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The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

Abstract: Epidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22∶6Δ4,7,10,13,16,19), a chemoprotective long-chain n-3 polyunsaturated fatty acid suppresses EGFR signaling. However, the mechanism underlying this phenotype remains unclear. Therefore, we used super-resolution microscopy techniques to investigate the mechanistic link between EGFR function and DHA-induced alterations to plasma membrane nanodomains. Using isogenic in vitro (YAMC and IMCE mouse colonic cell lines) and in vivo (Drosophila, wild type and Fat-1 mice) models, cellular DHA enrichment via therapeutic nanoparticle delivery, endogenous synthesis, or dietary supplementation reduced EGFR-mediated cell proliferation and downstream Ras/ERK signaling. Phospholipid incorporation of DHA reduced membrane rigidity and the size of EGFR nanoclusters. Similarly, pharmacological reduction of plasma membrane phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2) or cholesterol was associated with a decrease in EGFR nanocluster size. Furthermore, in DHA-treated cells only the addition of cholesterol, unlike PA or PIP2, restored EGFR nanoscale clustering. These findings reveal that DHA reduces EGFR signaling in part by reshaping EGFR proteolipid nanodomains, supporting the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane organization, to reduce EGFR signaling and cancer risk.

The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (Lo) phase typical of the raft structure, namely “cholesterol-rich Lo phase”, with a phase fully missing in Cav-1 KO that we named “caveolin-induced Lo phase”. Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a “caveolin-induced Lo phase”, as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources.

Membrane organization and fluidity research continues to expand and the understanding of membrane dynamics continues to be refined. Within this field of study, laurdan remains among the most popular, versatile, and established fluorescence probes. Fluorimetry and multiphoton microscopy techniques are standards for measuring laurdan fluorescence and continue to be refined. However, complications have arisen due to an amended membrane model, revised terms used for describing membrane phases, and wide variation in the selection of laurdan generalized polarization equation values. Here, in the context of the history and chemical properties of laurdan, discrepancies are highlighted and important recommendations are made to promote uniformity and ongoing progress. [ABSTRACT FROM AUTHOR]

Abstract: Based on the results of research conducted over last two decades, lysophospholipids (LPLs) were observed to be not only structural components of cellular membranes but also biologically active molecules influencing a broad variety of processes such as carcinogenesis, neurogenesis, immunity, vascular development or regulation of metabolic diseases. With a growing interest in the involvement of extracellular lysophospholipids in both normal physiology and pathology, it has become evident that those small molecules may have therapeutic potential. While lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been studied in detail, other LPLs such as lysophosphatidylglycerol (LPG), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE) or even lysophosphatidylcholine (LPC) have not been elucidated to such a high degree. Although information concerning the latter LPLs is sparse as compared to LPA and S1P, within the last couple of years much progress has been made. Recently published data suggest that these compounds may regulate fundamental cellular activities by modulating multiple molecular targets, e.g. by binding to specific receptors and/or altering the structure and fluidity of lipid rafts. Therefore, the present review is devoted to novel bioactive glycerol-based lysophospholipids and recent findings concerning their functions and possible signaling pathways regulating physiological and pathological processes. [Copyright &y& Elsevier]

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (Lo) phase typical of the raft structure, namely "cholesterol-rich Lo phase", with a phase fully missing in Cav-1 KO that we named "caveolin-induced Lo phase". Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a "caveolin-induced Lo phase", as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources. [ABSTRACT FROM AUTHOR]