Fig. 4. AtPIN and cytoskeleton in isolated Zea mays root cells. (A-D) Co-immunostaining of AtPIN with microtubules. AtPIN-Cy3-tubulin-FITC double labelling of cells in (A) interphase, (B) metaphase, (C) late anaphase, (D) telophase. Chromosomes are stained with Hoechst 33342 (blue). PIN proteins (red) are inserted within the newly formed plasma membrane at the cell plate. Notice the presence of PIN-labelled intracellular structures on each side of the cell plate. (E) A cell plate in a cell treated with BFA. Notice the strong labelling of the plasma membrane and the symmetrical repartition of BFA-aggregates on each side of the cell plate. (F-H) Effect of latrunculin-B on the colocalisation of AtPIN and the Golgi marker JIM84. (F) JIM84-FITC (green). Golgi stacks tend to form small aggregates in absence of actin whereas the plasma membrane remains labelled. (G) AtPIN-CY3 (red). A strong polar labelling of the plasma membrane at the cell base is still observed in absence of actin. However, intracellular compartments form labelled aggregates. (H) Merged image, AtPIN and JIM84 labelled membranes are often juxtaposed in these latrunculin-B-induced aggregates, PIN proteins being generally in the core of these aggregates. (I-K) Effect of BFA on latrunculin-B-induced aggregates. Maize root cells were immersed in latrunculin-B solution to which BFA was added after 150 minutes. (I) JIM84-FITC (green). Increase of the initial latrunculin-induced aggregates (compare with F). (J) AtPIN-CY3 (red). BFA induces the disappearance of polar staining (compare with G) and the latrunculin-B-induced aggregates become thicker. (K) Merged picture, showing that AtPIN- and JIM84-labelled membranes colocalise in these fluorescent structures. Bars, 8 µm.