Naslov
Methodology twist in finding original constituents of lipid rafts

Autori
Heffer-Lauc, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
Bridges in Life Sciences US-CEE Regional Networking Meeting IV

Mjesto i datum
Debrecen, Mađarska, 04.04.2009

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
lipid rafts; detergents; gangliosides; GPI-anchored proteins

Sažetak
Lipid rafts are defined as detergent insoluble domains of plasma membranes. Because molecular composition of these domains varies with percentage and chemical properties of detergent used, 1% Triton X-100 is preferable standard. In most studies cholesterol, sphingomyelin, glycolipids and glycosylphosphatidylinositol (GPI) - anchored proteins predominate in lipid ordered membrane phase (rafts) in comparison to lipid disordered phase. Lipid rafts are recognized as association of molecules involved in signal transduction bringing different molecules of signal cascade from both side of plasma membrane in close proximity. The problem of immunohistochemically non-accessible epitopes hidden by different molecular interactions in tissue sections appeared to be solved by using detergents that provide better accessibility to antibodies. However, we have found evidence that detergent itself artificially creates natively non-existing associations, especially if the investigated molecule has a GPI- or ceramide anchor. It is, therefore, particularly challenging to find real participants of a single signal pathway that involves lipid anchored molecules. Gangliosides are the most abundant glycolipids of the mammalian central nervous system. They are widely distributed in gray (gangliosides GD1a, GD1b and GT1b) and white (ganglioside GM1) matter. Cell culture studies showed that gangliosides GD1a and GT1b, beside Nogo receptor, are ligands for myelin associated protein (MAG), involved in inhibition of axonal outgrowth after injury. MAG is “ hidden” behind layers of myelin, on interface between axon and oligodendrocyte, accessible for immunohistochemical analysis by using detergents. On the other hand, fixed tissue sections exposed to detergent, go to excessive rearrangement of gangliosides GD1a and GT1b from gray to white matter, creating artifacts. Native arrangement of MAG and gangliosides in tissue sections can be reconstructed combining confocal microscopy and lipid extraction with tract tracing methods. Also, localization of gangliosides in inaccessible places, such as periaxonal space, could be revealed using detergents after immobilization of gangliosides with primary and secondary antibodies. Each of these methods has its own limitations demanding additional controls. Final result cannot be achieved in one image, but can be deduced from various experiments.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti

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