Application of NBD-Labeled Lipids in Membrane and Cell Biology

Haldar, Sourav; Chattopadhyay, Amitabha

doi:10.1007/4243_2012_43

Cited by 37 publications

(48 citation statements)

References 63 publications

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“…Fractions corresponding to the monomeric volume by guest, on May 10, 2018 www.jlr.org Downloaded from to a red shift in the excitation wavelength is termed red edge excitation shift (REES). The NBD fl uorophore is highly sensitive to its environment, and the REES of NBDlabeled lipids has been used to monitor a variety of biological and biophysical processes in cellular and/or artifi cial lipid membranes ( 16,17 ). Therefore, we next examined whether the NBD-Sph spectral changes induced by SphK1 or SphK2 activity were accompanied by a corresponding shift in fl uorescence emission.…”

Section: Protein Expression and Purifi Cationmentioning

confidence: 99%

A real-time high-throughput fluorescence assay for sphingosine kinases

Lima

Milstien

Spiegel

2014

Journal of Lipid Research

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Currently, there are a wide variety of assays that are useful for the characterization of SphK activity in cell extracts or of the recombinant enzymes. However, most lack the capacity to follow SphK activity in real time, precluding the convenient characterization of SphK mutants, competitors, or inhibitors in a high-throughput or "one-pot" approach. The most widely cited method was originally developed by our group and utilizes [ ␥ -32 P]ATP and D-erythroSph as substrates, and it requires differential organic extraction, TLC separation, and quantifi cation of the radiolabeled S1P product ( 4 ). This method is highly accurate but is labor intensive and not high throughput. More specialized variations of the radiolabeling assay have been developed. For example, those that utilize [ 3 H]Sph as a substrate ( 5 ) or biotinylated-Sph with [ ␥ -32 P]ATP and capture of the radiolabeled phosphorylated product with streptavidin ( 6 ), or a method that conveniently avoids organic extraction and TLC separation and quantifi es [ 32 P] S1P by scintillation proximity counting ( 7 ). However, as with the classic [ ␥ -32 P]ATP method, these are not useful for monitoring reactions in real time and also have the drawback of requiring radioactive materials.Fluorophore-labeled Sphs have also been extensively used as substrates for examining SphK activity ( 8, 9 ) and have been shown to be useful surrogates for Sph in the screening of inhibitors. However, these are not highthroughput assays because in order to measure enzymatic activity the phosphorylated products must be isolated by organic fractionation ( 10 ) or processed with specialized equipment by capillary electrophoresis ( 9 ) or HPLC ( 11 ).A bioluminescence assay has been developed to measure ADP produced when the ␥ -phosphate from ATP is transferred to Sph ( 12 ). However, this is an indirect measure of SphK activity through ATP hydrolysis rates. Among the most accurate methods are those that fractionate and Sphingosine kinases (SphK), consisting of two isoforms, SphK1 and SphK2, catalyze the formation of sphingosine-1-phosphate (S1P) by transferring the ␥ -phosphate from ATP to the primary hydroxyl of sphingosine (Sph). S1P is a potent bioactive lipid that can regulate many complex cellular processes, including growth, survival, migration, angiogenesis, and infl ammation, to name a few ( 1-3 ). Accordingly, abnormal regulation of SphKs that leads to elevated levels of S1P has been implicated in the etiology of cancer, as well as autoimmune and cardiovascular diseases ( 3 ). Hence, pharmacologically modulating SphK activity has important therapeutic potential, and technologies that can assist in the discovery of SphK inhibitors are critical tools for their development.

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Section: Protein Expression and Purifi Cationmentioning

confidence: 99%

A real-time high-throughput fluorescence assay for sphingosine kinases

Lima

Milstien

Spiegel

2014

Journal of Lipid Research

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“…Evidence for specific organization of cholesterol in membranes at low concentrations was obtained from three independent studies carried out by various groups ( [7,15,16,28]; reviewed in [10] Fig. 2.…”

Section: A Unique Transbilayer Organization Of Membrane Cholesterol Amentioning

confidence: 99%

Cholesterol: An evergreen molecule in biology

Kumar

Chattopadhyay

2016

BSI

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Abstract. Cholesterol, an essential component of higher eukaryotic membranes, was discovered more than two centuries ago. The development and progress of cholesterol research in the last 200 years has been truly fascinating, with elements of surprise, serendipity and intrigue. In this review, we trace this journey the way we see it, and follow it up with the role of membrane cholesterol in crucial areas of contemporary research (transbilayer domains, regulation of GPCR function and role in the entry of intracellular pathogens into host cells), with considerable footprint from our work. We believe that cholesterol will continue to surprise and fascinate future researchers, thereby justifying its evergreen nature. Keywords: Membrane cholesterol, transbilayer dimer, G protein-coupled receptor, pathogen entry Abbreviations 7-DHC7-dehydrocholesterol 7-DHCR 3β-hydroxy-steroid-7 -reductase 25-NBD-cholesterol 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol CRAC cholesterol recognition/interaction amino acid consensus DHE dehydroergosterol ( 5,7,9(11)22 -ergostatetraen-3β-ol) DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine FRAP fluorescence recovery after photobleaching GPCR G protein-coupled receptor NBD 7-nitrobenz-2-oxa-1,3-diazol-4-yl REES red edge excitation shift SLOS Smith-Lemli-Opitz syndrome

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“…From all commercially available amine-reactive fl uorophores, NBDF is the most reactive one facilitating effi cient and simple labeling (see Materials and Methods). Although the quantum yield and photostability of NBD compared with BODIPY are much less, they are suffi ciently sensitive and stable for FRET assays ( 20,69 ). Furthermore, in contrast to BODIPY and other fl uorophores, the NBD group is readily reducible by membrane-impermeable dithionite allowing us to test whether liposomes are mono-or multilamellar.…”

Section: Fluorescent Membrane-spanning Bipolar Tetraether Lipidsmentioning

confidence: 99%