spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 22 April 2008
doi: 10.1242/jcs.015958

Journal of Cell Science 121, 1587-1592 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Franch-Marro, X.
Right arrow Articles by Vincent, J.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Franch-Marro, X.
Right arrow Articles by Vincent, J.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

In vivo role of lipid adducts on Wingless

Xavier Franch-Marro1,*, Franz Wendler1,*, Janice Griffith2, Madelon M. Maurice2 and Jean-Paul Vincent1,{ddagger}

1 National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
2 Department of Cell Biology, University Medical Center Utrecht (UMCU), Rm G02.525, Heidelberglaan 100, 3584CX Utrecht, The Netherlands


Figure 1
View larger version (91K):
[in this window]
[in a new window]

  		Fig. 1. (A) Expression and post-translational modifications of [S239] and [C93A]Wingless. Drosophila S2 cells were transfected with plasmids expressing either the wild-type or the [C93A]Wingless protein and processed for immunoblotting (left lanes). Right-hand-side lane shows [S239]Wingless obtained from transgenic imaginal discs. Distinct bands representing different states of glycosylation can be detected in all three cases. (B) [C93A]Wingless has no significant signalling activity, and that of [S239]Wingless is strongly reduced. Drosophila S2 cells were transfected with the topflash reporter along with plasmids expressing Fz2 and Renilla luciferase. The cells were also transfected with equal amounts of plasmids expressing [S239]Wingless, [C93A]Wingless or wild-type Wingless. Luciferase activity was measured and normalised against Renilla activity for the two groups of cells. (C) Diagram of a wing imaginal disc showing the domain of Wingless expression in green and of ap-Gal4 in red. (D-F) Expression of wild-type Wingless (D), [S239]Wingless (E) and [C93A]Wingless (F) under the control of ap-Gal4. Unlike the wild-type protein, the mutant proteins cannot be detected in non-expressing cells (no punctate staining can be detected). Scale bar: 10 µm. (G-N) Extracellular staining versus total staining of various Wingless forms expressed under the control of ap-Gal4. In all panels, total staining is shown in red and extracellular staining in green. Scale bar: 50 µm. (G,K) Expression of wild-type Wingless. Note the presence of extracellular signal at the surface of expressing cells, in the dorsal compartment (square brackets). The pouch is enlarged because of excess activation of Wingless signalling. (H,L) Expression of [S239]Wingless. Strong extracellular staining can be detected in the expression domain. (I,M) Expression of [C93A]Wingless. No extracellular signal is detected. (J,N) Expression of NRT-[C93A]Wingless. Extracellular localisation is restored by the presence of a heterologous transmembrane domain.

 

Figure 2
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 2. Secretion of wild-type and mutated Wingless in vitro and in vivo. (A) Drosophila S2 cells were transfected to express wild-type, [S239] or [C93A]Wingless fused to Renilla luciferase (RL) protein. The graph shows luciferase activity as a percentage of the total activity present in cell extracts and in the supernatant. No significant difference is seen between the three samples. Cells also expressed cytoplasmic firefly luciferase. In all cases, less than 2% activity was detected in the medium, indicating very limited leakage. (B-F) `Donor' cells were transfected with either wild-type or [C93A]Wingless together with Renilla luciferase for normalisation. `Receiving' cells were transfected with Fz2 and topflash. A histone-GFP plasmid was also included in the transfection mix in order to recognise receiving cells in subsequent immunofluorescence experiments. (B) First, a subset of the cells was processed for the topflash assay and luciferase activity was normalised to Renilla activity. As expected, wild-type Wingless causes high reporter activity in this paracrine assay, whereas [C93A]Wingless does not induce detectable activity over background. (C-F) In parallel, both donor and receiving cells were mixed and processed for immunofluorescence. Anti-Wingless is shown in red and receiving cells are green. Wild-type Wingless can be seen in receiving cells both at the cell surface and in intracellular vesicles (arrowhead in C and E). (D,F) By contrast, no HA-[C93A]Wingless can be seen in receiving cells. Scale bar: 10 µm. (G-J) Fz2 overexpression causes mild accumulation of [C93A]Wingless at the cell surface. Fz2 (Flag) and [C93A]Wingless (HA) were co-expressed with ap-Gal4, and imaginal discs were stained with the extracellular protocol. [C93A]Wingless is now detectable at the cell surface (G; compare with Fig. 1M, in which Fz2 is not co-expressed). An optical cross-section (along the dorsoventral axis) shows that [C93A]Wingless tends to accumulate in the apical half of the cell (I), whereas Fz2 is present across the apicobasal axis (J). Scale bars: 50 µm.

 

Figure 3
View larger version (99K):
[in this window]
[in a new window]

  		Fig. 3. Subcellular localization of wild-type Wingless and [C93A]Wingless in ectodermal cells of third-instar larvae. (A-C) High-magnification view of a region of ectodermal cells co-expressing wild-type Wingless from two transgenes (one tagged with GFP and the other with HA) with the wg-Gal4 driver. Inset in A shows the whole cell. GFP is shown in green and HA in red. Extensive overlap is seen (yellow in A), including in large structures (arrowheads). (D-F) Co-expression of GFP-tagged wild-type Wingless (green) with HA-tagged [C93A]Wingless (red). Again, the whole cell is shown in the inset in D. Only partial overlap is seen between the mutant and wild-type proteins (D). Note, for example, the lack of colocalisation in large structures (arrowheads). Scale bars: 1 µm.

 

Figure 4
View larger version (131K):
[in this window]
[in a new window]

  		Fig. 4. [C93A]Wingless accumulates in the ER of wing disc cells and is absent from MVBs. (A,B) Portion of the apical surface of a disc cell. Empty space is the peripodial lumen (PL). HA-[C93A]Wingless and HA-Wingless (WT) expressed with dpp-Gal4 are visualized with anti-HA and a secondary antibody labelled with 15-nm gold particles. Arrowheads indicate examples of HA-positive ER areas. These are particularly abundant in the [C93A]Wingless sample. (C,D) Basolateral region of disc cells. Note again the relatively abundant labelling in the perinuclear and other ER areas of cells expressing [C93A]Wingless (C). See for comparison an equivalent area in wild-type-Wingless-producing cells (D). Arrowheads indicate examples of ER areas positive for Wingless labelling. (E,F) MVBs (*) in cells expressing high levels of [C93A]Wingless. Note the absence of label in these areas. (G,H) By contrast, anti-HA labelling is seen in the MVBs of cells expressing wild-type Wingless. G, Golgi unit; M, mitochondrion; N, nucleus; PL, peripodial lumen. ER is recognised by its characteristic morphology, as indicated from an independent preparation stained with anti-KDEL (see supplementary material, Fig. S1). Scale bars: 200 nm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008