Figure 5. Ultrastructural localization of GFP–GPI and GFP-Cdc42 on plasma membrane.

A–G) BHK cells were transfected (A–E) or co-transfected (F–G) with GFP–GPI and/or GFP-Cdc42 as indicated. Cells were pulsed for 2 min with 5 nm anti-GFP-gold antibodies (E–G) prior to making membrane sheets. Membrane sheets were subsequently labelled with 5-nm anti-GFP-gold antibodies (A–E) or 2-nm anti-GFP-gold (F–G). GFP-Cdc42 is distributed over the entire surface but is concentrated in specific areas (arrowheads) as shown at higher magnification in (B) and (C). Note the elongated morphology of the labelled structures (B and C) and how gold particles can be seen to follow a membrane surface in places (e.g. B). In cells expressing higher levels of GFP-Cdc42, similarly sized clusters are still evident (D). Note GFP–GPI label is present over the entire surface (E) with discrete clusters evident in some areas of the cell surface (E, inset). Double labelling of co-transfected cells shows clusters containing both GFP–GPI (large arrowheads) and GFP-Cdc42 (small arrowheads; F and G). Scale bars, 100 nm. H) FRαTb cells were transfected with GFP-Cdc42 and labelled with PLR to mark FR-GPIs on cell surface. Cells were imaged live under dual colour TIRF illumination. Images were acquired at 1.5-s time interval. Montage (taken from Video S1) shows regions in GFP-Cdc42 and PLR image that co-localize with each other. Scale bar, 2 μm.