Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment

Abstract

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 IIα, 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.

The Great Escape: how phosphatidylinositol 4-kinases and PI4P promote vesicle exit from the Golgi (and drive cancer)

“…In mammals, the PI4KIIα protein (140,141) is constitutively associated with TGN and endosomal membranes (5,57,60,142,(154)(155)(156)(157)(158) via cholesterol-dependent, post-translational palmitoylation (159,160) catalysed by Golgi-resident palmitoyl transferases (161). PI4KIIα is always membrane bound and its activity is very sensitive to alterations in membrane cholesterol levels (4,(161)(162)(163), which are mainly determined via by PI4P-cholesterol transfer proteins such as OSBP (164).…”

The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells

“…A dot-blot method previously described by us was used to detect lipid-raft-enriched membrane fractions [ 26 ]. Equal volume 1 µl samples from each subcellular fraction were dotted onto nitrocellulose membrane and allowed to dry at room temperature before being probed with probed with horse radish peroxidase (HRP)-conjugated cholera toxin B subunit (1:20,000).…”