Who's in, who's out? Re‐evaluation of lipid raft residents

Abstract

Lipid rafts, membrane microdomains enriched with (glyco)sphingolipids, cholesterol, and select proteins, act as cellular signalosomes. Various methods have been used to separate lipid rafts from bulk (non‐raft) membranes, but most often, non‐ionic detergent Triton X‐100 has been used in their isolation. However, Triton X‐100 is a reported disruptor of lipid rafts. Histological evidence confirmed raft disruption by Triton X‐100, but remarkably revealed raft stability to treatment with a related polyethylene oxide detergent, Brij O20. We report isolation of detergent‐resistant membranes from mouse brain using Brij O20 and its use to determine the distribution of major mammalian brain gangliosides, GM1, GD1a, GD1b and GT1b. A different distribution of gangliosides—classically used as a raft marker—was discovered using Brij O20 versus Triton X‐100. Immunohistochemistry and imaging mass spectrometry confirm the results. Use of Brij O20 results in a distinctive membrane distribution of gangliosides that is not all lipid raft associated, but depends on the ganglioside structure. This is the first report of a significant proportion of gangliosides outside raft domains. We also determined the distribution of proteins functionally related to neuroplasticity and known to be affected by ganglioside environment, glutamate receptor subunit 2, amyloid precursor protein and neuroplastin and report the lipid raft populations of these proteins in mouse brain tissue. This work will enable more accurate lipid raft analysis with respect to glycosphingolipid and membrane protein composition and lead to improved resolution of lipid–protein interactions within biological membranes.

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts

“…TfR distribution is concentrated in the bulk membrane fractions (75% for both WT and KO mice). These distributions are in concordance with the literature and confirm the successful lipid raft isolation [28][29][30].…”

“…Lipid rafts were isolated as previously described by discontinuous sucrose gradients ultracentrifugation [28,29,64]. Briefly, 70 ± 5 mg of brain cortical tissue was homogenized, the nuclear fraction removed, and the cell membrane pellet obtained by centrifugation (100,000× g at +4 • C, 30 min; ultracentrifuge Beckman Optima XL-80 K, Beckman Coulter, Inc., Brea, CA, USA).…”

“…Western blotting was performed as previously described [21,28] in order to assess the lipid raft and the bulk membrane distribution of proteins of interest. Briefly, equal volumes of isolated lipid raft fractions were loaded onto precast 4-12% Bis Tris gels (Thermo Fisher Scientific, Life Technologies Corporation, Carlsbad, CA, USA) and resolved in MOPS running buffer (Thermo Fisher Scientific, Life Technologies Corporation, Carlsbad, CA, USA).…”

“…Briefly, equal volumes of isolated lipid raft fractions were loaded onto precast 4-12% Bis Tris gels (Thermo Fisher Scientific, Life Technologies Corporation, Carlsbad, CA, USA) and resolved in MOPS running buffer (Thermo Fisher Scientific, Life Technologies Corporation, Carlsbad, CA, USA). Equal loading volumes opposed to equal protein concentrations were used to allow direct comparisons of protein distribution across fractions [28][29][30]. In order to check the amount of protein loaded and to ensure successful transfer of proteins onto the PVDF membrane (Thermo Scientific, Rockford, IL, USA), we used Stain-free technology [65].…”

Neuroplastin in human cognition: review of literature and future perspectives

“…The finding that implies effects of membrane lipid composition on functionality of Np has been recently supported by a study describing specific rearrangements of Np and gangliosides upon chronic stress and aging in the rat hippocampus [31]. Most recently, Np has been shown to preferentially segregate within lipid rafts [32], neighboring with specific synaptic proteins that reside in rafts such as synaptophysin, GABA receptors, glutamate receptors, and PMCA [33]. Exact mechanism of action and signaling cascade triggered by Np is yet to be resolved; however, its intramembrane clustering with molecular partners involved in synaptic organization, neurotransmission, and ion homeostasis makes it at least an additional important factor in myriad of (patho)physiological events occurring on the neuronal cell surface.…”