Plasmalemma lipid domains and their relationship with Nicotinic Acetylcholine Receptor cluster localization

KAMERBEEK C.; BORRONI M.V; PEDICONI M.F.; BARRANTES F.J

Buenos Aires

Workshop; 3rd South American Workshop on new trends in advance fluorescence microscopy techniques and International Gregorio Weber Conference; 2011

In the present work we calibrate an experimental paradigm to study antibody-induced nicotinic acetylcholine receptor (AChR) clusters and the physical state of the underlying cell membrane in living cells. For this purpose di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered and liquid-disordered phases in model membranes was used in combination with labeling of the AChR in CHO-K1/A5, a clonal cell line expressing adult muscle-type AChR. The so-called generalized polarization (?GP?) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with the AChR platforms clearly identified using conventional wide-field fluorescence microscopy. Under control conditions AChR clusters are roughly equally distributed among liquid-ordered and liquid-disordered domains. This distribution changes upon cyclodextrin-mediated cholesterol depletion or by Latrunculin disruption of the actin cytoskeleton. We also extend our studies to the early endocytic stages of AChR internalization induced by ligand binding. Association of AChR clusters with lipid domains with different biophysical properties may have consequences on AChR trafficking processes.