Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.
Loss of functional adenomatous polyposis coli (APC)*
Abbreviation used in this paper: APC, adenomatous polyposis coli.
A for schematic). The organ of Corti consists mainly of a strip of neuroepithelial tissue. Sensory hair cells and the adjacent supporting cells are arranged in rows that extend along the length of this strip (Lim, 1986; see Fig. 1 A). This organization together with the cytoskeletal organization of the supporting cells is crucial for efficient transmission of vibrations to the sensory hair cells and thus for auditory perception (Patuzzi, 1996). Supporting cells contain an extremely high number of microtubules organized in an apico-basal array that facilitates the detection of low abundance proteins that specifically associate with microtubule ends (Henderson et al., 1994; Tucker et al., 1992, 1995; Mogensen et al., 1997). In comparison, epithelial cells in the gut contain an order of magnitude fewer microtubules, making their preservation and the detection of microtubule ends significantly more difficult.
We found that APC concentrates near the basal plasma membrane of supporting cells where microtubule plus ends terminate in a dense matrix. Hook decoration was used to confirm that the microtubules in these highly polarized cells are oriented with their plus ends near the base and their minus ends near the apex. Ninein, a microtubule minus end binding and anchoring protein is found near the apex of the cell, further supporting this organization (Bouckson-Castaing et al., 1996; Mogensen et al., 2000; Piel et al., 2000). During the development and assembly of this highly structured, polarized microtubule array, APC associates with microtubules as they extend toward the cellular base. These data suggest that APC may play an important role in the stabilization of the microtubule arrays during their formation. This was further supported by our finding that in the cochlea of Min mice, which are heterozygous for APC and express reduced levels of full-length APC protein, these apico-basal microtubule arrays showed a significant reduction in the number of microtubules present in the parallel bundles when compared with wild-type litter mates.
Results And Discussion
Specificity of APC antibodies
To confirm the specificity of available antibodies against APC, cell lysates from human colonic tumor cells with wild-type (HCT116) and truncated APC (DLD1) (Rowan et al., 2000) and UE1 cultured mouse inner ear cells expressing full-length APC (Lawlor et al., 1999) were probed with a panel of three different polyclonal APC antibodies (Fig. 1, B and C). Affinity-purified antiserum raised against the middle domain of APC, and crude serum raised against the NH2-terminal domain (Midgley et al., 1997), detected APC as the major protein in all lysates (Fig. 1, B and C). A commercially available, affinity-purified anti-APC antiserum, N15 (raised against the NH2-terminal domain of APC) did not detect any APC protein in the lysates from human cells, even after prolonged exposure and only very faintly detected APC in mouse UE1 cells. Instead, proteins with molecular masses of 65–85 kD were detected as major bands by this antibody in human cells lysates, and the 65-kD protein was also detected in the mouse cells. After longer exposure (Fig. 1 C), a band that comigrates with full-length APC appeared in blots exposed to N15 in all samples including DLD1 cell lysates, although these cells do not contain full-length APC. It is important to note that the gels shown in Fig. 1 (B and C) were simply scanned and not processed any further. Additionally, the entire gel lanes are depicted showing all proteins above 35 kD. These data confirm that the N15 antibody is not suitable for detecting endogenous APC. For our studies, we used the affinity-purified anti–M-APC antibody. However, immunofluorescence staining with another anti–C-APC antibody (Midgley et al., 1997) gives identical results in cultured cells, and the staining pattern with either the anti–M-APC or anti–C-APC antiserum is independent of fixation conditions.
Microtubule positioning in supporting cells
The association of APC with microtubules in epithelial cells has been well documented and suggests that APC associates primarily with microtubule ends. However, microtubule polarity has only been inferred and never been directly demonstrated relative to APC accumulations (Näthke et al., 1996; Mimori-Kiyosue et al., 2000). Cultured colonic tumor cells, the subject of previous investigations, polarize their membrane domains when grown to confluency on glass coverslips, however, they rarely polarize their microtubule network under these conditions, making it impossible to identify specific microtubule ends and associated proteins unambiguously (unpublished data). To establish the localization of endogenous APC protein in polarized epithelial cells with a well-defined microtubule organization, we used supporting cells isolated from the organ of Corti (Fig. 1 A, schematic). These epithelial cells contain an apico-basal array of several thousand microtubules that provides a large target for end-associated proteins allowing their unambiguous detection.
We examined microtubule polarity in all three types of supporting cells in the organ of Corti: inner pillar cells, outer pillar cells, and Deiters cells. All three contain large microtubule arrays. Mature supporting pillar or Deiters cells contain two microtubule arrays whose ends are anchored at the apex and base of the cell. The apical end of one of the arrays in each cell is situated near the apical centrosome and its centrioles (Fig. 1 A, dark blue). The apical end of the other array is remotely located with respect to the centrosome (>10 μm distant; Fig. 1 A, light blue). The largest arrays occur in the pillar cells and include several thousand microtubules (Henderson et al., 1995; Tucker et al., 1995). In these cells, the microtubules splay at the cell base to either side of cone-shaped fibrous meshworks rather than terminating within them. The ends of many of these microtubules are situated within 0.5 μm of the basal membrane (see Fig. 4
The microtubules in inner and outer pillar cells are arranged in apico-basal arrays with the plus ends at the basal membrane
To determine the orientation of the microtubule array in the supporting cells we performed hook decoration experiments in the organ of Corti from three mature and one 6 d cochlea when assembly of the apico-basal array is still progressing (for a detailed description of these results see
We would like to thank Michel Bornens (Institue Curie, Paris) for the gift of the antininein antibody, Matthew Holley (University of Sheffield) for the UE1 cell line, Charles Patek (Western General Hospital, Edinburgh) for the gift of the Min mice, Birgit Lane for support and use of lab space for M.M. Mogensen, John James and Richard Evans-Gowing for assistance with TEM analysis of the Min mutants. We are grateful to Dina Dikovskaya for valuable comments on the manuscript.
This work was supported by grants from the Medical Research Council (Grant G9326558MB) to J.B. Tucker, J.B. Mackie, and M.M. Mogensen, the Wellcome Trust to A.R. Prescott and M.M. Mogensen (Grant 049616/Z/96/Z/WRE/MK/JAT). I.S. Näthke is supported by a Cancer Research United Kingdom Senior Research Fellowship and a Burroughs Wellcome Fund Career Development Award.
The online version of this article contains supplemental material.