Raft-like membranes from the trans-Golgi network and endosomal compartments

Waugh, Mark G.

doi:10.1038/nprot.2013.148

Cited by 18 publications

(23 citation statements)

References 104 publications

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“…Within 90 min, the OlyA-mCherry–labelled membranes reached the juxtanuclear region of the Golgi apparatus. These data indicate the enrichment of cholesterol and SM in the intracellular membrane compartments, and they are in agreement with the very recent findings showing the enrichment of raft-associated cholesterol in endosomal fractions and in the trans -Golgi network [60]. In the study by Bhat et al [28], where intracellular labelling of PlyA2-EGFP was studied for fixed and permeabilised HeLa cells, PlyA2-EGFP co-localisation was only seen with late endosome markers, and there was no co-localisation with early endosome and cis -Golgi markers.…”

Section: Discussionsupporting

confidence: 93%

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

et al. 2014

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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-β-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.

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Section: Discussionsupporting

confidence: 93%

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

et al. 2014

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“…It is possible that the requirement of palmitoylation for c-Met to exit the Golgi is related to trans-Golgi lipid raft structures [52–54]. In the absence of palmitoylation, c-Met may not integrate into these membrane domains, potentially required for Golgi exit.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation regulates the intracellular trafficking and stability of c-Met

et al. 2016

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c-Met is a receptor tyrosine kinase whose activity can promote both mitogenic and motogenic phenotypes involved in tissue development and cancer progression. Herein, we report the first evidence that c-Met is palmitoylated and that palmitoylation facilitates its trafficking and stability. Inhibition of palmitoylation reduced the expression of c-Met in multiple cancer cell lines post-transcriptionally. Using surface biotinylation, confocal microscopy, and metabolic labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized c-Met and causes accumulation at the Golgi. Acyl-biotin exchange and click chemistry-based palmitate labeling indicated the c-Met β-chain is palmitoylated, and site-directed mutagenesis revealed two likely cysteine palmitoylation sites. Moreover, by monitoring palmitoylation kinetics during the biosynthesis and trafficking of c-Met, we revealed that stable palmitoylation occurs in the endoplasmic reticulum prior to cleavage of the 170 kDa c-Met precursor to the mature 140 kDa form. Our data suggest palmitoylation is required for egress from the Golgi for transport to the plasma membrane. These findings introduce palmitoylation as a critical modification of c-Met, providing a novel therapeutic target for c-Met-driven cancers.

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“…The use of this assay to measure membrane cholesterol mass has been previously validated (Bate et al, 2008;Minogue et al, 2010;Nicholson and Ferreira, 2009). Ganglioside glycosphingolipids were detected by dot blotting of membrane fractions (Ilangumaran et al, 1996) and probing with HRP-conjugated cholera toxin B subunit as described previously (Ilangumaran et al, 1996;Mazzone et al, 2006;Waugh, 2013;Waugh et al, 2011a;Waugh et al, 2011b). Membranebound cholera toxin was visualized by incubation with chemiluminescence detection reagents and spots were quantified as described for the analysis of immunoblotting data (Waugh, 2013).…”

Section: Measurements Of Membrane Lipid Levelsmentioning

confidence: 99%

Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

2015

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Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIalpha, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.

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Raft-like membranes from the trans-Golgi network and endosomal compartments

Cited by 18 publications

References 104 publications

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

Palmitoylation regulates the intracellular trafficking and stability of c-Met

Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

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