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First published online 7 March 2006
doi: 10.1242/jcs.02847

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The plasma membrane recycling pathway and cell polarity in plants: studies on PIN proteins

¹ Laboratoire de Dynamique de la Compartimentation Cellulaire, Institut des Sciences du Végétal, CNRS UPR2355, 9 Gif-sur-Yvette CEDEX, France
² Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Versailles CEDEX, France

View larger version (16K): [in a new window]   Fig. 1. Western blot of protein extracts incubated with AtPIN. A major 70 kDa protein band is recognised by AtPIN. (A) Zea mays root. The minor band corresponds to protein degradation, a phenomenon commonly observed on western blots made of maize extracts (B) Left lane, Arabidopsis root; right lane, protoplasts from Arabidopsis cell culture.

View larger version (132K): [in a new window]   Fig. 2. AtPIN immuno-fluorescence staining in plant tissues. (A-C) Arabidopsis root expressing AtPIN1::GFP. (A) Staining pattern with AtPIN-Cy3 (red). (B) AtPIN1::GFP expression pattern (green). (C) Merged picture showing that AtPIN colocalises with AtPIN1::GFP (yellow) and stains additional cell lines (red). Nuclear staining with Hoechst 33342 (blue). (D) pin-formed1 mutant naked stem with AtPIN-Cy3. A PIN-like polar pattern is still observed (arrow); x indicates autofluorescence of xylem elements. (E) Longitudinal section of Zea mays root labelled with AtPIN-FITC with the typical ladder-pattern. (F-G) Longitudinal sections of BFA-treated Zea mays roots stained with AtPIN-FITC. (F) 90 minutes BFA, (G) 150 minutes BFA. Occurrence of `BFA compartments' and progressive disappearance of the polar PIN pattern. Bars 20 µm.

View larger version (61K): [in a new window]   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.

View larger version (102K): [in a new window]   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.

View larger version (22K): [in a new window]   Fig. 5. Diagram summarizing the BFA and latrunculin-B (LAT) effects on AtPIN- and JIM84 labelled compartments in maize root cells. (1) In control cells, PIN- and JIM84-labelled compartments are dispersed throughout the cytoplasm; strong AtPIN polar staining of the basal membrane. G, Golgi stacks labelled with JIM84. E, endosome-like structures labelled with AtPIN. (2) Under latrunculin-B treatment, both populations tend to mix and form small aggregates, suggesting a similar actin dependency for their distribution. However, AtPIN polar staining is still observed, suggesting that actin is not involved in the maintenance of cell polarity. (3) Treatment with brefeldin A results in the formation of typical BFA compartments, trapping both Golgi stacks and endosome-like membranes. These observations are the sum of two distinct dynamic events: first, the gathering of compartments in cytoplasmic subdomains, a phenomenom that is actin dependent and, second, a vesicularisation of compartments that is actin independent - as shown for Golgi membranes (Satiat-Jeunemaitre et al., 1992). (4) In latrunculin pre-treated cells, BFA still targets the BFA-sensitive compartments in an actin-independent process because the polar labelling of the membrane is lost and the small, induced latrunculin aggregates increase significantly in size, probably due to an actin-independent deconstruction process as described in (3).

View larger version (126K): [in a new window]   Fig. 6. AtPIN and microtubules. (A-C) AtPIN and microtubule labelling in ton mutants. (A) Microtubule arrays are disorganised. (B) AtPIN labelling in the ton2.1 mutant (weak allele). Polar pattern can still be recognised there is, however, some diffusion into lateral membranes. (C) AtPIN labelling in ton2.2 mutant (strong allele). The typical ladder-like pattern is lost, although AtPIN still labels sub-domains of the cell surface. (D-F) AtPIN and microtubule labelling after short-term treatment with oryzalin (3 hours) in isolated Zea mays root cells. AtPIN-Cy3-tubulin-FITC double labelling in (D) untreated cells, and cells treated with (E) 17 µM oryzalin, (F) 28 µM oryzalin. AtPIN polar staining is not affected by the progressive alterations of the microtubule network by oryzalin. (G-K) AtPIN labelling after long-term treatment with oryzalin (42 hours) in Arabidopsis and Zea mays roots. (G) AtPIN typical ladder-like staining pattern in untreated Arabidopsis root. (H-J) Longitudinal sections of Arabidopsis root apex treated with different concentrations of oryzalin (H) 1.7 µM, (I) 17 µM, (J) 28 µM. Initial basal polar staining pattern is lost and often replaced by labelling of the proximal side of the plasma membrane. (K) Transverse section illustrating the labelling shift to the proximal side of the cells, towards the central cylinder. (L) Longitudinal section Zea mays root treated with 17 µM of oryzalin. Relocation of AtPIN-labelling to proximal cell sides is also shown. Bars, 8 µm.

View larger version (101K): [in a new window]   Fig. 7. Redistribution of AtPIN1::GFP in BY-2 protoplasts and oryzalin-treated BY-2 cells. (A) Stably transformed BY-2 cells exhibit a polar pattern of AtPIN1::GFP because the radial surface (but not their longitudinal membranes) was clearly fluorescent. (B) High-magnification of the transverse membranes shows that the two sides of the cell-cell contact are indeed labelled. (C-H) Progressive relocation of AtPIN1::GFP-labelling during the process of making protoplasts. (C) Bottom cell becomes rounder and labelling begins to extend to the whole plasma membrane. Notice the more intense labelling when cells are still in contact (top cells). (D) The arrow marks the cell followed over time and shown in the two following sequences (E) and (G). (E) When the protoplast finally detaches from its neighbour, differential staining is still displayed for a few minutes on the plasma membrane (arrowhead). (F) Corresponding DIC image of (E). (G) Same protoplast at a later stage, AtPIN1::GFP labelling of plasma membrane is still conserved but it has extended to the whole surface. (H) Corresponding DIC image. (I-L) Progressive relocation of AtPIN1::GFP under oryzalin treatment. (I) After 10 minutes, cells begin to round up, labelling is still observed on cell plate and transverse cell surface. (J-L) Labelling at the cell surface is still observed as long as there is some cell-cell contact, regardless of the loss of the ribbon form of BY-2 cells (arrowheads). Bars, 16 µm.

View larger version (58K): [in a new window]   Fig. 8. Position of the p74 peptide chosen for raising antibodies AtPIN against AtPIN1. The p74 peptide is made of 16 aa (bold in grey box) placed in the a glycine-rich domain (asterisks), in the intracellular loops placed between two transmembrane domains (sequence shaded grey).

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