Differential sensitivity to detergents of actin cytoskeleton from nerve endings

Abstract

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.

Sphingolipids and lipid rafts: Novel concepts and methods of analysis

“…Using cytochalasin D and immunoaffinity isolation of Mg 2+- stabilized DRMs, a recent study found that two archetypical GPI-anchored raft proteins, Thy-1 and PrP, are distributed to actin-dependent and independent DRMs, respectively, suggesting that a combination of methods interfering with the cytoskeleton is useful to differentiate specific lipid rafts (Chen et al, 2009). As standard operating procedure (SOP) it is recommended to use two methods to isolate DRMs (1% Triton X-100 at 4 °C and 1% Brij98 at 37 °C) or the detergent-free carbonate method should be combined with cholesterol depletion using pre-incubation with MβCD and F-actin depolymerization (latrunculin A or cytochalasin D), followed by immunoaffinity isolation of DRMs to determine lipid and protein composition in different lipid raft fractions (Cubi et al, 2013). Raft fractions should be tested for presence raft or raft-associated proteins such as flotillin (not GPI anchored), alkaline phosphatase (GPI-anchored), caveolin (not GPI anchored, specific for caveolae-type rafts), and transferrin receptor as a non-raft protein.…”

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

“…The crude synaptosomal fractions (P2) were prepared from 4- to 6-week-old Sprague-Dawley rat brains, as described previously [ 45 ], with slight modifications. Animal samples were obtained according to a procedure approved by the Ethics Committee in Animal and Human Experimentation of the Universitat Autònoma de Barcelona (ref.…”

“…Detergent-resistant membranes (DRM) are an experimental model commonly used to study the so-called ‘membrane rafts’, which are cholesterol- and sphingolipid-rich dynamic membrane microdomains that incorporate specific proteins. DRM were extracted as described previously [ 45 ]. To obtain DRM, the medium was removed and the synaptosomal P2 fraction or MDCK cells were rinsed with cold PBS and homogenized at 4°C with sodium buffer containing 1% Triton X-100 by end-over-end mixing.…”

Do ABC transporters regulate plasma membrane organization?

“…One of the common criticisms expressed regarding the purification temperature could be addressed when other less stringent detergents (e.g. Brij98) made it possible to purify DRMs at physiological temperature [20][21][22][23], although showing some notable differences between the methods and the choice of the detergent [24]. Another method emerged, consisting of aggregating these raft lipids with cholera toxin, which binds specifically to GM1 gangliosides, preferentially partitioning in DRMs, and considered as markers of the lipid raft [25].…”