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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 inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins are as follows: none (X), LacZ (•), aPKCλkn (□), aPKCλwt (▵), and nPKCδkn (○). 2 d after cell seeding (1.5 × 105 cells/cm2) when the TER values reached a plateau, the cells were processed for adenovirus infection (time 0, left) for 2 h in LC medium. Immediately after virus infection, the medium was changed to NC growth medium to allow the cells to complete junctional structure formation before the expression of ectopic proteins. After TER measurement for 45 h, the cells were subjected to calcium switch by changing the medium to LC for 2 h, and then back to NC. The values given represent mean values (1 ± SD) of three parallel cultures. The background resistance obtained from empty filters was deducted. Note that only cells expressing aPKCλkn show a retardation in TER development after calcium switch. (Bottom) MDCK II cells were infected with adenovirus vectors encoding LacZ (•), aPKCλkn (□), or aPKCλwt (▵) as in a. In this case, the cells were kept in fresh LC medium to induce ectopic protein expression in the absence of cell–cell adhesion. 20 h after virus infection, the medium was changed to NC medium, and the development of TER was monitored for 48 h. Note that aPKCλkn-expressing cells show substantial suppression of TER development even 48 h after calcium switch. (b) Overexpression of aPKCλkn increases the paracellular diffusion of FITC-dextran in a molecular mass-dependent manner. Paracellular diffusion of FITC-dextran 40K (left) and 500K (right) across adenovirus-infected MDCK II cells was evaluated. FITC-dextran was added to the medium on the apical side of cells subjected to calcium switch 2 d before. After 3 h incubation at 37°C, the fluorescence intensity of the medium in the basolateral side was measured with a fluorometer. Values given represent the mean values (±SD) of three parallel cultures.
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Figure 5: Overexpression of aPKCλkn inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins are as follows: none (X), LacZ (•), aPKCλkn (□), aPKCλwt (▵), and nPKCδkn (○). 2 d after cell seeding (1.5 × 105 cells/cm2) when the TER values reached a plateau, the cells were processed for adenovirus infection (time 0, left) for 2 h in LC medium. Immediately after virus infection, the medium was changed to NC growth medium to allow the cells to complete junctional structure formation before the expression of ectopic proteins. After TER measurement for 45 h, the cells were subjected to calcium switch by changing the medium to LC for 2 h, and then back to NC. The values given represent mean values (1 ± SD) of three parallel cultures. The background resistance obtained from empty filters was deducted. Note that only cells expressing aPKCλkn show a retardation in TER development after calcium switch. (Bottom) MDCK II cells were infected with adenovirus vectors encoding LacZ (•), aPKCλkn (□), or aPKCλwt (▵) as in a. In this case, the cells were kept in fresh LC medium to induce ectopic protein expression in the absence of cell–cell adhesion. 20 h after virus infection, the medium was changed to NC medium, and the development of TER was monitored for 48 h. Note that aPKCλkn-expressing cells show substantial suppression of TER development even 48 h after calcium switch. (b) Overexpression of aPKCλkn increases the paracellular diffusion of FITC-dextran in a molecular mass-dependent manner. Paracellular diffusion of FITC-dextran 40K (left) and 500K (right) across adenovirus-infected MDCK II cells was evaluated. FITC-dextran was added to the medium on the apical side of cells subjected to calcium switch 2 d before. After 3 h incubation at 37°C, the fluorescence intensity of the medium in the basolateral side was measured with a fluorometer. Values given represent the mean values (±SD) of three parallel cultures.

Mentions: The defects in TJ formation in aPKCλkn-expressing cells was further demonstrated functionally by measuring TER to passive ion flow across a cell monolayer grown on permeable support (Fig. 5 a). Consistent with the immunostaining results shown in Fig. 1 a, none of the ectopically expressed proteins, including LacZ, nPKCδkn, or aPKCλwt, as well as aPKCλkn, affected TER if the experiments were arranged so as to induce the expression of the ectopic proteins after the completion of TJ biogenesis (Fig. 5 a, top, 0–45 h). This also suggests that the overexpression of these proteins, especially aPKCλkn, does not produce any artificial cytotoxic effects on TJ barrier function. On the other hand, if the cells were subsequently subjected to calcium switch (2-h incubation in LC medium followed by switching to NC medium), only cells expressing aPKCλkn showed a large retardation in TER development (Fig. 5 a, top, 45–70 h). If the preincubation in low calcium medium was prolonged to 20 h to ensure the dissociation of cell–cell attachments before calcium switch, the effect of aPKCλkn on TER development was more significant: similar to the immunostaining experiments shown in Fig. 1, the expression of ectopic proteins was induced in the cells cultured in LC medium, and calcium switch was applied 20 h after virus infection (Fig. 5 a, bottom). In this case, TER development of aPKCλkn-expressing cells was substantially suppressed until 48 h after calcium switch compared with LacZ- or aPKCλwt-expressing control cells, suggesting that the development of functional TJ is strongly suppressed in aPKCλkn-expressing cells.


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 inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins are as follows: none (X), LacZ (•), aPKCλkn (□), aPKCλwt (▵), and nPKCδkn (○). 2 d after cell seeding (1.5 × 105 cells/cm2) when the TER values reached a plateau, the cells were processed for adenovirus infection (time 0, left) for 2 h in LC medium. Immediately after virus infection, the medium was changed to NC growth medium to allow the cells to complete junctional structure formation before the expression of ectopic proteins. After TER measurement for 45 h, the cells were subjected to calcium switch by changing the medium to LC for 2 h, and then back to NC. The values given represent mean values (1 ± SD) of three parallel cultures. The background resistance obtained from empty filters was deducted. Note that only cells expressing aPKCλkn show a retardation in TER development after calcium switch. (Bottom) MDCK II cells were infected with adenovirus vectors encoding LacZ (•), aPKCλkn (□), or aPKCλwt (▵) as in a. In this case, the cells were kept in fresh LC medium to induce ectopic protein expression in the absence of cell–cell adhesion. 20 h after virus infection, the medium was changed to NC medium, and the development of TER was monitored for 48 h. Note that aPKCλkn-expressing cells show substantial suppression of TER development even 48 h after calcium switch. (b) Overexpression of aPKCλkn increases the paracellular diffusion of FITC-dextran in a molecular mass-dependent manner. Paracellular diffusion of FITC-dextran 40K (left) and 500K (right) across adenovirus-infected MDCK II cells was evaluated. FITC-dextran was added to the medium on the apical side of cells subjected to calcium switch 2 d before. After 3 h incubation at 37°C, the fluorescence intensity of the medium in the basolateral side was measured with a fluorometer. Values given represent the mean values (±SD) of three parallel cultures.
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Figure 5: Overexpression of aPKCλkn inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins are as follows: none (X), LacZ (•), aPKCλkn (□), aPKCλwt (▵), and nPKCδkn (○). 2 d after cell seeding (1.5 × 105 cells/cm2) when the TER values reached a plateau, the cells were processed for adenovirus infection (time 0, left) for 2 h in LC medium. Immediately after virus infection, the medium was changed to NC growth medium to allow the cells to complete junctional structure formation before the expression of ectopic proteins. After TER measurement for 45 h, the cells were subjected to calcium switch by changing the medium to LC for 2 h, and then back to NC. The values given represent mean values (1 ± SD) of three parallel cultures. The background resistance obtained from empty filters was deducted. Note that only cells expressing aPKCλkn show a retardation in TER development after calcium switch. (Bottom) MDCK II cells were infected with adenovirus vectors encoding LacZ (•), aPKCλkn (□), or aPKCλwt (▵) as in a. In this case, the cells were kept in fresh LC medium to induce ectopic protein expression in the absence of cell–cell adhesion. 20 h after virus infection, the medium was changed to NC medium, and the development of TER was monitored for 48 h. Note that aPKCλkn-expressing cells show substantial suppression of TER development even 48 h after calcium switch. (b) Overexpression of aPKCλkn increases the paracellular diffusion of FITC-dextran in a molecular mass-dependent manner. Paracellular diffusion of FITC-dextran 40K (left) and 500K (right) across adenovirus-infected MDCK II cells was evaluated. FITC-dextran was added to the medium on the apical side of cells subjected to calcium switch 2 d before. After 3 h incubation at 37°C, the fluorescence intensity of the medium in the basolateral side was measured with a fluorometer. Values given represent the mean values (±SD) of three parallel cultures.
Mentions: The defects in TJ formation in aPKCλkn-expressing cells was further demonstrated functionally by measuring TER to passive ion flow across a cell monolayer grown on permeable support (Fig. 5 a). Consistent with the immunostaining results shown in Fig. 1 a, none of the ectopically expressed proteins, including LacZ, nPKCδkn, or aPKCλwt, as well as aPKCλkn, affected TER if the experiments were arranged so as to induce the expression of the ectopic proteins after the completion of TJ biogenesis (Fig. 5 a, top, 0–45 h). This also suggests that the overexpression of these proteins, especially aPKCλkn, does not produce any artificial cytotoxic effects on TJ barrier function. On the other hand, if the cells were subsequently subjected to calcium switch (2-h incubation in LC medium followed by switching to NC medium), only cells expressing aPKCλkn showed a large retardation in TER development (Fig. 5 a, top, 45–70 h). If the preincubation in low calcium medium was prolonged to 20 h to ensure the dissociation of cell–cell attachments before calcium switch, the effect of aPKCλkn on TER development was more significant: similar to the immunostaining experiments shown in Fig. 1, the expression of ectopic proteins was induced in the cells cultured in LC medium, and calcium switch was applied 20 h after virus infection (Fig. 5 a, bottom). In this case, TER development of aPKCλkn-expressing cells was substantially suppressed until 48 h after calcium switch compared with LacZ- or aPKCλwt-expressing control cells, suggesting that the development of functional TJ is strongly suppressed in aPKCλkn-expressing cells.

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