Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain

Arthur, Julian R.; Heinecke, Karie A.; Seyfried, Thomas N.

doi:10.1194/jlr.m012633

Cited by 51 publications

(37 citation statements)

References 49 publications

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“…Filipin is a drug that binds to cholesterol, disrupts caveolae, perturbs the structure of lipid rafts, and interferes with many raftdependent cellular processes but does not remove cholesterol from cellular membranes (23,24). We saw no effect of filipin on EV1 infection at a concentration that, as we previously observed (12), was sufficient to inhibit infection by CVB3 (Fig.…”

Section: Ev1 Infects Polarized Caco-2 Cells By Binding To Vla-2 On Thsupporting

confidence: 48%

Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

Krieger

Kim

Zhang

et al. 2013

J Virol

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c Enteroviruses invade their hosts by crossing the intestinal epithelium. We have examined the mechanism by which echovirus 1 (EV1) enters polarized intestinal epithelial cells (Caco-2). Virus binds to VLA-2 on the apical cell surface and moves rapidly to early endosomes. Using inhibitory drugs, dominant negative mutants, and small interfering RNAs (siRNAs) to block specific endocytic pathways, we found that virus entry requires dynamin GTPase and membrane cholesterol but is independent of both clathrin-and caveolin-mediated endocytosis. Instead, infection requires factors commonly associated with macropinocytosis, including amiloride-sensitive Na ؉ /H ؉ exchange, protein kinase C, and C-terminal-binding protein-1 (CtBP1); furthermore, EV1 accumulates rapidly in intracellular vesicles with dextran, a fluid-phase marker. These results suggest a role for macropinocytosis in the process by which EV1 enters polarized cells to initiate infection.

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Section: Ev1 Infects Polarized Caco-2 Cells By Binding To Vla-2 On Thsupporting

confidence: 48%

Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

Krieger

Kim

Zhang

et al. 2013

J Virol

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“…Filipin was recently reported to under certain conditions also bind to GM1 as well as further membrane lipids [48]. In order to assure specificity of filipin to accurately reflect free cholesterol under the experimental settings used here (Figure S1), SphingoStrips (Echelon Biosciences) and MembraneLipidStrips (Echelon Biosciences) representing 26 different lipids were washed for 5 min in PBS before staining for 30 min with 50 µg/ul filipin III in PBS.…”

Section: Methodsmentioning

confidence: 99%

RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function

et al. 2013

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Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL–receptor levels and/or that overexpression as GFP–tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci.

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“…A polyene antibiotic, filipin, has been used frequently to visualize endogenous cholesterol distribution both by fluorescence microscopy and EM. Filipin has been reported to probe also GM1 (Arthur et al, 2011), but given that 5–7 mol% cholesterol required for filipin visualization in membranes (Behnke et al, 1984) and the scarcity of GM1 this should not be a problem in cell staining. However, membrane deformation caused by filipin-cholesterol complex formation does not indicate in which leaflet of the membrane cholesterol exists.…”

Section: Lipid Asymmetry In the Plasma Membranementioning

confidence: 99%

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

Fujimoto

Parmryd

2017

Front. Cell Dev. Biol.

116

114

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The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

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Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain

Cited by 51 publications

References 49 publications

Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

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