Redistribution of gangliosides accompanies thermally induced Na+, K+-ATPase activity alternation and submembrane localisation in mouse brain

Puljko, Borna; Stojanović, Mario; Ilić, Katarina; Hrvat, Nikolina Maček; Zovko, Ana; Damjanović, Vladimir; Mlinac-Jerkovic, Kristina; Kalanj-Bognar, Svjetlana

doi:10.1016/j.bbamem.2020.183475

Cited by 4 publications

(8 citation statements)

References 15 publications

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“…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].…”

Section: Content Of Neuroplastin and Pmcas Is Altered In Lipid Rafts ...supporting

confidence: 91%

“…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).…”

Section: Lipid Raft Isolationmentioning

confidence: 99%

“…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].…”

Section: Western Blottingmentioning

confidence: 99%

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

Ilić

Lin

Malci

et al. 2021

IJMS

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The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

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Section: Content Of Neuroplastin and Pmcas Is Altered In Lipid Rafts ...supporting

confidence: 91%

Section: Lipid Raft Isolationmentioning

confidence: 99%

Section: Western Blottingmentioning

confidence: 99%

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

Ilić

Lin

Malci

et al. 2021

IJMS

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“…Following a discontinuous sucrose density centrifugation, one ml of each lipid raft (LR) and bulk membrane (non‐lipid raft; nLR) fraction was pooled and used for ganglioside isolation according to the following protocol as reported previously (Puljko et al., 2021). Samples were subjected to short organic extraction (Schnaar, 1994; Svennerholm & Fredman, 1980) by adding 2.67 volumes of methanol followed by 1.33 volumes of chloroform to LR and nLR fractions.…”

Section: Methodsmentioning

confidence: 99%

“…Following a discontinuous sucrose density centrifugation, one ml of each lipid raft (LR) and bulk membrane (non-lipid raft; nLR) fraction was pooled and used for ganglioside isolation according to the following protocol as reported previously (Puljko et al, 2021).…”

Section: Ganglioside Analysesmentioning

confidence: 99%

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

Mlinac-Jerkovic

Ilić

Zjalić

et al. 2021

Journal of Neurochemistry

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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.

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Methodological Pitfalls of Investigating Lipid Rafts in the Brain: What Are We Still Missing?

Mlinac-Jerkovic,

Kalanj-Bognar,

Heffer

et al. 2024

Biomolecules

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The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative approaches. Lipid rafts are biochemically and biophysically different from the bulk membrane. A specific lipid environment within membrane domains provides a harbor for distinct raftophilic proteins, all of which in concert create a specialized platform orchestrating various cellular processes. Studying lipid rafts has proved to be arduous due to their elusive nature, mobility, and constant dynamic reorganization to meet the cellular needs. Studying neuronal lipid rafts is particularly cumbersome due to the immensely complex regional molecular architecture of the central nervous system. Biochemical fractionation, performed with or without detergents, is still the most widely used method to isolate lipid rafts. However, the differences in solubilization when various detergents are used has exposed a dire need to find more reliable methods to study particular rafts. Biochemical methods need to be complemented with other approaches such as live-cell microscopy, imaging mass spectrometry, and the development of specific non-invasive fluorescent probes to obtain a more complete image of raft dynamics and to study the spatio-temporal expression of rafts in live cells.

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Redistribution of gangliosides accompanies thermally induced Na+, K+-ATPase activity alternation and submembrane localisation in mouse brain

Cited by 4 publications

References 15 publications

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

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

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

Methodological Pitfalls of Investigating Lipid Rafts in the Brain: What Are We Still Missing?

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