Antibody-induced acetylcholine receptor clusters inhabit liquid-ordered and liquid-disordered domains

Authors
Kamerbeek, Constanza Belén; Borroni, Maria Virginia; Pediconi, Maria Filomena; Sato, Satoshi B.; Kobayashi, Toshihide; Barrantes, Francisco Jose
Publication Year
2013
Language
English
Format
article
Status
Published version
Description
The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (á-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity
Fil: Kamerbeek, Constanza Belén. Consejo Nacional de Investigaciones Cienti­ficas y Tecnicas. Centro Cientifico Tecnologico Bahi­a Blanca. Instituto de Investigaciones Bioquimicas Bahi­a Blanca (i); Argentina
Fil: Borroni, Maria Virginia. Consejo Nacional de Investigaciones Cienti­ficas y Tecnicas. Centro Cientifico Tecnologico Bahi­a Blanca. Instituto de Investigaciones Bioquimicas Bahi­a Blanca (i); Argentina
Fil: Pediconi, Maria Filomena. Consejo Nacional de Investigaciones Cienti­ficas y Tecnicas. Centro Cientifico Tecnologico Bahi­a Blanca. Instituto de Investigaciones Bioquimicas Bahi­a Blanca (i); Argentina
Fil: Sato, Satoshi B.. Lipid Biology Laboratory; Japón
Fil: Kobayashi, Toshihide. Lipid Biology Laboratory; Japón
Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Cienti­ficas y Tecnicas. Centro Cientifico Tecnologico Bahi­a Blanca. Instituto de Investigaciones Bioquimicas Bahi­a Blanca (i); Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Biomedicas; Argentina
Subject
Nicotinic receptor
lipid domains
di-4-ANEPPDHQ
generalized polarization
Biofísica
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/5260