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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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The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 104 cells/cm2) on cover slips and, 40 h later, subjected to immunofluorescent analysis. (Top) The antibodies used are indicated. Bar, 25 μm.
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Figure 3: The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 104 cells/cm2) on cover slips and, 40 h later, subjected to immunofluorescent analysis. (Top) The antibodies used are indicated. Bar, 25 μm.

Mentions: The calcium switch dependence of the effects of aPKCλkn suggests that this dominant-negative mutant is effective only when the cells develop junctional structures to establish epithelial cell polarity. In fact, we further observed the similar effects of aPKCλkn when cells form de novo cell–cell contacts and develop TJ under normal growth conditions. As shown in Fig. 3, where cells were sparsely reseeded immediately after viral infection and cultured for 40 h before immunofluorescent analysis, cells expressing LacZ or aPKCλwt show a normal appearance while aPKCλkn-expressing cells exhibit a flattened shape with prominent lamellipodia (Fig. 3 c) with many cell–cell boundaries negative for ASIP/PAR-3 and ZO-1 staining (Fig. 3, a and b). However, it should be noted that aPKCλkn-expressing cells still form islands and remain close to each other through cell–cell adhesions even in the absence of ASIP/PAR-3 or ZO-1 staining at the cell–cell boundary (Fig. 3 c). These results suggest that the effect of aPKCλkn on TJ formation is not the result of the complete disruption of cell–cell adhesions.


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)

The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 104 cells/cm2) on cover slips and, 40 h later, subjected to immunofluorescent analysis. (Top) The antibodies used are indicated. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199212&req=5

Figure 3: The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 104 cells/cm2) on cover slips and, 40 h later, subjected to immunofluorescent analysis. (Top) The antibodies used are indicated. Bar, 25 μm.
Mentions: The calcium switch dependence of the effects of aPKCλkn suggests that this dominant-negative mutant is effective only when the cells develop junctional structures to establish epithelial cell polarity. In fact, we further observed the similar effects of aPKCλkn when cells form de novo cell–cell contacts and develop TJ under normal growth conditions. As shown in Fig. 3, where cells were sparsely reseeded immediately after viral infection and cultured for 40 h before immunofluorescent analysis, cells expressing LacZ or aPKCλwt show a normal appearance while aPKCλkn-expressing cells exhibit a flattened shape with prominent lamellipodia (Fig. 3 c) with many cell–cell boundaries negative for ASIP/PAR-3 and ZO-1 staining (Fig. 3, a and b). However, it should be noted that aPKCλkn-expressing cells still form islands and remain close to each other through cell–cell adhesions even in the absence of ASIP/PAR-3 or ZO-1 staining at the cell–cell boundary (Fig. 3 c). These results suggest that the effect of aPKCλkn on TJ formation is not the result of the complete disruption of cell–cell adhesions.

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