Fig. 6. Localisation of GFP-Rab21 on Golgi and endosomal membranes by immunogold labelling and electron microscopy. HeLa cells transfected with GFP-Rab21 plasmids using calcium phosphate were fixed and prepared for cryosectioning and immunolabelling with anti-GFP, and anti-EEA1, TfR or Hrs antibodies. (A) Single labelling of GFP-Rab21 in a section across an elongated cell process showing the presence of GFP-Rab21 on the plasma membrane and the periphery of internal vesicles. (B) Peripheral membranes of a swollen endosome labelled with GFP-Rab21. (C,D) Colocalisation of GFP-Rab21 and EEA1 on endosomal membranes. Cryosections were double labelled with anti-GFP (arrowheads) and anti-EEA1 (arrows) antibodies. The images show examples of vesicular and tubular structures labelled with both antibodies. The localisation of Rab21 and EEA1 is most apparent on peripheral membranes of early endosomes (marked EE) close to the plasma membrane (marked PM). In D, both Rab21 and EEA1 are in close proximity to a caveolae invagination on the plasma membrane (marked Cv). (E-I) Examples of the extent of colocalisation of GFP-Rab21 with the two endosomal markers TfR and Hrs. Note in E, that Golgi (marked Go) labelling is unique to Rab21; however, endosomes (marked E) and tubular structures are labelled with both antibodies. (H,I) GFP-Rab21 and Hrs are often colocalised on endosomal structures close to the plasma membrane (marked PM). Note, however, the Hrs-positive, Rab21-negative early endosome (marked EE) in I. Some labelling of the plasma membrane (PM) was also observed with both antibodies.