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Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

Suzuki A, Yamanaka T, Hirose T, Manabe N, Mizuno K, Shimizu M, Akimoto K, Izumi Y, Ohnishi T, Ohno S - J. Cell Biol. (2001)

Bottom Line: Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells.On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells.These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

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Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and GC2AP (b). IgG means an equal amount of normal rabbit IgG used as a negative control. In b, the photograph was taken at higher sensitivity than in a to visualize weak cell–cell staining by GC2AP. (c) Confocal z-sectional view of MDCK cells doubly stained with anti–PAR-6 (GW2AP), anti–aPKCλ (λ1), or anti–ASIP antibodies (green), together with anti–ZO-1 antibody (red). The arrowhead indicates the position of the basal membrane. All three proteins colocalize with ZO-1 to the apical end of lateral membrane. (d) Immunostaining of a frozen section of mouse intestinal epithelium with anti–PAR-6 (GW2AP), anti–aPKCλ, or anti–ASIP antibodies (green). Only merged views with anti–ZO-1 staining (red) are shown. (e) Overexpression of aPKCλkn affects the junctional localization of PAR-6 as well as ZO-1. Bars, 25 μm (a, b, d, and e) and 10 μm (c).
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Figure 9: Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and GC2AP (b). IgG means an equal amount of normal rabbit IgG used as a negative control. In b, the photograph was taken at higher sensitivity than in a to visualize weak cell–cell staining by GC2AP. (c) Confocal z-sectional view of MDCK cells doubly stained with anti–PAR-6 (GW2AP), anti–aPKCλ (λ1), or anti–ASIP antibodies (green), together with anti–ZO-1 antibody (red). The arrowhead indicates the position of the basal membrane. All three proteins colocalize with ZO-1 to the apical end of lateral membrane. (d) Immunostaining of a frozen section of mouse intestinal epithelium with anti–PAR-6 (GW2AP), anti–aPKCλ, or anti–ASIP antibodies (green). Only merged views with anti–ZO-1 staining (red) are shown. (e) Overexpression of aPKCλkn affects the junctional localization of PAR-6 as well as ZO-1. Bars, 25 μm (a, b, d, and e) and 10 μm (c).

Mentions: To evaluate the physiological significance of the physical interaction between aPKCλ, ASIP/PAR-3, and PAR-6 in epithelial cells, we next examined the intracellular localization of PAR-6 in MDCK cells. As shown in Fig. 9 a, the anti–PAR-6 antibody, GW2AP, clearly stains the cell–cell boundary of confluent MDCK II cells. Since the similar result was obtained with the other independent antibody, GC2AP (Fig. 9 b), we concluded that these junctional stainings represent the genuine localization of endogenous PAR-6 in MDCK II cells. Closer inspection of the localization of endogenous PAR-6 by confocal z-sectioning revealed PAR-6 staining at the most apical end of the cell–cell contact region with ZO-1 (Fig. 9 c). Since, as previously suggested (Izumi et al. 1998), aPKCλ and ASIP/PAR-3 also localize to the corresponding region with ZO-1 (Fig. 9 c), these results strongly suggest that PAR-6 colocalizes with aPKCλ and ASIP/PAR-3 to the apical junctional complex of epithelial cells. To clarify further the localization of PAR-6 in epithelial cells, we next stained mouse intestinal epithelia, a typical tissue containing polarized epithelial cells. Similar to aPKCλ and ASIP/PAR-3, PAR-6 also localizes to the most apical end of the junctional complex with ZO-1 (Fig. 9 d). In addition, like ASIP/PAR-3, the junctional localization of PAR-6 is severely disturbed in aPKCλkn-expressing cells, showing complete colocalization with ZO-1 (Fig. 9 e). Taken together with the physical interactions among these three proteins, the above results provide evidence supporting the idea that PAR-6, aPKCλ, and ASIP/PAR-3 asymmetrically localize in the apical junctional complex in polarized epithelial cells as a ternary protein complex.


Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

Suzuki A, Yamanaka T, Hirose T, Manabe N, Mizuno K, Shimizu M, Akimoto K, Izumi Y, Ohnishi T, Ohno S - J. Cell Biol. (2001)

Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and GC2AP (b). IgG means an equal amount of normal rabbit IgG used as a negative control. In b, the photograph was taken at higher sensitivity than in a to visualize weak cell–cell staining by GC2AP. (c) Confocal z-sectional view of MDCK cells doubly stained with anti–PAR-6 (GW2AP), anti–aPKCλ (λ1), or anti–ASIP antibodies (green), together with anti–ZO-1 antibody (red). The arrowhead indicates the position of the basal membrane. All three proteins colocalize with ZO-1 to the apical end of lateral membrane. (d) Immunostaining of a frozen section of mouse intestinal epithelium with anti–PAR-6 (GW2AP), anti–aPKCλ, or anti–ASIP antibodies (green). Only merged views with anti–ZO-1 staining (red) are shown. (e) Overexpression of aPKCλkn affects the junctional localization of PAR-6 as well as ZO-1. Bars, 25 μm (a, b, d, and e) and 10 μm (c).
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Figure 9: Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and GC2AP (b). IgG means an equal amount of normal rabbit IgG used as a negative control. In b, the photograph was taken at higher sensitivity than in a to visualize weak cell–cell staining by GC2AP. (c) Confocal z-sectional view of MDCK cells doubly stained with anti–PAR-6 (GW2AP), anti–aPKCλ (λ1), or anti–ASIP antibodies (green), together with anti–ZO-1 antibody (red). The arrowhead indicates the position of the basal membrane. All three proteins colocalize with ZO-1 to the apical end of lateral membrane. (d) Immunostaining of a frozen section of mouse intestinal epithelium with anti–PAR-6 (GW2AP), anti–aPKCλ, or anti–ASIP antibodies (green). Only merged views with anti–ZO-1 staining (red) are shown. (e) Overexpression of aPKCλkn affects the junctional localization of PAR-6 as well as ZO-1. Bars, 25 μm (a, b, d, and e) and 10 μm (c).
Mentions: To evaluate the physiological significance of the physical interaction between aPKCλ, ASIP/PAR-3, and PAR-6 in epithelial cells, we next examined the intracellular localization of PAR-6 in MDCK cells. As shown in Fig. 9 a, the anti–PAR-6 antibody, GW2AP, clearly stains the cell–cell boundary of confluent MDCK II cells. Since the similar result was obtained with the other independent antibody, GC2AP (Fig. 9 b), we concluded that these junctional stainings represent the genuine localization of endogenous PAR-6 in MDCK II cells. Closer inspection of the localization of endogenous PAR-6 by confocal z-sectioning revealed PAR-6 staining at the most apical end of the cell–cell contact region with ZO-1 (Fig. 9 c). Since, as previously suggested (Izumi et al. 1998), aPKCλ and ASIP/PAR-3 also localize to the corresponding region with ZO-1 (Fig. 9 c), these results strongly suggest that PAR-6 colocalizes with aPKCλ and ASIP/PAR-3 to the apical junctional complex of epithelial cells. To clarify further the localization of PAR-6 in epithelial cells, we next stained mouse intestinal epithelia, a typical tissue containing polarized epithelial cells. Similar to aPKCλ and ASIP/PAR-3, PAR-6 also localizes to the most apical end of the junctional complex with ZO-1 (Fig. 9 d). In addition, like ASIP/PAR-3, the junctional localization of PAR-6 is severely disturbed in aPKCλkn-expressing cells, showing complete colocalization with ZO-1 (Fig. 9 e). Taken together with the physical interactions among these three proteins, the above results provide evidence supporting the idea that PAR-6, aPKCλ, and ASIP/PAR-3 asymmetrically localize in the apical junctional complex in polarized epithelial cells as a ternary protein complex.

Bottom Line: Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells.On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells.These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

Show MeSH