Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop

• Published in: PloS one 2019-01, Vol.14 (1), p.e0210704-e0210704
• Main Authors: Liu, Yilin, Rodriguez-Calvo, Ricardo, Wang, Shujin, Zhu, Xiaoqing, Broers, Jos L V, Glatz, Jan F C, Luiken, Joost J F P, Neumann, Dietbert
• Format: Article
• Language: English
• Subjects: Acidification; Adenosine triphosphatase; Arsenic; Biology and Life Sciences; Blotting, Western; Cardiomyocytes; Cardiomyopathy; CD36 antigen; CD36 Antigens - metabolism; Data processing; Diabetes; Dismantling; Endosomes; Endosomes - metabolism; Fatty acid metabolism; Fatty acids; Fluorescein; Fluorescence; Fluorescence microscopy; G proteins; H+-transporting ATPase; HEK293 Cells; Humans; Imaging; Immunoglobulins; Insulin resistance; Kinases; Labeling; Labelling; Lipids; Localization; Medicine and Health Sciences; Mutants; Myocytes, Cardiac - metabolism; Palmitic acid; Proteins; Research and Analysis Methods; Sarcolemma; Sarcolemma - metabolism; Translocation; Vacuolar Proton-Translocating ATPases - metabolism; Western blotting
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0210704

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. Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging. 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. 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. 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.