Figure 1.
Chemical hits induce the accumulation of GFP-TIP2;1 to an ER-like network or aberrant compartments.
Three-day-old seedlings expressing GFP-TIP2;1 and mCherry-HDEL were exposed to DMSO (control, A), 55 uM C834 (B), 62.34 μM C410 (C), 88 μM C755 (D), 79.14 μM C103 (E) or 80 μM C578 (F) for 48 h, and imaged under a confocal microscope. Signals from GFP-TIP2;1 (green), mCherry-HDEL (red) and merged image are shown. Insets in (E) show cells with vesiculated structures. Arrows indicate sites of co-localization at the ER network. Arrowheads indicate vesiculated ER structures (E) or cytoplasmic staining (F). Bar = 20 μm.
Figure 2.
GFP-TIP2;1 accumulates in the Endoplasmic Reticulum in C834-treated cells.
Three-day-old seedlings expressing GFP-TIP2;1 and mCherry-HDEL were exposed to DMSO (control, A, B) or 55 uM C834 (C, D) for 48 h, and imaged under a confocal microscope. Signals from GFP-TIP2;1 (green), mCherry-HDEL (red) and merged image are shown. Scatter plots showing pixel intensity in the red (Y axis) or green (X axis) channel for each image is also shown. While signal in the DMSO treated cells are distributed equally (A, B), the pixels in the C834-treated cells show strong correlation between the two channels (C, D). Both cortical (A, C) and medial (B, D) sections of the same cells are shown. Bar = 10 μm.
Figure 3.
C834 uncouples two pathways for tonoplast proteins trafficking.
Three-day-old seedlings expressing GFP-TIP2;1 (A, F), TIP3;1-YFP (B, C, G) or TIP1;1-YFP (D, E, H, I) were exposed to DMSO (control, A–E) or 55 μM C834 (F–I) for 48 h and imaged under a confocal microscope. ER network localization (arrows) was observed in C834-treated GFP-TIP2;1 and TIP3;1-YFP seedlings, but not in TIP1;1-YFP. Cortical sections are shown in B, D and F-H. Medial sections are shown in A, C, E and I. Arrowheads indicate vacuolar “bulbs” labeled with TIP1;1-YFP. Bar = 20 μm.
Table 1.
Effects of chemical hits on three TIP proteins and mCherry-HDEL.
Figure 4.
C834 is a strong inhibitor of root hair elongation.
Plants were grown in the light in media containing DMSO or 55 μM C834 for 7 days prior to imaging. Bar = 5 mm (A) or 1 mm (B).
Figure 5.
Tonoplast trafficking of TIP2;1 and TIP3;1 is insensitive to BFA.
Three-day old seedlings expressing GFP-TIP2;1 (A, B), TIP3;1-YFP (C, D), TIP1;1-YFP (E, F), NAG1-GFP (G, H) and VHA-a1-GFP (I, J) were incubated in the presence of DMSO (-BFA, A, C, E, G, I) or 75 μM BFA (+BFA, B, D, F, H, J) for 3 h. Hypocotyl cells from treated plants were imaged by confocal microscopy. ER network localization is shown in the inset (F) or indicated with arrows (H). Internal BFA compartments labeled with TIP1;1-YFP (F), NAG1-GFP (H inset) and VHA-a1-GFP (J inset) are indicated with arrowheads. Bar = 20 μm.
Figure 6.
C834 enhances the vacuolar targeting and degradation of PIN2-GFP in the dark.
Four-day-old seedlings expressing PIN1-GFP, PIN2-GFP, PIN3-GFP, PIN4-GFP or PIN7-GFP were transferred to either DMSO or 55 μM C834 for 18 h in the light (A–D, I–N) or the dark (E–H, O–T). All images of each marker were taken at the same microscope settings. Bar = 10 μm.
Figure 7.
Proposed model for two pathways for TIP protein trafficking to the vacuole.
A Golgi-dependent pathway may be used by TIP1;1 and is sensitive to BFA but insensitive to C834. A Golgi-independent pathway may be used by TIP3;1 and TIP2;1 and is BFA-independent and sensitive to C834. The pathway for PIN2 trafficking to the vacuole in the dark may merge with the Golgi-independent pathway at an intermediate pre-vacuolar compartment.