Dataset Information

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

ABSTRACT: Ostreolysin A (OlyA) is an ?15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-?-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

SUBMITTER: Skocaj M

PROVIDER: S-EPMC3963934 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Skočaj Matej M Resnik Nataša N Grundner Maja M Ota Katja K Rojko Nejc N Hodnik Vesna V Anderluh Gregor G Sobota Andrzej A Maček Peter P Veranič Peter P Sepčić Kristina K

PloS one 20140324 3

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholest ...[more]

PMID: 24664106

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Project description:Lateral segregation and the formation of lateral domains are well-known phenomena in ternary lipid bilayers composed of an unsaturated (low gel-to-liquid phase transition temperature (Tm)) phospholipid, a saturated (high-Tm) phospholipid, and cholesterol. The formation of lateral domains has been shown to be influenced by differences in phospholipid acyl chain unsaturation and length. Recently, we also showed that differential interactions of cholesterol with low- and high-Tm phospholipids in the bilayer can facilitate phospholipid segregation. Now, we have investigated phospholipid-cholesterol interactions and their role in lateral segregation in ternary bilayers composed of different unsaturated phosphatidylcholines (PCs) with varying acyl chain lengths, N-palmitoyl-D-erythro-sphingomyelin (PSM), and cholesterol. Using deuterium NMR spectroscopy, we determined how PSM was influenced by the acyl chain composition in surrounding PC environments and correlated this with the affinity of cholestatrienol (a fluorescent cholesterol analog) for PSM in the different PC environments. Results from a combination of time-resolved fluorescence measurements of trans-parinaric acid and Förster resonance energy transfer experiments showed that the relative affinity of cholesterol for phospholipids determined the degree to which the sterol promoted domain formation. From Förster resonance energy transfer, deuterium NMR, and differential scanning calorimetry results, it was clear that cholesterol also influenced both the thermostability of the domains and the degree of order in and outside the PSM-rich domains. The results of this study have shown that the affinity of cholesterol for both low-Tm and high-Tm phospholipids and the effects of low- and high-Tm phospholipids on each other influence both lateral structure and domain properties in complex bilayers. We envision that similar effects also contribute to lateral heterogeneity in even more complex biological membranes.

Dataset Information

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Publications

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Similar Datasets

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