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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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Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other TJ components (a) or adherens junction components (b) by double staining as indicated. For E-cadherin and rhodamine-phalloidin staining, the data for LacZ-expressing cells are also presented for reference. Arrows indicate the positions of aberrant small ring junctional structures, while arrowheads indicate the regions where discrepant staining is observed between ZO-1 and the compared TJ component. Bar, 25 μm. (c and d) Western blot analysis of several junctional proteins in adenovirally infected MDCK II cells. Cells were subjected to calcium switch, lysed 6 h later, and processed for Western blot analysis using the antibodies indicated (left). (c) Typical results of Western blot analysis are presented. The infected viruses are indicated (top). The total amount of protein in each lane was finely adjusted based on the quantitative results of Coomassie brilliant blue staining of the blot membrane (data not shown). Quantified results based on three independent experiments examining the amounts of the indicated proteins in MDCK cells expressing LacZ (white bar), aPKCλwt (hatched bar), or aPKCλkn (black bar) are presented in d. Chemiluminescent signals from the immunostained bands for each junctional protein were quantified using a luminescence Image Analyzer.
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Figure 4: Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other TJ components (a) or adherens junction components (b) by double staining as indicated. For E-cadherin and rhodamine-phalloidin staining, the data for LacZ-expressing cells are also presented for reference. Arrows indicate the positions of aberrant small ring junctional structures, while arrowheads indicate the regions where discrepant staining is observed between ZO-1 and the compared TJ component. Bar, 25 μm. (c and d) Western blot analysis of several junctional proteins in adenovirally infected MDCK II cells. Cells were subjected to calcium switch, lysed 6 h later, and processed for Western blot analysis using the antibodies indicated (left). (c) Typical results of Western blot analysis are presented. The infected viruses are indicated (top). The total amount of protein in each lane was finely adjusted based on the quantitative results of Coomassie brilliant blue staining of the blot membrane (data not shown). Quantified results based on three independent experiments examining the amounts of the indicated proteins in MDCK cells expressing LacZ (white bar), aPKCλwt (hatched bar), or aPKCλkn (black bar) are presented in d. Chemiluminescent signals from the immunostained bands for each junctional protein were quantified using a luminescence Image Analyzer.

Mentions: When aPKCλkn was expressed in MDCK II cells maintained under normal calcium conditions, the junctional localization of ASIP/PAR-3 was not impaired even in cells showing extremely high levels of aPKCλkn expression (Fig. 1 a, −CS). However, if cells expressing aPKCλkn were subjected to calcium switch to induce a disruption–regeneration process of cell–cell adhesion, then the junctional staining of ASIP/PAR-3 was significantly affected (Fig. 1 a, +CS). Despite the apparently normal restoration of a confluent monolayer as indicated by phase contrast observations (data not shown), ASIP/PAR-3 did not develop a complete junctional distribution over the entire cell circumference, even 6 h after calcium switch (see Fig. 4 a), which is achieved <2 h after calcium switch in control cells. This fragmentary staining of the cell–cell boundaries remains unchanged until 20 h after calcium switch (Fig. 1 a, +CS), suggesting that the junctional localization of ASIP/PAR-3 is significantly inhibited by aPKCλkn. Interestingly, the junctional localization of ZO-1, a TJ marker, in cells was similarly disturbed in a calcium switch-dependent manner, suggesting that TJ formation itself is severely affected (Fig. 1 a, right). Table summarizes the statistical results of ZO-1 mislocalization observed 20 h after calcium switch. While most (>99%) of the cells infected with adenovirus vectors carrying LacZ as well as aPKCλwt display normal ZO-1 staining (Fig. 1 c, and Table ), >60% infected with an aPKCλkn-encoding adenovirus vector exhibit partial or complete mislocalization of ZO-1. The severity of ZO-1 mislocalization correlates well with the level of aPKCλkn expression (Table , and Fig. 1 b); for example, >50% of cells with a high fluorescent signal (+++) exhibit a complete disappearance of ZO-1, whereas the rate is <11% in cells with a low fluorescent signal (+). On the other hand, the data also indicate that >40% of cells apparently negative for an aPKCλ fluorescent signal (−/+) also show fragmentary ZO-1 distribution. Considering the low sensitivity of the anti–aPKCλ antibody used, and the proportional correlation between fluorescent signal intensity and phenotype severity (Fig. 1 b), we infer that the actual infection efficiency is higher than estimated and some cells negative for a fluorescent signal also express levels of aPKCλkn that are undetectable but still sufficient to affect ZO-1 distribution.


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)

Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other TJ components (a) or adherens junction components (b) by double staining as indicated. For E-cadherin and rhodamine-phalloidin staining, the data for LacZ-expressing cells are also presented for reference. Arrows indicate the positions of aberrant small ring junctional structures, while arrowheads indicate the regions where discrepant staining is observed between ZO-1 and the compared TJ component. Bar, 25 μm. (c and d) Western blot analysis of several junctional proteins in adenovirally infected MDCK II cells. Cells were subjected to calcium switch, lysed 6 h later, and processed for Western blot analysis using the antibodies indicated (left). (c) Typical results of Western blot analysis are presented. The infected viruses are indicated (top). The total amount of protein in each lane was finely adjusted based on the quantitative results of Coomassie brilliant blue staining of the blot membrane (data not shown). Quantified results based on three independent experiments examining the amounts of the indicated proteins in MDCK cells expressing LacZ (white bar), aPKCλwt (hatched bar), or aPKCλkn (black bar) are presented in d. Chemiluminescent signals from the immunostained bands for each junctional protein were quantified using a luminescence Image Analyzer.
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Related In: Results  -  Collection

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Figure 4: Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other TJ components (a) or adherens junction components (b) by double staining as indicated. For E-cadherin and rhodamine-phalloidin staining, the data for LacZ-expressing cells are also presented for reference. Arrows indicate the positions of aberrant small ring junctional structures, while arrowheads indicate the regions where discrepant staining is observed between ZO-1 and the compared TJ component. Bar, 25 μm. (c and d) Western blot analysis of several junctional proteins in adenovirally infected MDCK II cells. Cells were subjected to calcium switch, lysed 6 h later, and processed for Western blot analysis using the antibodies indicated (left). (c) Typical results of Western blot analysis are presented. The infected viruses are indicated (top). The total amount of protein in each lane was finely adjusted based on the quantitative results of Coomassie brilliant blue staining of the blot membrane (data not shown). Quantified results based on three independent experiments examining the amounts of the indicated proteins in MDCK cells expressing LacZ (white bar), aPKCλwt (hatched bar), or aPKCλkn (black bar) are presented in d. Chemiluminescent signals from the immunostained bands for each junctional protein were quantified using a luminescence Image Analyzer.
Mentions: When aPKCλkn was expressed in MDCK II cells maintained under normal calcium conditions, the junctional localization of ASIP/PAR-3 was not impaired even in cells showing extremely high levels of aPKCλkn expression (Fig. 1 a, −CS). However, if cells expressing aPKCλkn were subjected to calcium switch to induce a disruption–regeneration process of cell–cell adhesion, then the junctional staining of ASIP/PAR-3 was significantly affected (Fig. 1 a, +CS). Despite the apparently normal restoration of a confluent monolayer as indicated by phase contrast observations (data not shown), ASIP/PAR-3 did not develop a complete junctional distribution over the entire cell circumference, even 6 h after calcium switch (see Fig. 4 a), which is achieved <2 h after calcium switch in control cells. This fragmentary staining of the cell–cell boundaries remains unchanged until 20 h after calcium switch (Fig. 1 a, +CS), suggesting that the junctional localization of ASIP/PAR-3 is significantly inhibited by aPKCλkn. Interestingly, the junctional localization of ZO-1, a TJ marker, in cells was similarly disturbed in a calcium switch-dependent manner, suggesting that TJ formation itself is severely affected (Fig. 1 a, right). Table summarizes the statistical results of ZO-1 mislocalization observed 20 h after calcium switch. While most (>99%) of the cells infected with adenovirus vectors carrying LacZ as well as aPKCλwt display normal ZO-1 staining (Fig. 1 c, and Table ), >60% infected with an aPKCλkn-encoding adenovirus vector exhibit partial or complete mislocalization of ZO-1. The severity of ZO-1 mislocalization correlates well with the level of aPKCλkn expression (Table , and Fig. 1 b); for example, >50% of cells with a high fluorescent signal (+++) exhibit a complete disappearance of ZO-1, whereas the rate is <11% in cells with a low fluorescent signal (+). On the other hand, the data also indicate that >40% of cells apparently negative for an aPKCλ fluorescent signal (−/+) also show fragmentary ZO-1 distribution. Considering the low sensitivity of the anti–aPKCλ antibody used, and the proportional correlation between fluorescent signal intensity and phenotype severity (Fig. 1 b), we infer that the actual infection efficiency is higher than estimated and some cells negative for a fluorescent signal also express levels of aPKCλkn that are undetectable but still sufficient to affect ZO-1 distribution.

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