Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Abstract

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

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Abbreviations

CalA:

Calyculin A

DF-BSA:

Defatted-bovine serum albumin

FRAP:

Fluorescence recovery after photobleaching

LatB:

Latrunculin B

mβCD:

Methyl-β-cyclodextrin

MLVs:

Multilamellar vesicles

PC:

Phosphatidylcholine

PKC:

Protein kinase C

PMA:

Phorbol 12-myristate 13-acetate

RBC:

Red blood cell

SEM:

Scanning electron microscopy

SM:

Sphingomyelin

SMase:

Sphingomyelinase

theta*:

His-mCherry-theta-D4

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Acknowledgments

We thank Drs. A. Miyawaki, M. Abe and T. Kobayashi at Riken Brain Science Institute (Saitama, Japan) as well as H. Mizuno (KU Leuven, Belgium) for generously supplying the Dronpa-theta-D4 plasmid, Dr. A. De Matteis (Naples, Italy) for mCherry-Rab5 plasmid and P. Gailly for C2C12 myoblasts; P. Henriet, H. Emonard and J. Lorent for help in producing toxin* and liposomes and T. Lac and N. Chevalier for technical support (UCL, Belgium). This work was supported by grants from UCL (FSR), the F.R.S.-FNRS, Salus Sanguinis foundation, ARC, IUAP and the Région Wallonne. DT and MVdC are Research associates at Fonds de la Recherche Scientifique (F.R.S.-FNRS).

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Correspondence to Donatienne Tyteca.

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Carquin, M., Conrard, L., Pollet, H. et al. Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation. Cell. Mol. Life Sci. 72, 4633–4651 (2015). https://doi.org/10.1007/s00018-015-1951-x

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Keywords

  • His-mCherry-theta-D4
  • 125I-toxin
  • Lateral membrane heterogeneity
  • Vital confocal imaging
  • Membrane tension
  • Temperature
  • C2C12 myoblasts
  • BODIPY-sphingomyelin