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

Yilin Liu, Ricardo Rodriguez-Calvo, Shujin Wang, Xiaoqing Zhu, Jos L. V. Broers, Jan F. C. Glatz, Joost J. F. P. Luiken, Dietbert Neumann*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Web of Science)

Abstract

Context

Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 trans location results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V-1-subcomplex from the membrane-integrated V-0-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 V-0/V-1 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 wild type. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V-1 co-localization with CD36 upon high-palmitate culturing. Conversely, V-0 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 V-0/V-1 disassembly in high-palmitate-treated cells.

Original languageEnglish
Article number0210704
Number of pages14
JournalPLOS ONE
Volume14
Issue number1
DOIs
Publication statusPublished - 23 Jan 2019

Keywords

  • RAT CARDIAC MYOCYTES
  • CONTRACTILE DYSFUNCTION
  • SARCOLEMMAL FAT/CD36
  • INSULIN
  • PROTEIN
  • TRANSLOCATION
  • METABOLISM
  • DIET

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