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Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells.

Takaishi K, Sasaki T, Kotani H, Nishioka H, Takai Y - J. Cell Biol. (1997)

Bottom Line: The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton.ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines.Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan.

ABSTRACT
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell-cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell- cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell- cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell- cell adhesion.

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Localization of ZO-1 in sMDCK-RhoDA-5, -RacDA-1,  and RacDN-2 cells. Wild-type MDCK cells (a), sMDCK-RhoDA-5  cells (b), sMDCK-RacDA-1 cells (c), and sMDCK-RacDN-2  cells (d) were stained with the anti–ZO-1 mAb and analyzed by  confocal microscopy. Confocal images are shown at the junctional levels. Note that the scale in RacDN panels (d) is different  from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 μm.
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Figure 6: Localization of ZO-1 in sMDCK-RhoDA-5, -RacDA-1, and RacDN-2 cells. Wild-type MDCK cells (a), sMDCK-RhoDA-5 cells (b), sMDCK-RacDA-1 cells (c), and sMDCK-RacDN-2 cells (d) were stained with the anti–ZO-1 mAb and analyzed by confocal microscopy. Confocal images are shown at the junctional levels. Note that the scale in RacDN panels (d) is different from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 μm.

Mentions: ZO-1 is one of the structural proteins of tight junctions (Woods and Bryant, 1993) but is also partly colocalized with E-cadherin in MDCK cells (Howarth et al., 1994). We analyzed the localization of ZO-1 in sMDCK-RhoDA, -RacDA, -RacDN, -Cdc42DA, and -Cdc42DN cells. The staining patterns of ZO-1 in sMDCK-RhoDA-5, -RacDA-1, and -RacDN-2 cells were apparently indistinguishable from those in wild-type MDCK cells (Fig. 6). The staining patterns of ZO-1 in the other MDCK cell lines stably expressing V14RhoA, V12Rac1, or N17Rac1 were also similar to that in wild-type MDCK cells (data not shown). The staining patterns of ZO-1 in all the clones of sMDCK-Cdc42DA and -Cdc42DN were also similar to that in wild-type MDCK cells (data not shown). These results indicate that neither the Rho, the Rac, nor the Cdc42 subfamily affects the localization of ZO-1.


Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells.

Takaishi K, Sasaki T, Kotani H, Nishioka H, Takai Y - J. Cell Biol. (1997)

Localization of ZO-1 in sMDCK-RhoDA-5, -RacDA-1,  and RacDN-2 cells. Wild-type MDCK cells (a), sMDCK-RhoDA-5  cells (b), sMDCK-RacDA-1 cells (c), and sMDCK-RacDN-2  cells (d) were stained with the anti–ZO-1 mAb and analyzed by  confocal microscopy. Confocal images are shown at the junctional levels. Note that the scale in RacDN panels (d) is different  from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Localization of ZO-1 in sMDCK-RhoDA-5, -RacDA-1, and RacDN-2 cells. Wild-type MDCK cells (a), sMDCK-RhoDA-5 cells (b), sMDCK-RacDA-1 cells (c), and sMDCK-RacDN-2 cells (d) were stained with the anti–ZO-1 mAb and analyzed by confocal microscopy. Confocal images are shown at the junctional levels. Note that the scale in RacDN panels (d) is different from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 μm.
Mentions: ZO-1 is one of the structural proteins of tight junctions (Woods and Bryant, 1993) but is also partly colocalized with E-cadherin in MDCK cells (Howarth et al., 1994). We analyzed the localization of ZO-1 in sMDCK-RhoDA, -RacDA, -RacDN, -Cdc42DA, and -Cdc42DN cells. The staining patterns of ZO-1 in sMDCK-RhoDA-5, -RacDA-1, and -RacDN-2 cells were apparently indistinguishable from those in wild-type MDCK cells (Fig. 6). The staining patterns of ZO-1 in the other MDCK cell lines stably expressing V14RhoA, V12Rac1, or N17Rac1 were also similar to that in wild-type MDCK cells (data not shown). The staining patterns of ZO-1 in all the clones of sMDCK-Cdc42DA and -Cdc42DN were also similar to that in wild-type MDCK cells (data not shown). These results indicate that neither the Rho, the Rac, nor the Cdc42 subfamily affects the localization of ZO-1.

Bottom Line: The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton.ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines.Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan.

ABSTRACT
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell-cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell- cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell- cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell- cell adhesion.

Show MeSH
Related in: MedlinePlus