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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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Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II cell monolayers infected with the indicated adenovirus vectors and subjected to calcium switch were labeled for 10 min on ice with BODIPY-sphingomyelin. The cells were then immediately mounted (0 min) or left for an additional 60 min on ice after extensive washing. The distribution of the fluorescent lipid was then analyzed by taking z-sectional views using a confocal microscope. The upper and lower arrowheads indicate the positions of the apical and basal membranes, respectively. Bar, 25 μm. (b and c) Effect of aPKCλkn on the asymmetric distribution of epithelial polarity markers. Adenovirally infected MDCK II cells were doubly immunostained with anti–aPKCλ and anti–Na+, K+-ATPase (b) or anti–gp135 antibodies (c) 20 h after calcium switch. Representative xz-sectional views obtained by confocal microscopic analysis are presented. Note that aPKCλkn-expressing cells (arrowheads) exhibit disturbed localization of NKA as well as gp135, proteins that show polarized distributions in LacZ-expressing control cells. Bars, 25 μm.
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Figure 6: Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II cell monolayers infected with the indicated adenovirus vectors and subjected to calcium switch were labeled for 10 min on ice with BODIPY-sphingomyelin. The cells were then immediately mounted (0 min) or left for an additional 60 min on ice after extensive washing. The distribution of the fluorescent lipid was then analyzed by taking z-sectional views using a confocal microscope. The upper and lower arrowheads indicate the positions of the apical and basal membranes, respectively. Bar, 25 μm. (b and c) Effect of aPKCλkn on the asymmetric distribution of epithelial polarity markers. Adenovirally infected MDCK II cells were doubly immunostained with anti–aPKCλ and anti–Na+, K+-ATPase (b) or anti–gp135 antibodies (c) 20 h after calcium switch. Representative xz-sectional views obtained by confocal microscopic analysis are presented. Note that aPKCλkn-expressing cells (arrowheads) exhibit disturbed localization of NKA as well as gp135, proteins that show polarized distributions in LacZ-expressing control cells. Bars, 25 μm.

Mentions: TJ have been suggested to contribute to the establishment of epithelial cell surface polarity by acting as fences for the diffusion of lipids in the outer leaflet of the plasma membrane between the apical and basolateral membrane domains (van Meer and Simons 1986). Therefore, the finding that functional depletion of aPKC blocks the development of the epithelia-specific junctional structures, including TJ, suggests the possibility that aPKCkn expression also results in the disruption of epithelial cell surface polarity. To confirm this possibility, we examined the two-dimensional diffusion of ectopically introduced fluorescent lipids in aPKCλkn-expressing cells using confocal microscopy. In Fig. 6 a, the apical membranes were labeled with BODIPY-sphingomyelin for 10 min at 4°C, and left for an additional 60 min on ice. Confocal microscopic analysis of the x–z sections of these cells demonstrated that only aPKCλkn-expressing cells show markedly enhanced labeling of the lateral membrane after a 60-min chase, suggesting a reduced diffusion fence between the apical and basolateral membranes.


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)

Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II cell monolayers infected with the indicated adenovirus vectors and subjected to calcium switch were labeled for 10 min on ice with BODIPY-sphingomyelin. The cells were then immediately mounted (0 min) or left for an additional 60 min on ice after extensive washing. The distribution of the fluorescent lipid was then analyzed by taking z-sectional views using a confocal microscope. The upper and lower arrowheads indicate the positions of the apical and basal membranes, respectively. Bar, 25 μm. (b and c) Effect of aPKCλkn on the asymmetric distribution of epithelial polarity markers. Adenovirally infected MDCK II cells were doubly immunostained with anti–aPKCλ and anti–Na+, K+-ATPase (b) or anti–gp135 antibodies (c) 20 h after calcium switch. Representative xz-sectional views obtained by confocal microscopic analysis are presented. Note that aPKCλkn-expressing cells (arrowheads) exhibit disturbed localization of NKA as well as gp135, proteins that show polarized distributions in LacZ-expressing control cells. Bars, 25 μm.
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Figure 6: Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II cell monolayers infected with the indicated adenovirus vectors and subjected to calcium switch were labeled for 10 min on ice with BODIPY-sphingomyelin. The cells were then immediately mounted (0 min) or left for an additional 60 min on ice after extensive washing. The distribution of the fluorescent lipid was then analyzed by taking z-sectional views using a confocal microscope. The upper and lower arrowheads indicate the positions of the apical and basal membranes, respectively. Bar, 25 μm. (b and c) Effect of aPKCλkn on the asymmetric distribution of epithelial polarity markers. Adenovirally infected MDCK II cells were doubly immunostained with anti–aPKCλ and anti–Na+, K+-ATPase (b) or anti–gp135 antibodies (c) 20 h after calcium switch. Representative xz-sectional views obtained by confocal microscopic analysis are presented. Note that aPKCλkn-expressing cells (arrowheads) exhibit disturbed localization of NKA as well as gp135, proteins that show polarized distributions in LacZ-expressing control cells. Bars, 25 μm.
Mentions: TJ have been suggested to contribute to the establishment of epithelial cell surface polarity by acting as fences for the diffusion of lipids in the outer leaflet of the plasma membrane between the apical and basolateral membrane domains (van Meer and Simons 1986). Therefore, the finding that functional depletion of aPKC blocks the development of the epithelia-specific junctional structures, including TJ, suggests the possibility that aPKCkn expression also results in the disruption of epithelial cell surface polarity. To confirm this possibility, we examined the two-dimensional diffusion of ectopically introduced fluorescent lipids in aPKCλkn-expressing cells using confocal microscopy. In Fig. 6 a, the apical membranes were labeled with BODIPY-sphingomyelin for 10 min at 4°C, and left for an additional 60 min on ice. Confocal microscopic analysis of the x–z sections of these cells demonstrated that only aPKCλkn-expressing cells show markedly enhanced labeling of the lateral membrane after a 60-min chase, suggesting a reduced diffusion fence between the apical and basolateral membranes.

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
Related in: MedlinePlus