Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop.
PLoS One
; 14(1): e0210704, 2019.
Article
em En
| MEDLINE
| ID: mdl-30673728
ABSTRACT
CONTEXT Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 translocation results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V1-subcomplex from the membrane-integrated V0-subcomplex of vacuolar-type H+-ATPase. OBJECTIVE:
Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging.METHODS:
Three human CD36 (hCD36) mutants were constructed via insertion of a tetracysteine motif at different positions within the extracellular domain. Constructs were lentivirally transduced for subsequent CD36 labeling with fluorescein-arsenical hairpin-binder (FlAsH). Cell imaging was combined with V0/V1 immunostaining and Western blotting.RESULTS:
Transduction of hCD36-wildtype and mutants yielded corresponding proteins in HL-1 cardiomyocytes. Tetracysteine mutant-2 (hCD36-TC2) showed similar fatty acid uptake to wildtype. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V1 co-localization with CD36 upon high-palmitate culturing. Conversely, V0 consistently co-localized with CD36.CONCLUSION:
hCD36-TC2 is a possible candidate for application of biarsenical dyes in live imaging studies pending further investigation. Our data is compatible with V0/V1 disassembly in high-palmitate-treated cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Antígenos CD36
Limite:
Humans
Idioma:
En
Revista:
PLoS One
Ano de publicação:
2019
Tipo de documento:
Article