A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains

Kimberly J. Krager, Mitul Sarkar, Erik C. Twait, Nancy L. Lill, John G. Koland

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

Original languageEnglish (US)
Pages (from-to)2214-2225
Number of pages12
JournalJournal of Lipid Research
Volume53
Issue number10
DOIs
StatePublished - Oct 1 2012

Fingerprint

Membrane Microdomains
Cell membranes
Cell Membrane
Electrons
Lipids
Imaging techniques
Biotin
Green Fluorescent Proteins
Electron microscopy
Electron Microscopy
Streptavidin
Gold
Labeling
Peptides
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
Membranes
Molecules
Avidin
Cell Surface Receptors
Epidermal Growth Factor Receptor

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

Krager, Kimberly J. ; Sarkar, Mitul ; Twait, Erik C. ; Lill, Nancy L. ; Koland, John G. / A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains. In: Journal of Lipid Research. 2012 ; Vol. 53, No. 10. pp. 2214-2225.
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A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains. / Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

In: Journal of Lipid Research, Vol. 53, No. 10, 01.10.2012, p. 2214-2225.

Research output: Contribution to journalArticle

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