Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains
We describe here the principles of this procedure and its application in the imaging of plasma membrane inner leaflet lipid rafts. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity
Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification o...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity
Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity
Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha
Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KII&alp...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Fluorescent Assays for Ceramide Synthase Activity
Ceramides are the central lipid metabolite of the sphingolipid family, and exert a potent influence over cell polarity, differentiation, and survival through their biophysical properties and their specific interactions with cell signaling proteins. Literature on the importance of ceramides in physiology and pathological conditions continues to grow, with ceramides having been identified as central effectors in major human pathologies such as diabetes and neurodegenerative conditions. In mammals, ceramide synthesis from a sphingoid base and a variable length fatty acid is catalyzed by a family of six ceramide synthases (CER...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry
Sphingolipids are one the major lipid families in eukaryotes, incorporating a diverse array of structural and signaling lipids such as sphingomyelin and gangliosides. The core lipid component for all complex sphingolipids is ceramide, a diacyl lipid consisting of a variable length fatty acid linked through an amide bond to a long chain base such as sphingosine or dihydrosphingosine. This reaction is catalyzed by a family of six ceramide synthases (CERS1-6), each of which preferentially catalyzes the synthesis of ceramides with different fatty acid chain lengths. Ceramides are themselves potent cellular and physiological si...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Assay for CDP-Diacylglycerol Generation by CDS in Membrane Fractions
CDP-DAG is a liponucleotide formed by the condensation of CTP with the phospholipid phosphatidic acid in a reaction catalyzed by CDP-DAG synthase (CDS). CDP-DAG is required for the synthesis of phosphatidylinositol; the parent molecule whence all seven phosphoinositides including the signaling molecules PI4P, PI(4,5)P2, and PI(3,4,5)P3 are derived. This protocol describes a highly sensitive radiometric assay to detect the generation of CDP-DAG on isolated biological membrane fractions. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Measuring Phosphatidylinositol Generation on Biological Membranes
Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange re...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format
Lipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we desc...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Single-Molecule Imaging of Signal Transduction via GPI-Anchored Receptors
Lipid rafts have been drawing extensive attention as a signaling platform. To investigate molecular interactions in lipid rafts, we often need to observe molecules in the plasma membranes of living cells because chemical fixation and subsequent immunostaining with divalent or multivalent antibodies may change the location of the target molecules. In this chapter, we describe how to examine dynamics of raft-associated glycosylphosphatidylinositol (GPI)-anchored receptors and interactions of the receptors with downstream signaling molecules by single-particle tracking or single-molecule imaging techniques. (Source: Springer ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions
Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651–657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells
Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [3H]Serine
Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Guidelines for the Use of Protein Domains in Acidic Phospholipid Imaging
Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the y...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
