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.
Highlights
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
To facilitate the gold labeling of lipid raft membrane microdomains in EM imaging studies, we developed an Lck-based lipid raft reporter construct designated LckBAP-green fluorescent protein (GFP) (Fig. 1)
The biotin acceptor peptide (BAP) module is efficiently biotinylated by endogenous biotin ligases upon its expression in mammalian cells [15, 26] and confers to proteins an ability to be detected with avidin-based reagents, such as streptavidin-HRP or streptavidin-gold particle conjugates
Summary
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. 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 LckBAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Double-immunogold labeling allows an assessment of the colocalization of distinct signaling molecules as well as their potential localization within membrane microdomains, given the availability of a suitable marker such as a resident lipid raft or caveolar protein [12, 13]. Considering our criteria for a raft marker, the extent to which various raft constituents have been characterized, and our interest in noncaveolar lipid rafts, we identified the Src family protein tyrosine kinase Lck as an ideal candidate and generated an Lck-derived reporter that could be readily labeled with avidin-biotin technology
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