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Figure 1. Structures of GFP fusion proteins with full-length APC and deletion mutants of APC. Xenopus APC (2829 aa) was tentatively divided into two portions: COOH-terminal (aa 2159–2829) and the remaining portions (aa 1–2158). Based on these portions, two deletion mutants of APC were constructed and fused with GFP. The numbers indicate the amino acid positions.
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Figure 3. Subcellular distribution of endogenous APC and GFP-tagged full-length APC in stable A6 transfectants. (a) Parental A6 cells were double stained with anti-APC pAb (red) and anti-tubulin mAb (green), and observed with DeltaVision microscope. (b and c) A6 transfectants expressing fAPC-GFP (clone A1; see Fig. 2) were fixed and immunofluorescently stained with anti-tubulin mAb, and observed by confocal microscopy. Expressed fAPC-GFP and MTs were visualized as green and red fluorescence, respectively. In b, fAPC-GFP was concentrated at the tip regions of some cellular extensions, where fAPC-GFP was associated with MTs to form thick bundles (inset). In thin lamellae (c), where MTs spread out toward the cellular edges (toward right), fAPC-GFP was visualized as granules on the distal ends of individual MTs. Note that the subcellular distribution of fAPC-GFP in b was very similar to that of endogenous APC in a. Bars: (a and b) 20 μm; (c) 10 μm; (insets) 5 μm.
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Figure 2. Expression levels of endogenous APC and exogenous GFP-tagged full-length APC. To separate the band of endogenous APC (lower arrowhead) from that of exogenous GFP-tagged APC (upper arrowhead), cell lysates of parental A6 cells and stable clones (A1, A4, and B4 for fAPC-GFP–expressing transfectants, and A4, B1, and C1 for fAPC-mGFP–expressing transfectants) were preincubated with λ-protein phosphatase followed by SDS-PAGE and immunoblotting with anti-GFP pAb (GFP) or anti-APC mAb, APC(Ab-1), (APC). In each lane, the cell lysate from 1 × 106 cells was applied. The amount of endogenous APC in parental A6 cells (Q1) and GFP-tagged APC in stable clones (Q2) were quantified as described in Materials and Methods, and their Q2/Q1 ratios were calculated.
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Figure 4. Immunolabeling of fAPC-GFP aggregates with anti-GFP pAb in A6 transfectants at the electron microscopic level. A6 transfectants expressing large amounts of fAPC-GFP (clone B4; see Fig. 2) were permeabilized with digitonin and incubated with rabbit anti-GFP pAb, followed by incubation with 5-nm gold-conjugated anti-rabbit IgG pAb. Abnormal thick bundles of MTs were occasionally observed (arrows in a). Boxed areas in a, b, and e were enlarged in b, d, and f, respectively. Both longitudinal (b) and transverse (c) sectional images of these thick MT bundles revealed that in these bundles, MTs were embedded in fuzzy matrices, which were directly labeled with anti-GFP pAb. Single microtubules surrounded by immunolabeled fuzzy structures were also occasionally observed (d–f). Note that no membranous vesicles were labeled with immunogold particles. Bars: (a) 1,000 nm; (b and e) 200 nm; (c, d, and f) 100 nm.
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Figure 5. The behavior of full-length APC in live cells (clone C1; see Fig. 2) during cell spreading and wound healing. (a) A6 transfectants stably expressing fAPC-mGFP were replated on glass-bottomed dishes, and a series of fluorescence microscopic images were obtained. The aggregates of fAPC-mGFP were visualized as green fluorescence. Plasma membranes of these cells were labeled with PKH26 in red (some endocytotic vesicles were also labeled in red). Cells were replated 1 h after trypsinization and recording of the images was started 15 min after replating. Only the GFP signal in boxed areas in color plates were selected in corresponding black-and-white plates at larger magnification. See details in the text. Elapsed time was indicated at top right (in min:s). QuickTime video is available at http://www.jcb.org/cgi/content/full/148/3/505/DC1. (b) Confluent cultures of A6 transfectants expressing fAPC-mGFP were manually scratched with sharp needles. The top panel shows a phase-contrast image of cells at the edge of the wound shortly after wounding. Second to fourth panels (times 00:00:00–02:02:03) show fluorescence images of fAPC-mGFP with the same scale and position as the top phase-contrast panel. Fifth to last panels (times 02:02:03–02:49:07) show a series of magnified images of the boxed area in the fourth panel. See details in the text. Elapsed time is indicated at the bottom (in h:min:s). (c) A6 transfectants expressing fAPC-mGFP were incubated with 33 μM nocodazole to disassemble MTs for 10 min, 30 min, and 3 h, then fixed and immunofluorescently stained with anti-tubulin mAb in red. The fAPC-mGFP was visualized as green fluorescence. See details in the text. Bars: (a, b, top, and c) 10 μm; (b, bottom) 5 μm.
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Figure 6. The behavior of full-length APC in live cells at the distal ends of MTs. (a and b) A series of time-lapse images was obtained from a thin lamella of a A6 cell expressing fAPC-GFP (clone A1; see Fig. 2) or fAPC-mGFP (transient expression), respectively. In these thin lamellae, individual MTs were spread out toward the leading edge of the cell, which was directed to the left. Since GFP-tagged molecules were not only concentrated at the distal ends of MTs but also evenly covered individual MTs, imaging only by GFP fluorescence allowed us to observe the dynamic behavior of fAPC-GFP/fAPC-mGFP as well as MTs simultaneously. Asterisks and arrowheads indicate the rearward movement of the bending points of MTs and the granules dropped off from shortening MT ends, respectively. See details in the text. Microtubule-A/B and -C in b were colored in yellow and pink on the computer, respectively. (c) Another series of time-lapse images of clone B4 (see Fig. 2) showing the continuous flow of fAPC-GFP along MTs (arrows and arrowheads) toward their distal ends. This continuous flow is clearly visualized in the video. In a–c, elapsed time is indicated at bottom (in min:s). QuickTime videos for a–c are available at http://www.jcb.org/cgi/content/full/148/3/505/DC1. Bars: 3 μm.
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Figure 7. The behavior of the COOH-terminal region of APC in live cells. A6 cells transiently expressing GFP-cAPC (see Fig. 1) were observed. (a) In A6 cells expressing relatively low level of GFP-cAPC, GFP-cAPC was distributed along the entire length of all MTs, but was significantly concentrated at the distal ends of MTs. (b) The dynamic behavior of GFP-cAPC at the distal ends of two MTs (A and B) in the lamellae, which was directed to the top. Only when these MTs continued to grow, the distal ends of MTs were highlighted by the GFP signal (arrowheads). When MTs were switched from growth to shortening phase, the GFP-cAPC concentrated at ends of MTs abruptly disappeared (arrows). (c) Effects of low concentration of nocodazole (100 nM), which affects the assembly/disassembly dynamics of MTs at their plus ends without changing their polymer mass, on the behavior of GFP-cAPC at the ends of MTs. The boxed area in a was enlarged in c. Within 1-min incubation with nocodazole, the concentration of GFP-cAPC at MT ends became undetectable, leaving evenly distributed GFP-cAPC along the entire length of MTs. (d) Quantitative analyses of the relationship between the length of MT-A and the degree of concentration of GFP-cAPC on their ends in b. The upper panel is a plot of MT length and the lower panel represents changes of MT length (open squares; right-hand scale) and the ratios of fluorescence intensities between the MT end and the proximal segments (closed circles, left-hand scale; see Materials and Methods). This quantification clearly showed the specific association of the GFP-cAPC concentration with growing MTs but not with shortening MTs. Elapsed time is indicated at top left (in min:s). Bars: (a) 10 μm; (b) 3 μm; (c) 5 μm. QuickTime videos for b and c are available at http://www.jcb.org/cgi/content/full/148/3/505/DC1.
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Figure 8. Subcellular distribution and the behavior of the deletion mutant of APC lacking its COOH-terminal region in A6 transfectants. (a and b) A6 cells expressing ΔcAPC-GFP (see Fig. 1) were fixed and stained with anti-tubulin mAb in red. Expressed ΔcAPC-GFP formed granular structures (green) with various diameters scattered around the cytoplasm. These granules occasionally clustered in the tip region of some, but not all, of the cellular extensions (arrows in a). In thin lamellae (b), these granules were shown to be aligned along MTs (arrowheads in b). (c) A series of time-lapse fluorescence images was obtained from cellular extensions of live A6 cells expressing ΔcAPC-GFP. Since, differently from fAPC-mGFP and GFP-cAPC, ΔcAPC-GFP did not decorate MTs, MTs were not visible in these images. A large granule moving linearly toward the cell edge is indicated by an arrowhead. A QuickTime video is available at http://www.jcb.org/cgi/content/full/148/3/505/DC1. (d) Movements of the ΔcAPC-GFP granules in permeabilized cells. A6 cells expressing ΔcAPC-GFP were replated, cultured for 4 h, and then permeabilized with digitonin in the presence of 10 μM taxol. Addition of 0.2 mM ATP induced linear movements of the granules toward the cell periphery (times −01:50–01:45, arrows). Further addition of 1 mM AMP-PNP at time 00:00 blocked these movements (times 01:45–05:35, arrowheads). Elapsed time is indicated at top left (in min:s). Bars: (a and b) 10 μm; (c) 5 μm; (d) 3 μm.
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