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

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

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ContextUpon 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 Spring Kit of vacuolar-type H+-ATPase.ObjectiveDevelop a CD36 fluorescent labeling technique as initial step towards live cell imaging.

MethodsThree 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.

ResultsTransduction 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.ConclusionhCD36-TC2 is a possible candidate for application of biarsenical dyes Propellers in live imaging studies pending further investigation.

Our data is compatible with V0/V1 disassembly in high-palmitate-treated cells.