Correspondence to: Shoichiro Tsukita, Department of Cell Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606-8315, Japan. Tel:81 75-753-4372 Fax:81 75-753-4660 E-mail:htsukita{at}mfour.med.kyoto-u.ac.jp.
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Abstract |
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Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.
Key Words: adenomatous polyposis coli, green fluorescent protein, microtubules, microtubule-associated proteins, epithelial cells
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Introduction |
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Microtubules (MTs)1 are polarized tubular structures which are produced by linear polymerization of /ß-tubulin heterodimers, with the plus end favored for assembly over the minus end (
In migrating cells, MTs are generally polarized along the axis of cell movement with their plus ends facing the leading edge (
From these viewpoints, the subcellular localization of adenomatous polyposis coli (APC) protein in cultured MDCK epithelial cells is intriguing (
The APC gene is mutated during the progression of sporadic colorectal tumors as well as in patients with familial adenomatous polyposis (for reviews see -helix domain that forms a parallel coiled-coil dimer (
-catenin (
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In the present study, to further clarify the relationship between APC protein and MTs, we examined the dynamic behavior of APC protein in live cells: we constructed a green fluorescent protein (GFP) fusion protein with Xenopus APC protein, and introduced it into cultured Xenopus A6 epithelial cells. Furthermore, we constructed GFP fusion proteins with deletion mutants of APC protein, and compared them with full-length APC protein in terms of their behavior in live cells. These analyses uncovered peculiar interactions between APC protein and MTs. The results of this study will allow us to consider certain unknown function for the APC gene product.
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Materials and Methods |
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Antibodies and Cells
Rabbit anti-GFP pAb (Clontech or MBL), rabbit anti-APC pAb specific for COOH-terminal 20 amino acids of human APC (APC(C-20); Santa Cruz Biotechnology, Inc.), mouse anti-APC mAb specific for NH2-terminal 35 amino acids of human APC (APC(Ab-1); Oncogene), and mouse anti-tubulin mAb (DM1A; Sigma) were purchased from the sources shown. The Xenopus kidney epithelial cell line A6 was grown at 23°C without CO2 atmosphere in 50% Leivobitz's L-15 medium (GIBCO BRL) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 0.2 mg/ml kanamycin).
Plasmid Construction and Transfection
The expression vector (pQBI25) for the bright red-shifted GFP variant was purchased from Quantumn Biotechnologies, Inc. This vector was used to express recombinant proteins with a GFP tag at their COOH termini. Another expression vector (pGFP-C(NheI)) to express recombinant proteins with a GFP tag at their NH2 termini was produced by modifying pQBI25. In this recombinant protein, two glycine residues were inserted between GFP and the rest of the fusion protein as a flexible spacer.
The Xenopus APC cDNA was cloned from a Xenopus oocyte cDNA library (Uni-ZAP XR cDNA library; Stratagene) by PCR using gene-specific primers with a 5' NheI site. The PCR product was subcloned into pGEM-T Easy vector (Promega), and the construct was verified by sequencing. As summarized in Fig 1, the following expression vectors for GFP-tagged full-length or deletion mutants of APC were constructed: pGFP-C(NheI)/APC(1-8490) for GFP-fAPC; pQBI25/APC(1-8487) for fAPC-GFP; pQBI25/APC(1-6474) for cAPC-GFP; pGFP-C(NheI)/APC(6475-8490) for GFP-cAPC. To construct the expression vector for fAPC-mGFP, the GFP coding sequence was inserted into the SpeI site (nucleotide 6475) of APC cDNA.
Lipofectin (GIBCO BRL) was used for transfection of cDNAs, according to the manufacturer's protocol. Drug-resistant clones were selected in the presence of 0.6 or 0.75 mg/ml genetisin (GIBCO BRL or Calbiochem) and screened by detecting GFP signals with fluorescent microscope.
Quantification of Expression Levels of Endogenous and Exogenous APC
Expression levels of GFP-tagged full-length APC were analyzed by SDS-PAGE followed by immunoblotting. To separate the band of endogenous APC from that of GFP-tagged APC, 50 µl of Triton X-100 lysates of 1 x 107 transfectants (three clones expressing fAPC-GFP and three clones expressing fAPC-mGFP; see Fig 2) were incubated with 1,000 U of -protein phosphatase (New England Biolabs Inc.) at 30°C for 30 min. The sample buffer containing 6 M urea was then added, and the samples containing lysates of 1 x 106 cells were separated by SDS-PAGE (28 M Urea, 35% acrylamide gradient gels), and electrophoretically transferred to PVDF membranes, which were incubated with anti-GFP pAb (1:1,000) or APC(Ab-1) mAb (1:100). The antibodies were detected with a blotting detection kit (Amersham).
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For quantification of expression levels of endogenous and exogenous APC, we first expressed a NH2 terminal portion (amino acids [aa] 1284) of APC fused with GST at its COOH terminus in E. coli, and purified it. The amount of this purified GST fusion protein was determined densitometrically by comparing the intensity of its band after Coomassie brilliant blue staining with those of various amounts of BSA. We then compared the intensity of immunoblotted endogenous APC and exogenous GFP-tagged APC in -protein phosphatase-treated cell lysates with those of immunoblotted bands of various amounts of purified APC(1-284)-GST. These bands were digitized by scanning with ScanJet IIcx (Hewlett Packard) and processed using Adobe Photoshop software. The intensity of the band was measured by densitometry using the NIH image software.
Immunofluorescence Staining
Cells (12 x 105 cells/cm2) were cultured on coverslips for 2448 h, washed with 70% Dulbecco's PBS(+) and fixed with 3.7% formaldehyde in the same PBS for 3060 min. After washing with Dulbecco's PBS(-), cells were permeabilized with 0.1% Triton X-100 for 5 min, followed by incubation with 10% FBS. Samples were then incubated with the following primary antibodies for 1 h; anti-APC pAb, C-20 (x1,000) and anti-tubulin mAb, DM1A (x200). After washing several times, cells were labeled with secondary antibodies for 1 h. Fluorescein-conjugated sheep anti-mouse Ig antibody (Amersham), rhodamine-conjugated donkey antirabbit IgG antibody (Chemicon), and Texas redconjugated sheep anti-mouse Ig antibody (Amersham) were used as secondary antibodies. Samples were then washed several times and mounted in Mowiol (Calbiochem). For visualization of plasma membranes, live cells were stained using a PKH26 red fluorescent cell linker kit (Sigma) according to the manufacturer's protocol.
Fluorescence Microscopy and Image Analysis
To observe live cells, cells were cultured on glass-bottomed dishes with No.1S cover glasses (Matsunami) in culture medium without phenol red at 2325°C. Cells were cultured at low density (12 x 105 cells/cm2) except for wound healing experiments. For wounding, cells cultured on glass-bottomed dishes at high density were wounded by scraping with a needle.
Images of fixed cells were acquired with an MRC-1024 laser confocal microscope (BioRad) or DeltaVision optical sectioning microscope (Version 2.00 or 2.10; Applied Precision, Inc.), equipped with Zeiss Axioplan2 (Plan Apochromat 63x/1.40 NA oil immersion objective) or Olympus IX70 (PlanApo 60x/1.40 NA or PlanApo 100x/1.40 NA oil immersion objective) microscope, respectively. Images from live cells were collected with a DeltaVision through a cooled CCD camera (Quantix-LC, Photometrics) with appropriate exposure time and time intervals. Fluorescence signals were visualized using appropriate ND filters, quad beamsplitter, and either of the following excitation and emission filters (Chroma): for GFP, 490/20 nm and 525/50 nm; for FITC, 490/20 nm and 528/38 nm; for rhodamine and PKH26, 555/28 nm and 617/50 nm, respectively.
Pixel positions, distances, and intensity on digital images were measured using the analysis function of DeltaVision. Images used for quantitative analyses were acquired with x100 objective and effective pixel size was 0.067 x 0.067 µm. To quantify fluorescence intensities, mean background fluorescence obtained from an area of 11 x 25 pixels close to the targeted area was subtracted from mean intensity of the targeted area. In Fig 7 d, the background-subtracted fluorescence intensities were obtained from 3 x 3 pixels at MT end (F1) and 2 x 5 pixels along the MT at 3 µm apart from its end (F2) for each time-lapse image. The values were imported into Microsoft Excel, and F1/F2 ratio and changes of MT length were calculated for a plot.
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Treatment of Cells with Various Reagents
To disassemble MTs, cultured cells were incubated with 33 µM nocodazole (Sigma) at room temperature, whereas 100 nM nocodazole (
For permeabilization of cells, cells cultured on glass-bottomed dishes were washed briefly with 70% Dulbecco's PBS(+), and rinsed in the permeabilization buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, and 0.1 mM PMSF). Cells were then extracted with permeabilization buffer containing 35 µg/ml digitonin, which had been diluted immediately before use, 10 µM taxol and protease inhibitor cocktail (1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin) for 5 min at room temperature. After washing with permeabilization buffer containing 10 µM taxol and protease inhibitor cocktail, the dishes were filled with 1 ml of the same buffer. ATP or 5'-adenylylimidodiphosphate (AMP-PNP; Nakarai Tesque) diluted in 100 µl of the same buffer was added to a final concentration of 0.2 mM or 1 mM, respectively, during time-lapse recording (see Fig 8).
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Immunoelectron Microscopy
Cells cultured on collagen-coated dishes were permeabilized with 53 µg/ml digitonin in permeabilization buffer containing 10 µM taxol and protease inhibitor cocktail at room temperature for 10 min. Cells were then washed with the same buffer and fixed with 3.7% formaldehyde at room temperature for 1 h. After washing in Dulbecco's PBS(-), cells were incubated with rabbit anti-GFP pAb for 3 h. Samples were washed several times and then incubated with 5-nm gold-conjugated anti-rabbit IgG antibody (Amersham) for 4 h. Cells were then carefully scraped off the dishes and collected by centrifugation. The pellets were fixed with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) followed by post-fixation with 1% OsO4. Samples were embedded in Epon, and ultrathin sections were cut with a diamond knife, doubly stained with 4% uranyl acetate and 0.4% lead citrate, and viewed with a JEM1010 transmission electron microscope (JEOL). Electron micrographs were digitized with a FILM SCANNER (Nikon; LS-4500AF).
Online Supplemental Material
Seven video files (see below) corresponding to Fig 5 a; 6, ac; 7, b and c; and 8 c are available through the JCB online (http://www.jcb.org/cgi/content/full/148/3/505/DC1). QuickTime videos with a longer time duration are also available at http://www.tsukita.jst.go.jp/kiyosue/apc.html. Time-lapse images collected using DeltaVision were processed with Adobe Photoshop software, and converted to QuickTime videos (JPEG compression) with software installed on a Silicon Graphics O2 computer. All videos contain the time-lapse images from the first to the last panel in the corresponding figures. In the videos for Fig 6 c and 7, b and c, the areas represented in the printed figures are boxed in the first frame of the video.
Fig 5 a Video.
A6 transfectants expressing fAPC-mGFP (green) were stained with PKH26 (red), and images of cells were recorded from 15 min after replating at 1-min intervals. The GFP signal was gradually concentrated at extending cell edges.
Fig 6 a Video.
Time-lapse images of a cell expressing fAPC-GFP (clone A1) were collected at 5-s intervals. Note the clear visualization of a MT itself by fAPC-GFP labeling, the dynamic movement of APC granular structures around MT ends, and the flow of fAPC-GFP signals along the MT.
Fig 6 b Video.
Time-lapse images of a cell transiently expressing fAPC-mGFP were collected at 5-s intervals. Note the dynamic movement of APC granular structures around MT ends.
Fig 6 c Video.
Time-lapse images of a cell expressing fAPC-GFP (clone B4) were collected at 5-s intervals. Note the flow of fAPC-GFP signals along MTs.
Fig 7 b Video.
Time-lapse images of a cell expressing GFP-cAPC were collected at 5-s intervals. GFP-cAPC was concentrated at growing MT ends.
Fig 7 c Video.
Images were collected at 5-s intervals. The cell expressing GFP-cAPC was treated with a low concentration of nocodazole at the timing of 9th frame (indicated in the video) to perturb dynamics of MT plus ends. The concentration of GFP-cAPC at MT ends disappeared within 1 min.
Fig 8 c Video.
Time-lapse images of a cell transiently expressing cAPC-GFP were collected at 5-s intervals. In the cellular extension directing toward the bottom of the image, GFP-positive granular structures moved linearly toward the cell edge.
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Results |
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Expression Level of GFP-tagged Full-Length APC in Cultured A6 Cells
A GFP-tagged Xenopus APC protein (APC), fAPC-GFP, in which GFP was fused to the COOH terminus of full-length APC protein (Fig 1), was constructed, and introduced into Xenopus A6 epithelial cells. Three independent clones stably expressing fAPC-GFP were then isolated (Fig 2). The COOH-terminal -TSV motif of APC was reported to directly bind to the PDZ domain of hdlg (
In these stable clones, we examined the degree of overexpression of fAPC-GFP and fAPC-mGFP relative to endogenous APC. Since both endogenous and exogenous APC proteins exhibited fairly broad bands in SDS-PAGE, it was very difficult to resolve these two bands by immunoblotting from the total cell lysate of transfectants. However, when the cell lysate was treated with -protein phosphatase before SDS-PAGE, these bands converged to form relatively sharp bands, which allowed quantitative comparison between endogenous and exogenous APC proteins by immunoblotting. As shown in Fig 2, the ratios of exogenous APC/endogenous APC in transfectants were distributed from ~1 to ~25. For the time-lapse microscopic observation, we mainly used A1 clone of fAPC-GFP-expressing cells (ratio = 3.3) and C1 clone of fAPC-mGFP-expressing cells (ratio = 2.7), and no significant difference was detected between these two clones in terms of the dynamic behavior of APC-GFP fusion proteins along MTs in live cells.
Subcellular Distribution of GFP-tagged Full-Length APC in Cultured A6 Cells
In parental A6 cells, immunofluorescence microscopy revealed that endogenous APC proteins were concentrated along distal end segments of MTs at the tip regions of cellular extensions (Fig 3 a), similarly to APC proteins in MDCK cells (
Before examination of the dynamic behavior of APC in live cells, fAPC-GFP condensed around MTs was observed by immunoelectron microscopy in A6 transfectants expressing large amounts of fAPC-GFP (clone B4; see Fig 2). In this clone, at the immunofluorescence microscopic level, fAPC-GFP was detected along the entire length of a subset of MTs, and the fAPC-GFP-associated MTs showed a tendency to be bundled and to run into the cellular extensions, the tips of which showed concentration of fAPC-GFP (data not shown). For immunoelectron microscopy, expressed fAPC-GFP was labeled with anti-GFP pAb after permeabilization of cells with digitonin (Fig 4). As shown in Fig 4 a, abnormal thick bundles of MTs were occasionally seen to run through the cytoplasm, and their surfaces were specifically and heavily labeled with immunogold particles. Close inspection revealed that in these bundles parallel MTs were embedded in fuzzy matrices (Fig 4b and Fig c). Single MTs decorated by these fuzzy structures were also observed in association with thick bundles (Fig 4 d) or as scattered structures in the cytoplasm (Fig 4e and Fig f). These fuzzy structures, but not MTs per se or membranous structures, were labeled with immunogold particles. Therefore, these findings indicated that these fuzzy structures surrounding MTs were the ultrastructural counterparts for the GFP signals detected in A6 transfectants expressing fAPC-GFP (see Fig 3).
Dynamic Behavior of GFP-tagged Full-Length APC during Cell Spreading and Wound Healing
We first followed the dynamic behavior of fAPC-mGFP to concentrate at the tip regions in live A6 transfectants (clone C1; see Fig 2) after replating dissociated cells on glass-bottomed dishes (Fig 5 a; see video). In trypsinized single cells, the GFP signal was detected as small granules or along filaments, probably MTs (data not shown). At the initial phase of spreading, fAPC-mGFP began to associate with the entire length of radially arranged MTs rather evenly (time 00:00). Within the next several minutes, fAPC-mGFP gradually accumulated around the edges of the cell periphery as granular structures (arrowheads; time 08:09). Subsequently, these GFP-positive granular structures increased in number and began to cluster in several specific regions of the cell periphery (time 20:26). Some of these clusters of APC granular structures continued to grow (arrows), while some disappeared (arrowheads). The emerging of clusters appeared to be associated with the formation/growth of the cellular extensions, resulting in the concentration of the APC granular structures at the tip regions of the extensions (times 34:2047:43).
Similar polarized clustering of APC granular structures was observed during the process of wound healing. Confluent cultures of A6 transfectants expressing fAPC-mGFP were manually scratched with sharp needles as shown in the phase-contrast image in Fig 5 b. Within 2 h after wounding, at the front row of the wound, fAPC-mGFP began to gradually concentrate at the distal ends of a subset of MTs, which were laterally associated to form thick bundles (times 00:30:5202:02:03). These fAPC-mGFP-concentrated MT bundles appeared to continuously grow toward the wounded region (arrows and arrowheads; times 02:02:0302:49:07).
These findings as well as images shown in Fig 3 and Fig 4 suggested that the peculiar polarized behavior of APC is dependent on MTs. Then, A6 transfectants expressing fAPC-mGFP were treated with a high concentration of nocodazole (33 µM) to disassemble MTs, and at 10-, 30-min, or 3-h incubation, cells were fixed and immunofluorescently stained with anti-tubulin mAb (Fig 5 c). At 10-min incubation, almost all MTs in the cell periphery were disassembled (time 10 min). However, the APC granular structures remained localized at the original positions, i.e., the tip regions of cellular extensions, and were clustered at the level of the basal plasma membrane cortex. These granular structures gradually moved toward the center of cells (~0.4 µm/min) remaining at the level of the basal plasma membrane cortex (time 30 min), and were finally distributed along the central portion of the basal plasma membrane cortex in a striped pattern (time 3 h). This pattern was complementary with that of rhodamine phalloidin-stained stress fibers (data not shown). Taking into consideration that the plasma membrane cortex continuously moves rearward in the migrating lamellae and lamellipodia (reviewed in
Dynamic Behavior of GFP-tagged Full-Length APC at the Distal Ends of Microtubules
Next, we examined the dynamic behavior of fAPC-GFP or fAPC-mGFP at the distal ends of MTs at higher magnification with shorter time intervals in live cells. Fig 6 a shows a series of time-lapse images of the GFP signal (fAPC-GFP) in a lamella traveling toward the left (clone A1; see Fig 2; see video). Since fAPC-GFP was not only concentrated at the tips of MTs but also diffusely covered the distal segments of these MTs (see Fig 3 c), imaging only by the GFP fluorescence allowed to observe the dynamic behavior of fAPC-GFP as well as MTs simultaneously. In this series of time-lapse images, MT itself was continuously transported rearward (toward the right) in the lamellae (
In the series of Fig 6 a, some continuous flow of the GFP signals along MTs toward their distal ends was clearly detected (see video). This flow of APC was more clearly visualized in A6 transfectants expressing relatively large amounts of fAPC-GFP (clone B4; see Fig 2; Fig 6 c, see video). Therefore, we were led to conclude that APC molecules move continuously on the surface of a subset of MTs toward plus ends, and that only when MTs are growing they accumulate at their plus ends as granules. To further understand this peculiar and complicated behavior of APC molecules, we next constructed GFP-tagged deletion mutants of APC (see Fig 1), and examined their dynamic behavior in live A6 cells.
Concentration of COOH-terminal Segment of APC at Growing Ends of Microtubules
Since the COOH-terminal 671 amino acids sequence contained the MT-binding domain of APC in vitro (aa 22192580 of human APC;
Active Transport of APC Mutant Lacking Its COOH-terminal Region along Microtubules toward Their Plus Ends
We next examined the subcellular distribution and the dynamic behavior of the APC mutant lacking its COOH-terminal MT-binding domain with a GFP tag at its COOH terminus, cAPC-GFP, in live A6 cells (see Fig 1). First, A6 transfectants expressing
cAPC-GFP were fixed and immunofluorescently stained with anti-tubulin mAb (Fig 8 a). Expressed
cAPC-GFP formed granular structures scattered around the cytoplasm with various diameters. Interestingly, these granules occasionally clustered in the tip region of some of the cellular extensions similarly to fAPC-mGFP (see Fig 3). In thin lamellae, it was clear that these granules were aligned along MTs (Fig 8 b), indicating that
cAPC-GFP lacking the conventional MT-binding site could still interact with MTs directly or indirectly. This type of MT-associated distribution of
cAPC-GFP was observed clearly up to 1 day after cells were replated.
When these cells were observed by time-lapse microscopy at low magnification, the cAPC-GFP granules were seen to continuously move toward the cell periphery. Fig 8 c shows one series of time-lapse images indicating the movement of these granules at higher magnification (see video). Since, differently from fAPC-mGFP and GFP-cAPC,
cAPC-GFP did not decorate MTs diffusely, MTs were not visible in these images, but a large granule was found moving linearly toward the tip of cellular extensions over a long period (arrowheads). During the course of traveling to the tip, granules occasionally changed their shapes, divided into smaller fragments, or fused to each other, suggesting that they have a flexible liquid-like nature. Their movements were predominantly unidirectional with a peak velocity of ~10 µm/min. When cells were treated with 33 µM nocodazole to disassemble MTs, the movements of the granules became vibrational with no net translocation (data not shown). These observations suggested that the
cAPC-GFP granules move along MT tracks. Then, to check the ATP dependency, the movement of these
cAPC-GFP granules was examined in permeabilized A6 transfectants (Fig 8 d). Permeabilization with digitonin did not affect the shape of granules, but completely stopped their directional movement. When these permeabilized cells were incubated with 0.2 mM ATP, the movement of some granules was induced. The
cAPC-GFP granules moved unidirectionally and linearly toward the tips of cellular extensions (arrows; times -01:5001:45). When 1 mM AMP-PNP, a nonhydrolyzable analogue of ATP, was added, the movement stopped within a minute (arrowheads; times 01:4505:35). These findings indicated that the motility of the
cAPC-GFP granules requires ATP hydrolysis.
APC was reported to form dimers through its NH2-terminal -helical coiled-coil region (
cAPC-GFP bound to endogenous APC to form heterodimers in A6 transfectants, and that endogenous APC in the heterodimers mediated the localization and movement of
cAPC-GFP. However, when A6 transfectants expressing
cAPC-GFP were stained with a pAb against COOH-terminal peptide of APC, in the tip region of cellular extensions only a small number of the GFP-positive granules (34%) were stained with this pAb (not shown). Therefore, it is likely that the subcellular localization and the dynamic behavior of
cAPC-GFP observed in this study was attributable to the intrinsic nature of
cAPC-GFP itself.
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Discussion |
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This is essentially a descriptive study, in which the localization of GFP-tagged APC molecules was detected in live Xenopus A6 epithelial cells. In this description, we first reported the peculiar dynamic behavior of GFP-APC molecules within cells. It is reasonable to consider that this behavior of GFP-tagged full-length APC can be regarded as that of endogenous APC, partly because the expression level of GFP-APC was approximately threefold that of endogenous APC (see Fig 2), and partly because the subcellular localization of GFP-APC was very similar to that of endogenous APC in fixed cells at the immunofluorescence microscopic level (see Fig 3). Immunoelectron microscopy revealed the expressed GFP-tagged full-length APC as fuzzy structures decorating MTs (see Fig 4). Observations in live cells (see Fig 6 and corresponding videos) uncovered the following behavior of GFP-tagged full-length APC; (a) these GFP-APC-based fuzzy structures move in a stream along a subset of MTs toward their plus ends, (b) when GFP-APC molecules arrived at the plus ends of growing MTs, these molecules remain and accumulate at the ends to form granular structures, (c) when MTs began to shorten, these GFP-APC granules dropped off from the ends of MTs, maintaining their structural integrity, and (d) these APC granules were occasionally reloaded onto the same or another MT that passed by the granules. Through these behaviors, APC granules appeared to be transported to and accumulated at the tip regions of the cellular extensions. This concentration of APC was previously reported by conventional immunofluorescence microscopy (
The molecular mechanism behind this peculiar behavior of APC remains unclear, but the present observations on the behavior of GFP-cAPC and cAPC-GFP (see Fig 1) in live cells provide clues to understanding the mechanism. Similar to full-length APC, GFP-cAPC were also concentrated at the distal ends of growing MTs in live cells (see Fig 7), but there were several clear differences in their manner of concentration. First, continuous flow of the full-length APC was observed on MTs toward their distal ends (see Fig 6, a and c), whereas such flow was not detected for GFP-cAPC (see Fig 7 c). Second, when MTs were upon transition from growth to the shortening phase, the full-length APC granular structures at the ends of MTs dropped off from MTs, while maintaining granular structural integrity (see Fig 6, a and b), whereas the GFP-cAPC concentrated at the ends of MTs abruptly disappeared (see Fig 7 b). GFP-cAPC did not accumulate at MT ends as large granular structures. When MTs were totally disassembled with nocodazole (33 µM), GFP-cAPC was distributed diffusely in the cytoplasm without forming detectable granular structures, and was easily extracted by treatment with digitonin (data not shown). These findings suggested that, in contrast to full-length APC, GFP-cAPC molecules are associated with MTs without forming aggregates under the equilibrium with a soluble pool of GFP-cAPC in the cytoplasm.
The behavior and characteristics of GFP-cAPC are very similar to those of CLIP-170, which was recently reported using the GFP-CLIP170 fusion protein (
The behavior of cAPC-GFP was also peculiar (see Fig 8). In contrast to GFP-cAPC,
cAPC-GFP did not decorate MTs, which appeared to be consistent with previous studies that full-length APC interacts with MTs at its COOH-terminal domain (
cAPC-GFP formed granular structures in various sizes that were aligned and moved along MTs toward their distal ends in an ATP-dependent manner. These findings clarified two characteristic properties of
cAPC. First,
cAPC has an ability to aggregate to form polymers. To date, the first 45 amino acids of APC were reported to be necessary for APC dimerization (
cAPC as well as full-length APC can aggregate to form large granular structures. Second,
cAPC also interacts with MTs. If some kinesin-like motors are postulated to be associated with the
cAPC granules, the
cAPC/MT interaction as well as the ATP-dependent movement of granules along MTs toward their plus ends can be explained. However, other molecular mechanisms could also be responsible for this interaction and movement, and identification of
cAPC-binding proteins as well as in vitro motility analyses are required in the future studies.
In summary, among peculiar behaviors of full-length APC in live cells, its COOH-terminal domain (cAPC) and the remaining portion (cAPC) appear to be responsible for the high affinity to the ends of growing MTs and granular formation as well as translocation along MTs toward the plus ends, respectively. In addition to these behaviors, as shown in Fig 5, during cell spreading and wound healing, APC granules clustered in specific regions of the cell periphery with concomitant formation of cellular extensions, resulting in the accumulation of APC granules at the tip regions of cellular extensions, i.e., in these cells, a subset of MTs were selected to be decorated by full-length APC. The ability to select MTs appeared to be attributed not to the COOH-terminal domain (cAPC) but to the remaining portion of APC (
cAPC); GFP-cAPC labeled all MTs (see Fig 7), whereas
cAPC-GFP granules showed a tendency to concentrate at some cellular extensions (see Fig 8 a). These findings favor the notion that APC plays some important role in polarized cellular morphogenesis through the stabilization of cellular extensions (
Much attention has been focused on the function of APC that pertains to its ability to regulate ß-catenin activity (for reviews see
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: aa, amino acids; AMP-PNP, 5'-adenylylimidodiphosphate; APC, adenomatous polyposis coli; GFP, green fluorescent protein; MAP, microtubule-associated protein; MT, microtubule; pAb, polyclonal antibody.
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Acknowledgements |
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We thank Drs. A. Nagafuchi (Kyoto University) and H. Oda (Tsukita Cell Axis Project) for valuable discussions. Our thanks are also due to Dr. S. Yonemura (Kyoto University) for technical help with electron microscopy, to Dr. A. Asano (Tsukita Cell Axis Project) for critically reading the manuscript, and to all the other members of our laboratory for helpful discussions and technical assistance.
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References |
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