Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope.
Membrane perturbing effects have been described in neurodegenerative process like Alzheimer's disease and prion disorders. For example, non fibrillar amyloid-beta peptides (Abeta) implicated in Alzheimer's disease may exert its toxicity via membrane perturbation. Membrane organisation can be evaluated by its influence on lateral diffusion of lipids, which itself can be measured by FRAP (fluorescence recovery after photobleaching). We used this technique to study the effects of Abeta on membrane fluidity (Pillot et al., manuscript in preparation). We propose here a simple adaptation of FRAP using standard confocal laser scanning microscopy (CLSM). As a test experiment, we analysed the lateral diffusion of a fluorescent analogue of sphingomyelin and were able to demonstrate its increase upon cholesterol depletion induced by methyl-beta-cyclodextrin (cdx).
Klein
Christophe
C
Service Commun d'Imagerie et Cytometrie, INSERM IFR-58, Institut Biomédical des Cordeliers, 15 Rue de l'Ecole de Médecine, 75006 Paris, France. christophe.klein@ifr58.bhdc.jussieu.fr
Pillot
Thierry
T
Chambaz
Jean
J
Drouet
Beatrice
B
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D001921:Brain / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology*; D002784:Cholesterol / Q000172:deficiency; D003505:Cyclodextrins / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008560:Membrane Fluidity / Q000502:physiology*; D008563:Membrane Lipids / Q000378:metabolism*; D018613:Microscopy, Confocal; D019636:Neurodegenerative Diseases / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D013109:Sphingomyelins; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D008563:Membrane Lipids; C041065:N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan