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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Streszczenie

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane's propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

Wprowadzenie

The plasma membrane is composed of a lipid bilayer enriched with various membrane proteins, including transmembrane receptors and ion channels. Lipid domains within the membrane have been elucidated through detergent-soluble and insoluble regions identified in detergent-resistant membrane (DRM) fractionation experiments1. The insoluble fractions were characterized by being enriched in cholesterol, tightly packed sphingomyelins and saturated phospholipids, exhibiting higher melting points, in contrast to the soluble fractions that predominantly consist of lower melting temperature and loosely packed unsaturated phospholipids. The tightly pa....

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Protokół

1. Preparation of Methyl-α-CD solution

  1. Add 20 mL of phosphate-buffered saline (PBS) to ~10 g of MαCD powder in a glass bottle. Incubate in a warm water bath (45 °C) to dissolve, stirring occasionally until well dissolved.
    NOTE: The solution may still be cloudy due to insoluble CD species.
  2. Pass solution through a 0.22 µm syringe filter. The solution will become clear.
  3. Use a refractometer to determine the exact concentration of MαCD.
    1. Place 10 µL of sample on the sample area of a refractometer, illuminate it using a white incandescent bulb, and record the solution's refr....

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Wyniki

To demonstrate the observable change in cellular lipid composition after the exchange, we performed HP-TLC on CHO IR cells following brain SM (bSM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exchange (Figure 1). In cases where sphingomyelins like bSM are being used for exchange, an increase in the SM band intensity is apparent, along with a decrease in PC band intensity relative to the untreated control. Conversely, when exchanging phosphatidylcholines like DOPC, the PC band becomes.......

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Dyskusje

Since the conceptualization of the existence of lipid rafts in the cell membrane there have been numerous attempts to visualize them in cells and study lipid and receptor association. Experiments involving microscopy11 in cells used fluorescently tagged biomarkers, usually, proteins and lipids known to associate with rafts, to visually study the localization of ordered lipid domains in the cell12. However, the cell membrane is full of folds13,

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Ujawnienia

The authors declare no conflicts of interest.

Podziękowania

Funding was provided by NIH grant GM 122493. CHO IR cells were a kind gift from Dr Jonathan Whittaker (Case Western Reserve University).

....

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Materiały

NameCompanyCatalog NumberComments
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)Avanti Polar Lipids850335
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)Avanti Polar Lipids850345
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)Avanti Polar Lipids850375
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)Avanti Polar Lipids850355
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)Avanti Polar Lipids850365
1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC)Avanti Polar Lipids850457
Anti-insulin receptor β antibodyCell Signaling TechnologyCST3025
Anti-pYpY1162/1163 Insulin receptor antibodyR&D Systems Inc.AF2507
Anti-rabbit IgG, HRP-linked AntibodyCell Signaling Technology7074
Borosilicate glass test tubes (12 x 75 mm)Thermo Fisher Scientific14-961-26
Brain sphingomyelin (bSM)Avanti Polar Lipids860062
Egg sphingomyelin (eSM)Avanti Polar Lipids860061
Fetal bovine serum (FBS)Corning35-016-CV
G418 disulfate saltSigma AldrichA1720
Gibco Antibiotic-antimycotic solution (100x)Thermo Fisher Scientific15240062
Gibco Dulbecco’s modified eagle medium (DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate)Thermo Fisher Scientific11965092
Gibco ham’s F12 mediaThermo Fisher Scientific11765054
Gibco L-glutamineThermo Fisher Scientific25030032
Gibco MEM Non-Essential Amino Acids Solution (100X)Thermo Fisher Scientific11140050
Gibco phosphate buffered saline (PBS) without calcium and magnesium (0.144 g/L KH2PO4, 9 g/L NaCl, 0.795 g/L Na2- HPO4 (anhydrous))Thermo Fisher Scientific10010023
Gibco Trypsin-EDTA (0.05%), phenol redThermo Fisher Scientific25300054
High performance thin layer chromatography (HP-TLC)MerckHP-TLC Silica Gel 60 plates
Immobilon-P PVDF MembraneMilliporeIPVH00010
MethotrexateSigma Aldrich454126
Methyl-α-cyclodextrin (MαCD)AraChemCDexA076/BR
Pierce ECL Western Blotting SubstrateThermo Fisher Scientific32106
Sodium orthovanadate, ActivatedSigma Aldrich5.08605

Odniesienia

  1. Brown, D. A., London, E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 14, 111-136 (1998).
  2. Brown, D. A. Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology. 21, 430-439 (2006).
  3. Schroeder, R....

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Lipid Exchange AssayLipid RaftsPlasma MembraneMembrane ProteinsCyclodextrinsPhospholipidsTransmembrane Protein ActivityLipid SubstitutionMammalian CellsHP TLCCellular ViabilityOuter LeafletLipid Constitution

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