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Recognition of the importance of lipid signaling in cellular function has led to rapid progress in the technology of lipid analysis. Measurements of lipid species changes are central to defining the networks of cell signaling (e.g., receptor activation by hormones or drugs) and lipids are involved in many biochemical and pathological processes. During the last several years our laboratory has focused...
Mammalian cell lipid analyses using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labeling, permit unparalleled access to membrane phospholipid molecular species compositions and turnover. Lipidomic data from isolable compartments of lipid second messenger generation, such as membrane-free nuclei, can provide dynamic insights into the topology of phospholipid...
The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily...
Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities...
Recent discoveries that provide a link between inositol phosphate (IP) signaling and fundamental cellular processes evoke many exciting new hypotheses about IP function, and underscore the importance of understanding how IP synthesis is regulated. Central to studies of IP metabolism is the essential development of efficient, fast, and reproducible methods for quantitative analysis of IPs in systems...
In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light...
Phospholipid asymmetry is a fundamental feature of the plasma membrane of most eukaryotic cells and its regulation is linked to diverse physiological processes such as apoptosis and blood clotting [P. Williamson, R.A. Schlegel, Biochim. Biophys. Acta 1585 (2002) 53–63; R.F. Zwaal, A.J. Schroit, Blood 89 (1997) 1121–1132]. In addition, the phospholipid translocases (flippases) that are thought to establish...
There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring...
Surface sensitive optical techniques based on surface plasmon resonance have become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces. We use surface plasmon resonance spectroscopy (SPS) in combination with surface plasmon enhanced fluorescence spectroscopy (SPFS) for the characterization of interaction processes associated with biomembranes. The...
A primary goal of cell biology is to uncover the mechanisms of cellular processes. A detailed structural understanding of the organelles and subcellular structures involved in these processes has often formed the foundation for the elucidation of their function. Electron tomography is a powerful technique for characterizing subcellular architecture and structural details in three dimensions. Electron...
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