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Fig. 3. AtPIN and endomembranes in isolated Zea mays root cells. (A-D) Colocalisation of AtPIN (AtPIN-Cy3) with markers for the Golgi network (JIM84-FITC). (A) Optical section of a cell with AtPIN-Cy3, showing the characteristic polar pattern at the cell surface and punctuate intracellular staining. (B) Golgi staining of the same cell with JIM84-FITC. (C) Merged image revealing a partial colocalisation of the two populations (yellow). (D) Statistical analysis of colocalisation events using Metamorph software (see Materials and Methods). In n=73 cells, the modal value of colocalisation events was 10-29%. (E-G) Colocalisation of AtPIN (AtPIN-Cy3) with markers for the pre-vacuolar compartment (mRab-FITC). (E) Optical section of a cell stained with AtPIN-Cy3. (F) Pre-vacuolar-compartment staining with mRab-FITC of the same cell. (G) Merged image, notice the absence of colocalisation between the two populations. (H-F) AtPIN-Cy3-JIM84-FITC double labelling of BFA-treated cells. (H) Optical section of a cell stained with AtPIN-Cy3. Notice the occurrence of BFA-compartments and the decrease in polar staining. (I) Golgi staining of the same cell with JIM84-FITC, showing the characteristic BFA-compartments. (J) Merged picture, the two labelled populations mixed in BFA compartments with PIN proteins more concentrated within the core of the compartments. (K-M) AtPIN-Cy3-mRab-FITC double labelling of BFA-treated cells. (K) Optical section of a cell stained with AtPIN-Cy3. Notice the BFA-induced fluorescent aggregates and the loss of polar labelling. (L) Pre-vacuolar-compartment staining of the same cell with mRab-FITC. No fluorescent aggregates are observed. (M) Merged image, no colocalisation events. Bars, 8 µm.

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