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

doi:10.1371/journal.pone.0210704

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 journal › Article › Academic › peer-review

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 language	English
Article number	0210704
Number of pages	14
Journal	PLOS ONE
Volume	14
Issue number	1
DOIs	https://doi.org/10.1371/journal.pone.0210704
Publication status	Published - 23 Jan 2019

Keywords

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

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10.1371/journal.pone.0210704Licence: CC BY

Cite this

@article{b8e7c49fabb441ee8b045cf8e87552c3,

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

abstract = "ContextUpon 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.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 V-0/V-1 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 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.ConclusionhCD36-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.",

keywords = "RAT CARDIAC MYOCYTES, CONTRACTILE DYSFUNCTION, SARCOLEMMAL FAT/CD36, INSULIN, PROTEIN, TRANSLOCATION, METABOLISM, DIET",

author = "Yilin Liu and Ricardo Rodriguez-Calvo and Shujin Wang and Xiaoqing Zhu and Broers, {Jos L. V.} and Glatz, {Jan F. C.} and Luiken, {Joost J. F. P.} and Dietbert Neumann",

note = "Funding Information: Y.L. and X.Z. received support from the Chinese Scholarship Council. D.N. is recipient of a Vidi Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW grant no. 864.10.007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: {\textcopyright} 2019 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

year = "2019",

month = jan,

day = "23",

doi = "10.1371/journal.pone.0210704",

language = "English",

volume = "14",

journal = "PLOS ONE",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "1",

}

TY - JOUR

T1 - Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop

AU - Liu, Yilin

AU - Rodriguez-Calvo, Ricardo

AU - Wang, Shujin

AU - Zhu, Xiaoqing

AU - Broers, Jos L. V.

AU - Glatz, Jan F. C.

AU - Luiken, Joost J. F. P.

AU - Neumann, Dietbert

N1 - Funding Information: Y.L. and X.Z. received support from the Chinese Scholarship Council. D.N. is recipient of a Vidi Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW grant no. 864.10.007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2019 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2019/1/23

Y1 - 2019/1/23

N2 - ContextUpon 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.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 V-0/V-1 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 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.ConclusionhCD36-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.

AB - ContextUpon 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.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 V-0/V-1 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 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.ConclusionhCD36-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.

KW - RAT CARDIAC MYOCYTES

KW - CONTRACTILE DYSFUNCTION

KW - SARCOLEMMAL FAT/CD36

KW - INSULIN

KW - PROTEIN

KW - TRANSLOCATION

KW - METABOLISM

KW - DIET

U2 - 10.1371/journal.pone.0210704

DO - 10.1371/journal.pone.0210704

M3 - Article

C2 - 30673728

SN - 1932-6203

VL - 14

JO - PLOS ONE

JF - PLOS ONE

IS - 1

M1 - 0210704

ER -