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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02968

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Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis

Department of Biology, Bowdoin College, Brunswick, ME 04011, USA

View larger version (34K): [in a new window]   Fig. 1. (A) Confocal images of WAK1-GFP localization in Arabidopsis protoplasts. 35S CaMV WAK1-GFP expressed from pCambia 1302 was transformed into leaf protoplasts, and cells were incubated in W5 for 1 day and then transferred to regeneration medium for additional times as indicated. GFP was detected in the green channel and chlorophyll in the red. Shown are single optical sections through representative cells. BAK-GFP and WAKFIN-GFP (lacking extracellular domain) were also expressed from pCambia 1302. Bar, 10 µm. (B) Quantification of cells on indicated day post transformation of WAK1-GFP, BAK-GFP or WAKFIN-GFP location; 1, cytoplasmic localization; 5, surface localization. (C) Ethidium-bromide staining of an agarose gel of RT-PCR from WAK-GFP mRNA with equal amounts of RNA isolated from WAK-GFP transformed protoplasts incubated for the indicated time.

View larger version (64K): [in a new window]   Fig. 2. Localization of WAK-GFP and pectin. Pectin was detected using Jim5 and cy3 anti-rat IgG serum (red) and WAKGFP by GFP fluorescence (green), and visualized by confocal microscopy. Jim5 (No WAKGFP): single optical section of protoplast not expressing WAK-GFP but stained with Jim5. Cells transformed with WAK1-GFP were fixed after 36 hours incubation, and stained with anti-pectin Jim5 antibody, and visualized by confocal microscopy. Jim5 (WAKGFP), pectin signal alone; WAKGFP, GFP signal alone; Jim5 and WAKGFP, merged signals; Jim5 and BAKGFP, cells transformed with BAK-GFP were fixed after 5 hours incubation, and stained with anti-pectin Jim5 antibody. Magnification, x450.

View larger version (57K): [in a new window]   Fig. 3. WAK1-GFP colocalizes with Golgi markers. Protoplasts were transformed with WAK-GFP (green) and the indicated RFP fusion (SYP31 and SYP41, Golgi markers; SRC2, protein-storage-vacuole marker; TIP, lytic and central vacuole marker; ARA7, endosomal marker) or stained with FM4-64 (endocytotic marker, red). Single optical sections were observed by confocal microscopy. Top two rows show single-channel fluorescence and merged images, bottom two rows show merged images only. Chlorophyll is false-colored blue. Magnification, x450.

View larger version (44K): [in a new window]   Fig. 4. Calcofluor staining of cellulose in transformed protoplasts. (A) single protoplasts transformed with WAK1-GFP were stained with 1 µg/ml calcofluor and the cellulose (calcofluor) or the GFP fluorescence was captured on a standard fluorescence microscope 1 hour or 3 days after transformation. Cells were incubated in RM. (B) Protoplasts were incubated in RM (0) or RM plus 2.5 µM isoxaben (+isox) for 3 days, and then stained with calcofluor and visualized by standard fluorescence microscopy for calcofluor. Red, chlorophyll; green, GFP; blue, calcofluor. Magnification, x300.

View larger version (21K): [in a new window]   Fig. 5. WAK1-GFP movement to the surface is slowed down by isoxaben. (A) Protoplasts were transformed with WAK1-GFP or BAK-GFP and incubated for the indicated times in 2.5 µM isoxaben. Shown are representative confocal optical sections. Green, GFP; red, chlorophyll. See Fig. 1 for comparison. (B) Quantification of WAK1-GFP or BAK-GFP location on indicated day post transformation. 1, cytoplasmic localization; 5, surface localization. Magnification, x300.

View larger version (37K): [in a new window]   Fig. 6. Detection of Triton-X-100-soluble and -insoluble WAK1-TAP in protoplasts. Equal amounts of protoplasts transformed with WAK-TAP after 1, 2 or 3 days (as indicated) were extracted with 1% Triton X-100, centrifuged at 10,000 g for 5 minutes, and supernatant (S) and pellet (P) were boiled in loading buffer and either run on an SDS-PAGE (western blot) or (in a separate experiment) were slot-blotted onto nitrocellulose (slot blot). Then, both were probed with anti-TAP serum. pSmGFP, cells transformed with plasmid expressing only GFP.

View larger version (36K): [in a new window]   Fig. 7. mur1-1 does not influence WAK1-TAP insolubility. Equal amounts of wild-type (col+) and mur1-1 cells transformed with WAK1-TAP after 1 or 3 days (as indicated) were extracted with 1% Triton X-100 and centrifuged at 10,000 g for 5 minutes. The supernatant was run in an SDS-PAGE (A), or (in a separate experiment) the supernatant and the pellet were boiled in gel loading buffer and slot-blotted onto nitrocellulose (B). Pellet and supernatant were then both probed with anti-TAP serum. isx; 2.5 µM isoxaben added for 1 or 3 days. Wild-type cells were also transformed with GFP alone (GFP).

View larger version (26K): [in a new window]   Fig. 8. (A) surface transport of WAK1-GFP is speeded up by mur1-1. Protoplasts from wild-type (+) and mur1-1 plants were transformed with WAK-GFP and after 1 day were visualized by confocal microscopy. mur1-1 cells were also treated with 2.5 µm isoxaben 1 hour post transformation. Red, chlorophyll; green, GFP. (B) Quantification of WAK1-GFP location in cells on indicated day post transformation. 1, cytoplasmic localization; 5, surface localization. Magnification, x300.

View larger version (118K): [in a new window]   Fig. 9. WAK-GFP accumulates in cytoplasmic compartments of the leaf bud. Single optical section of an emerging leaf bud of an Arabidopsis plant transformed with WAK-GFP (green). Chlorophyll was detected in the red channel. Bar, 10 µm.