Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

TLDR

Membrane microdomains enriched in GPI‑anchored proteins, glycosphingolipids, and cholesterol are implicated in trafficking and signaling, yet have not been visualized by light or electron microscopy. The study aimed to detect GPI‑anchored protein microdomains in intact cell membranes using imaging FRET. Imaging FRET, a digital microscopy technique extending fluorescence resolution to <100 Å, was employed to measure energy transfer between donor and acceptor antibodies against 5′ nucleotidase on MDCK apical membranes. Significant energy transfer between donor and acceptor antibodies against 5′ nucleotidase was observed, with efficiency correlating with acceptor density and matching two‑dimensional random‑distribution predictions, indicating that most 5′ NT molecules are randomly distributed rather than constitutively clustered, thereby constraining lipid‑raft models.

Abstract

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&amp;lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.