- Research article
- Open Access
Intracellular shuttling of a Drosophila APC tumour suppressor homolog
© Cliffe et al; licensee BioMed Central Ltd. 2004
Received: 01 July 2004
Accepted: 30 September 2004
Published: 30 September 2004
The Adenomatous polyposis coli (APC) tumour suppressor is found in multiple discrete subcellular locations, which may reflect sites of distinct functions. In Drosophila epithelial cells, the predominant APC relative (E-APC) is concentrated at the apicolateral adherens junctions. Genetic analysis indicates that this junctional association is critical for the function of E-APC in Wnt signalling and in cellular adhesion. Here, we ask whether the junctional association of E-APC is stable, or whether E-APC shuttles between the plasma membrane and the cytoplasm.
We generated a Drosophila strain that expresses E-APC (dAPC2) tagged with green fluorescent protein (GFP-E-APC) and we analysed its junctional association with fluorescence recovery after photobleaching (FRAP) experiments in live embryos. This revealed that the junctional association of GFP-E-APC in epithelial cells is highly dynamic, and is far less stable than that of the structural components of the adherens junctions, E-cadherin, α-catenin and Armadillo. The shuttling of GFP-E-APC to and from the plasma membrane is unaltered in mutants of Drosophila glycogen synthase kinase 3 (GSK3), which mimic constitutive Wingless signalling. However, the stability of E-APC is greatly reduced in these mutants, explaining their apparent delocalisation from the plasma membrane as previously observed. Finally, we show that GFP-E-APC forms dynamic patches at the apical plasma membrane of late embryonic epidermal cells that form denticles, and that it shuttles up and down the axons of the optic lobe.
We conclude that E-APC is a highly mobile protein that shuttles constitutively between distinct subcellular locations.
The Adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colonic epithelium . A key function of this highly conserved protein is to antagonize Wnt signalling, by constitutively downregulating the transcriptional activity of β-catenin/Armadillo, a key effector of the Wnt signalling pathway . Loss of this function is thought to be critical in the initiation of colorectal tumorigenesis as it causes a transcriptional switch in the intestinal epithelium towards actively dividing crypt progenitor cells [3–5]. APC proteins are highly conserved among vertebrates and flies, and flies encode two APC proteins with overlapping roles in Wnt signalling during development [6, 7].
However, APC proteins have additional functions in connection with the actin and microtubule cytoskeletons that appear to be separate from their function in controlling Wnt signalling [8, 9]. One of these functions is a role of APC in facilitating cellular adhesion, as indicated by studies in Drosophila tissues  and in mammalian colorectal cancer cells . This function in cellular adhesion is likely to be conferred by the subcellular pool of APC protein that is associated with adherens junctions (AJs) in Drosophila [12, 13] and in polarised mammalian cells . The mechanism by which APC facilitates cellular adhesion is unknown.
In order to explore this mechanism, we asked whether Drosophila E-APC (also called dAPC2) might have a structural role at AJs. If so, E-APC would be expected to be stably associated with AJs, similarly to the structural components of the adhesive complex. As in mammalian epithelia [15, 16], the main functional components of this complex in Drosophila are the transmembrane protein E-cadherin, a calcium-dependent trans-membrane adhesion molecule, and the catenins (Armadillo and α-catenin) that link E-cadherin to the actin cytoskeleton at the cytoplasmic side [17–22]. We thus conducted photobleaching experiments on live embryos expressing E-APC or structural AJ components tagged with green fluorescent protein (GFP) [23–25], to compare their relative mobility. These experiments revealed that GFP-E-APC is less stably associated with AJs than their structural components. We also found that GFP-E-APC is remarkably mobile in neurons.
Results and discussion
We also conducted FRAP experiments with structural AJ components, namely E-cadherin-GFP, Armadillo-GFP and α-catenin-GFP. In these cases, we can only recover a small fraction of the initial fluorescence within the time frame of the experiment (Fig. 4a,4b,4c,4d,4e; note that these experiments cannot be extended beyond ~6 minutes, due to the extensive cell shape changes during this developmental stage). Furthermore, the rate of recovery is slower than that observed with GFP-E-APC, with estimated half-times of >3 minutes (α-catenin-GFP and of E-cadherin-GFP; Fig. 4b,4d,4e). This also appears to be true for Armadillo-GFP (Fig. 4c,4e), though we cannot estimate its half-time of recovery with confidence, given that its fluorescence levels are considerably lower than that of the other GFP-tagged protein examined in this study.
We conclude that E-APC is significantly more mobile than the structural AJ components. This suggests that E-APC shuttles either within the cortex, along the zonula adherens, or that it shuttles from the cytoplasm to the plasma membrane (as previously proposed; ). Interestingly, the observed rates of recovery of GFP-E-APC were much slower than the estimated rate of free diffusion (e.g. ; the rate of recovery of GFP alone was <10 seconds, i.e. too fast to be measured by our experimental setup). This suggests that the movements of GFP-E-APC are primarily determined by the kinetics of its binding to ligands. One of these could be Axin which associates with E-APC in Drosophila cells to from large dot-like structures . Similarly, Axin associates with APC in mammalian cells to form large molecular weight protein complexes . Our observations argue against a structural role of E-APC in cellular adhesion. However, they are consistent with a catalytic role of E-APC in facilitating cellular adhesion, for example by maintaining the junctional pool of Armadillo [10, 29, 33]. In support of this, recent evidence suggests that there is rapid exchange of β-catenin within the junctional cadherin complex, and that APC is required for this process .
It has been reported that E-APC and Armadillo are required for anchoring mitotic spindles in the cortex of dividing blastoderm cells in the early Drosophila embryo . We cannot measure the kinetics of GFP-E-APC association with the cortex in these early embryonic cells, because of insufficient expression levels at this stage. However, assuming that these kinetics do not change radically during embryonic development, our observations from the later embryos (Fig. 3,4,5) suggest that E-APC has a catalytic role in capturing microtubules in the cellular cortex, rather than providing a structural tether .
The subcellular distribution of E-APC and its accumulation at the adherens junctions is unchanged in other mutants of the Wingless signalling pathway (including wg, axin, dsh and signalling-defective arm mutants; ; F. Hamada, X. Yu and M. B., unpublished observations). We thus did not expect any of these mutants to affect the shuttling behaviour of GFP-E-APC to and from the plasma membrane. In support of this, preliminary FRAP experiments indicated that the kinetics of fluorescence recovery are unaffected in dsh null mutant embryos (not shown). Taken together with our results from the sgg mutants, this suggests that the kinetic association of GFP-E-APC with the plasma membrane is unaffected by Wingless signalling.
Our FRAP experiments provided evidence that E-APC is a cytoplasmic shuttling protein whose association with the adherens junctions is highly dynamic. The speed of its shuttling to and from the plasma membrane appears to be constitutive and does not require GSK3 activity. The dynamic association of E-APC with the plasma membrane is consistent with a catalytic role of E-APC, and argues against a structural or tethering role in the cell cortex.
Fly lines transformed with UAS.GFP-E-APC (full length E-APC tagged with GFP at its N-terminal end, inserted into pUAST ) were generated by R. Rosin-Arbesfeld (see also [7, 28]). The GAL4 driver lines arm.GAL4 and GMR.GAL4 (FlyBase) were used to express GFP-E-APC throughout the embryonic epidermis  and in the larval eye disc, respectively. All fly strains were cultured at 25°C.
zw3 M11-1 and dsh v26 mutant embryos lacking maternal and zygotic gene function were generated as described . We did not detect any differences in the subcellular localisation of GFP-E-APC or α-catenin between zygotic null and paternally rescued sgg mutants (identified with an RFP-marked X chromosome ). For Western blot analysis, 10–16 hours old wild-type and sgg mutant embryos were hand-picked (from timed egg collections) under the dissecting microscope, and separated into GFP-positive and GFP-negative embryos; unfertilised embryos were discarded.
Analysis of fixed embryos and Western blots
Antibody staining of fixed embryos and analysis by confocal microscopy were described previously . The following primary and secondary antibodies were used: rabbit anti-E-APC , rabbit anti-GFP , rat anti-α-catenin ; goat anti-rat IgG Alexa Fluor 568, goat anti-rabbit IgG Alexa Fluor 488 (Molecular Probes).
The following primary and secondary antibodies were used for Western blotting: rabbit anti-E-APC ; mouse monoclonal anti-GFP IgG2a (Santa Cruz Biotechnology); mouse anti-α-tubulin (clone B-5-1-2, Sigma), as internal control; goat anti-mouse and anti-rabbit HRP IgG (Santa Cruz Biotechnology). The enhanced chemiluminescence (ECL) Western blotting system (Amersham) was used for detection .
Live imaging of embryos
For live imaging, embryos were dechorionated in 50% bleach for 1–2 minutes and washed. Embryos were transferred to a moistened black filter (Schleicher and Schüll). Embryos were adhered to coverslips with heptane glue, made by mixing heptane and clear sellotape (Sellotape Ltd). Embryos were mounted in Voltalef oil (10S). For short term imaging (<30 minutes), embryos were mounted on a glass slide with small coverslips as supports. For longer term imaging, e.g. for bleaching of pre-denticle patches, embryos were mounted in oil and placed on Bio-foil gas permeable membrane (Sartorius Ltd) mounted on a perspex frame .
Photobleaching of live embryos
FRAP experiments were performed using a Bio-Rad Radiance confocal microscope with a 40× NA 1.3 objective lense. Imaging was performed with a 488 nm argon laser at 5% laser power and the following confocal settings: iris at 4 mm, 50% gain, zoom 10, scan speed 500 lps, box size 512 × 512 pixels. These conditions were found to give minimal photobleaching over the observed time.
For each FRAP experiment, a pre-bleach image was recorded by selecting a focal plane and taking a Z-series, consisting of 3 0.5 μm steps either side of the desired focal plane (from -1.5 μm to +1.5 μm). The LaserSharp software was used to define several regions of interest (ROIs) for bleaching. A maximum of one bleach ROI was placed in any cell and several cells were always left unbleached. Typically, 3–5 ROIs were bleached in one field of view on one embryo. These regions were bleached at 100% laser power (scanning at 500 lps). 10 bleach scans were found to produce the best results for all constructs. After bleaching, a Z-series was recorded every 15 seconds for 5 minutes. At the time of these experiments, the LaserSharp software did not contain a function for performing this type of 4D bleaching experiment. This problem was overcome by manually switching between imaging and bleaching settings and manually saving pre bleach images and starting the time course. As a result of this, there was usually a 30–60 second delay between the pre-bleach image and the post-bleach images.
Data sets were analysed with the Bio-Rad LaserPix software. For each time point, the total pixel intensity distribution was compared to the pre-bleach image to select the corresponding region. The two images were then compared by eye to confirm that they did correspond to the same focal plane. The coordinates for the bleach ROIs were used to accurately locate the bleach spots on the pre bleach image, and the mean fluorescence intensity for each ROI was calculated. Several equivalent sized ROIs were also placed on unbleached cells to measure any change in fluorescence due to photobleaching or movement.
To track movement of the cells, an acetate sheet was placed over the computer monitor and each ROI was marked on it as well as the shapes of the cells surrounding it. By aligning the sheet with the appropriate cell shapes, the ROI could be appropriately positioned for each time point. This process was used to position each ROI on the appropriate image for each time point.
Once all ROIs had been placed on the image, the mean fluorescence intensities were calculated for each ROI, and their positions were saved on a copy of the image (See Fig 2.1). Data was exported to Microsoft Excel for analysis. Relative fluorescence was calculated for each bleach area by dividing fluorescence at time (t) by pre-bleach fluorescence. The change in fluorescence was plotted on a graph with Excel. For each construct tested, the data from multiple bleach experiments from multiple embryos were averaged to give the approximate rate of recovery.
Data sets were discarded for any of the following reasons. First, if movement of the embryo in the Z axis took the sample outside the range of the Z-series in any time point. Second, if movement in the X/Y axis was sufficient to move significant numbers of the bleach boxes outside of the observed region. Third, if an ROI ever left the field of view, all data points for that ROI was discarded. Fourth, all data sets were discarded if the intensities of the control ROIs changed dramatically at any point in the experiment, or showed a large general increase or decrease.
Pre-denticle structures were bleached in a similar manner to junctional E-APC described above.
Live imaging of the larval optic stalk
GFP-E-APC was expressed in eye imaginal discs by the GAL4 system, using the driver line GMR.GAL4 (described in FlyBase). Eye discs and brains were dissected from crawling third instar larvae in PBS. Eye discs were teased away from the brain and inverted to reveal the optic stalk. Whole disc/brains were mounted in a drop of PBS under a cover slip, supported by two smaller cover slips. Each disc was observed for no more than 30 minutes.
Photobleaching of the larval optic stalk
FRAP experiments were performed using a Bio-Rad Radiance confocal microscope and Bio-Rad LaserSharp software, using the 100× NA 1.4 objective lens. A narrow strip was bleached across the whole field of view by adjusting the size of the scanning area. These experiments were performed before a FRAP program was available for LaserSharp so bleaching was performed manually, leading to somewhat variable intervals between each stage of the experiment. The region was bleached with the 488 nm line of an argon laser for approximately 20 scans. Time courses were recorded after each bleaching experiment for 5 minutes.
We thank R. Rosin-Arbesfeld, H. Oda and M. Peifer for fly strains expressing GFP-tagged proteins, and F. Hamada for help with the Western blots. J. M. is supported by a grant from the Association for International Cancer Research (no. 03–275) awarded to M.B.
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