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Comment
. 2011 Mar;23(3):846-9; author reply 849-50.
doi: 10.1105/tpc.110.082099. Epub 2011 Mar 15.

ARF1 localizes to the golgi and the trans-golgi network

David G Robinson, David Scheuring, Satoshi Naramoto, Jirí Friml
Comment

ARF1 localizes to the golgi and the trans-golgi network

David G Robinson et al. Plant Cell. 2011 Mar.
No abstract available

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Figures

Figure 1.
Figure 1.
ARF1 Localization, MVBs, and PVC Maturation. (A) and (B) Immunogold electron microscopy with ARF1 antibodies. Bars = 500 nm in (A) and 100 nm in (B). (A) Tokuyasu cryosection of high-pressure frozen/freeze-substituted Arabidopsis roots. Thawed sections were stained with 1-nm gold particles and silver enhanced Nanogold according to Stierhof and El-Kasmi (2010). Label is present over both the Golgi stack and TGN. (Micrograph courtesy of York-Dieter Stierhof, Tübingen.) (B) Conventional cryosection of a maize (Zea mays) root cap cell (for details, see Pimpl et al., 2000). Gold particles (5 nm) are seen on vesicles (arrows) at both the cis- (c) and trans- (t) faces of the Golgi stack. (C) Ultrastructure of an MVB. Section prepared from a high-pressure frozen/freeze-substituted/plastic-embedded sample of an Arabidopsis root. Note the almost perfect circular profile lacking vesicle budding profiles, as well as an electron-dense putative clathrin plaque (arrowheads) at the surface. Bar = 100 nm. (Micrograph courtesy of York-Dieter Stierhof, Tübingen.) (D) to (G) Double immunofluorescence with antibodies directed against proteins indicated in each panel in 5-d-old root cells of the Arabidopsis seedlings. In all images, the GFP fusion proteins are shown in green and the endogenous ARF1 ([D], [F], and [G]) or COPI (E) proteins in red; merged signals are yellowish. The rabbit polyclonal anti-ARF1 antibody and anti-γ-COP antibody were diluted 1:800, and mouse monoclonal anti-GFP antibody was diluted 1:600. The secondary antibodies anti-rabbit CY3 (Sigma-Aldrich) and anti-mouse Alexa 488 (Invitrogen) were diluted 1:600. Bars = 4 μm. (D) Endogenous ARF1 (detected by ARF1 antibody) colocalizes with signal from GFP antibody in a line expressing At-ARFA1c fused to enhanced GFP (ARF1-GFP). (E) ARF1-GFP (detected by GFP antibody) colocalizes with COPI (detected by antibody against the Golgi marker γ-COP; Pimpl et al., 2000). (F) ARF1 (detected by ARF1 antibody) also colocalizes with an anti-GFP signal in a line expressing the TGN marker VHA-a1-GFP. (G) No significant colocalization was observed between the PVC/MVB marker GFP-ARA7 (detected by GFP antibody) and endogenous ARF1. (H) to (J) Transient expression of fluorescently tagged TGN (YFP-AtSYP61) and PVC/MVB (AtVSR2-RFP) markers in tobacco mesophyll protoplasts. After short expression periods (6 h), both markers show complete colocalization. However, the two markers becoming increasingly separate with increasing length of expression. This can be interpreted as demonstrating the formation of the PVC/MVB at the TGN and its gradual maturation away from the TGN. Bars = 5 μm.
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References

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