

β 2-Adrenoceptors (β 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that β 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of β 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited β 2AR production of cAMP in lipid raft domains without affecting β 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.

A Role for Lipid-Lipid Interactions in Vitamin E's Function as a Membrane Antioxidant

Vitamin E (α-tocopherol) is the principle lipid soluble antioxidant in cell membranes. Its purpose is to protect membrane lipids from oxidative damage. Whether unequal affinity for different lipids optimizes the proximity of vitamin E to polyunsaturated phospholipids, the lipid species most susceptible to oxidation, is the question that we address with MD simulations on lipid bilayers. Our studies suggest cholesterol, ubiquitous in the cell membranes of animals, excludes vitamin E from saturated raft-like domains enriched in the sterol. Preferential affinity for polyunsaturated phospholipids is not indicated - vitamin E, like polyunsaturated phospholipids, is pushed towards non-raft regions depleted in cholesterol. The binding energy measured for vitamin E in umbrella sampling AA (all-atom) simulations is greater for SM (sphingomyelin) than PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine). Adding cholesterol to SM eliminates the differential in binding energy. CG (coarse-grained) simulations run on PDPC/SM/cholesterol mixtures in the presence of vitamin E indicate the vitamin locates at the boundary between SM-rich/sterol-rich (raft-like) and PDPC-rich/sterol-poor (non-raft) domains. CG simulations run on mixtures replacing PDPC by DDPC (1,2-didocosahexanoyl-sn-glycero-3-phosphocholine) will reveal details of the molecular interactions of vitamin E at the distinct boundary between raft-like and non-raft domains that forms with the di-polyunsaturated phospholipid. The results of these studies will be presented.

Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane

The EphA4 receptor tyrosine kinase is well-known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on Förster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensor can produce a robust and specific FRET response upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRET responses for the non-raft targeting biosensor than for the raft targeting biosensor, suggesting that stronger EphA4 activation may occur in non-raft regions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensor could serve as a powerful tool to visualize and investigate EphA4 activation and signaling in specific subcellular compartments of single live cells.

Hypoxia-mediated alteration in cholesterol oxidation and raft dynamics regulates BDNF signalling and neurodegeneration in hippocampus.

Brain-derived neurotrophic factor (BDNF) which is primarily associated with neuronal survivability, differentiation and synaptic plasticity has been reported to mediate neurodegeneration in hypoxia through its p75 Neurotrophin receptors (p75NTR). The molecular events promoting BDNF-mediated pro-death signalling in hypoxia, however, still remain an enigma. This study attempts towards deciphering the signalling cascades involved in alteration of BDNF isoforms and its cognate receptor subtypes leading to neurodegeneration in hypoxia. Adult Sprague-Dawley rats were exposed to global hypobaric hypoxia simulating an altitude of 7620m at standard temperature and humidity. Chronic hypoxic exposure for 7days resulted in higher expression of pro-BDNF and alteration in N-linked glycosylation in hippocampus along with increased expression of endoplasmic reticulum stress markers viz., glucose-regulated protein (Grp78), calnexin and changes in the endoplasmic reticulum morphology. Our findings reveal enriched expression of p75NTR in lipid rafts and higher expression of tyrosine receptor kinase β (Trkβ) in non-raft regions following hypoxic exposure. Further investigations on membrane properties revealed decline in membrane fluidity along with increased cholesterol oxidation resulting in reduced translocation of Trkβ from non-raft to raft regions. Supplementation of vitamin E during hypoxic exposure on the other hand reduced cholesterol oxidation and increased translocation of Trkβ from non-raft to raft regions and promoted neuronal survival. Hence, our findings suggest a novel mechanism of cholesterol oxidation-induced alteration in raft dynamics which is promotes p75 receptor-mediated death signalling in hippocampal neurons during chronic hypoxia.

Transience of antidepressant‐induced Gαs signaling potentiation revealed by high‐throughput real‐time cAMP accumulation

Gαs is found at high concentrations in cholesterol rich membrane microdomains known as lipid rafts. These domains suppress coupling between Gαs and effectors such as adenylyl cyclase (AC), leading to decreased cAMP. Antidepressant drugs with diverse mechanisms of action have reliably shown the ability to promote the translocation of Gαs out of lipid rafts in cellular and animal models, leading to increased cAMP, pCREB and BDNF. In cells from depressed subjects Gαs is found at higher concentrations in lipid rafts. Importantly, this difference is eliminated in individuals who respond positively to antidepressant drug treatment, while in non‐responders the increased raft localization of Gαs is maintained. This differential localization may allow Gαs translocation out of lipid rafts to serve as a novel biomarker to screen for effective antidepressants.Although antidepressant drugs have been shown to gradually accumulate in lipid rafts, the relevant binding site, or sites, remain unknown. Notably, the S‐stereoisomer of the selective serotonin reuptake inhibitor citalopram binds to these raft regions, even in cells lacking the serotonin transporter, while the non‐antidepressant R‐stereoisomer does not. This suggests a novel, stereospecific protein target for antidepressant drugs may exist within lipid rafts. Little is known about the reversibility of this process, or whether antidepressant drug withdrawal itself produces unique signaling effects.In this study we use a combination of techniques including live cell cAMP accumulation analysis, fluorescence recovery after photobleaching (FRAP) of GFP tagged Gαs, and detection of antidepressant drug concentration via mass spectrometry. This allows determination of both the effects of extended antidepressant treatment on Gαs localization and signaling, as well as the effect of antidepressant withdrawal. Agents that increase cAMP concentrations, such as forskolin, produce greater effects following antidepressant treatment due to Gαs translocation out of lipid rafts and into closer association with AC. Gαs mobility within the membrane is also inhibited following antidepressant treatment due to formation of this Gαs/AC complex, and this reduction in mobility is observed as an increased FRAP half‐time with GFP tagged Gαs. The localization of the antidepressant drugs themselves are also quantified via mass spectrometry following separation of raft vs. non‐raft membrane regions.Analyzing these effects both during antidepressant treatment and following removal of the drug provides insight not only into the ways these drugs regulate cellular processes, but also how the cell responds to their withdrawal. It appears that antidepressant drug removal not only reverses the effects seen with extended treatment, but can also produce a lasting suppression of these processes. This work provides the next steps towards determining a binding site for antidepressants within lipid rafts, and will lead to the development of a more realistic screen for antidepressant drug development.Support or Funding InformationFunding: NIH R01AT009169; VA BX001149This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Membrane cholesterol depletion as a trigger of Nav1.9 channel-mediated inflammatory pain.

Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage-gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol-rich lipid rafts to cholesterol-poor non-raft regions of the membrane. Low-cholesterol environment enhances voltage-dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.

Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells.

Previous studies indicate that prostaglandin EP2 receptors selectively couple to AC2 in non-lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP-dependent responses. The goal of the present study was to identify the cellular microdomains where EP2 receptors stimulate cAMP production. FRET-based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2-camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2-MyrPalm and Epac2-CAAX were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane respectively. Epac2-NLS was used to monitor responses at the nucleus. Activation of AC with forskolin or β2 -adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 receptors with butaprost produced cAMP responses that were most readily detected by the non-raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains. EP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while β2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.

Sphingomyelins and ceramides with VLCPUFAs are excluded from low-density raft-like domains in differentiating spermatogenic cells

Rat spermatogenic cells contain sphingomyelins (SMs) and ceramides (Cers) with very long-chain PUFAs (VLCPUFAs) in nonhydroxylated (n-V) and 2-hydroxylated (h-V) forms. How these atypical species distribute among membrane fractions during differentiation was investigated here using a detergent-free procedure to isolate a small light raft-like low-density fraction and a large heavy fraction, mostly derived from the plasma membrane of spermatocytes, round spermatids, and late spermatids. The light fraction contained cholesterol, glycerophospholipids (GPLs), and SM with the same saturated fatty acids in all three stages. In the heavy fraction, as PUFA increased in the GPL and VLCPUFA in SM from spermatocytes to spermatids, the concentration of cholesterol was also augmented. The heavy fraction had mostly n-V SM in spermatocytes, but accumulated h-V SM and h-V Cer in spermatids. A fraction containing intracellular membranes had less SM and more Cer than the latter, but in both fractions SM and Cer species with h-V increased over species with n-V with differentiation. This accretion of h-V was consistent with the differentiation-dependent expression of fatty acid 2-hydroxylase (Fa2h), as it increased significantly from spermatocytes to spermatids. The non-raft region of the plasma membrane is thus the main target of the dynamic lipid synthesis and remodeling that is involved in germ cell differentiation.

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss.

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β.

Abstract 2457: Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region

Abstract K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. Kras is frequently mutated in cancer. K-Ras4B modulates downstream signaling at different lipid microdomains. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. HVR farnesylation plays an important role in Ras signaling, but the structural mechanisms and consequences of farnesyl-induced membrane binding are poorly understood and the basis for Ras localization to certain parts of plasma membrane is unknown. Application of confocal microscopy, surface plasmon resonance, and molecular dynamics (MD) simulations revealed that K-Ras4B is distributed between rigid lipid rafts and disordered non-raft regions of the plasma membrane. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains containing unsaturated phospholipids, while the rigid microdomains composed of tightly packed saturated lipids restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion towards the cell interior allows oligomerization of K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids, but the farnesyl's membrane orientation. Phosphorylation of Ser181 prohibits spontaneous farnesyl membrane insertion. We speculate that if the farnesyl group is not inserted into the membrane, it may serve in cooperative binding of other K-Ras4B molecules, thus leading to dimerization. In this case, the protein binds the membrane using electrostatic interaction between its positively charged HVR and the negatively charged membrane phospholipids. In the case of disordered phospholipid domains, the farnesyl group burial in the phospholipids tends to hinder its interactions. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting to microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. Elucidating the mechanism for K-Ras4B localization at different membrane microdomains is important for in-depth understanding of Ras-effector interactions mediating signaling pathways. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Sherwin J. Abraham, Tanmay S. Chavan, Ben Hitchinson, Lyuba Khavrutskii, Nadya I. Tarasova, Ruth Nussinov, Vadim Gaponenko. Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2015-2457