Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues

Grassi, Sara; Giussani, Paola; Mauri, Laura; Prioni, Simona; Prinetti, Alessandro

doi:10.1007/978-1-0716-0814-2_1

Cited by 4 publications

(9 citation statements)

References 153 publications

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“…As detailed in Chapter 2, we confirmed the presence of IDE in Triton-X100-resistant membrane domains, with only one antecedent in the literature in neurons (Bulloj et al, 2008). Some studies suggest that the different composition of lipid rafts obtained by using different detergents reflects the existence of biochemically distinct lipid membrane domains within the plasma membrane of the same cell (Grassi et al, 2021). Therefore, we explored other methods for lipid rafts isolation.…”

Section: Ide Molecular Evolution and Subcellular Localizationsupporting

confidence: 69%

“…Triton-X100-resistant membrane domains were isolated following the method described by Grassi and coworkers (Grassi et al, 2021), with minor modifications. The protein/detergent ratio turned out to be a critical parameter for the correct Triton-X100-resistant membranes isolation since an excess of detergent solubilizes even membrane domains with a high lateral order.…”

Section: Triton-x100mentioning

confidence: 99%

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The insulin-degrading enzyme: from molecular evolution and subcellular localization to new roles in microglial physiology

Gómez¹

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under our supervision, within the doctoral programme 1 DOCTORADO EN INVESTIGACIÓN BIOMÉDICA, under the modality of INTERNATIONAL DOCTOR MENTION, after taking into account the following considerations 2 : The candidate has fulfilled all the requirements and the level of training necessary to aspire to the Doctor's Degree with International Mention by the University of Valladolid, by performing an original research project. She has significantly contributed to the design of the scientific questions to be solved, framed at the interface of several disciplines, has designed, executed and analysed the experiments, and has performed data presentations in different formats (writing published work and presenting at scientific meetings), contributing to the dissemination of her results to the scientific community.

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Section: Ide Molecular Evolution and Subcellular Localizationsupporting

confidence: 69%

Section: Triton-x100mentioning

confidence: 99%

The insulin-degrading enzyme: from molecular evolution and subcellular localization to new roles in microglial physiology

Gómez¹

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“…A typical protocol of an LR isolation [59] would include a homogenization step, the choice of which depends on whether cultured cells or tissue are used. That could be achieved by mechanically disrupting tissue in a homogenizer, sonication, passing the sample through a needle, etc.…”

Section: Methodology Behind Lipid Rafts: Biochemical Methodsmentioning

confidence: 99%

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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“…Triton-X100: The protein/detergent ratio is a critical parameter for correct Triton-X100-resistant membranes isolation [ 40 ]. This parameter was empirically determined for the BV-2 cell line as 400 μg proteins/μL Triton-X100.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells

Corraliza-Gomez

Lillo

Cózar‐Castellano

et al. 2022

Cells

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The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

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Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues

Cited by 4 publications

References 153 publications

The insulin-degrading enzyme: from molecular evolution and subcellular localization to new roles in microglial physiology

The insulin-degrading enzyme: from molecular evolution and subcellular localization to new roles in microglial physiology

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

Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells

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