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© The Rockefeller University Press, 0021-9525/1998//85 $5.00
The Journal of Cell Biology, Volume 142, Number 1, , 1998 85-100

Articles

Effects of Regulated Expression of Mutant RhoA and Rac1 Small GTPases on the Development of Epithelial (MDCK) Cell Polarity



Tzuu-Shuh Jou and W. James Nelson

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345

MDCK cells expressing RhoA or Rac1 mutants under control of the tetracycline repressible transactivator were used to examine short-term effects of known amounts of each mutant before, during, or after development of cell polarity. At low cell density, Rac1V12 cells had a flattened morphology and intact cell–cell contacts, whereas Rac1N17 cells were tightly compacted. Abnormal intracellular aggregates formed between Rac1N17, F-actin, and E-cadherin in these nonpolarized cells. At all subsequent stages of polarity development, Rac1N17 and Rac1V12 colocalized with E-cadherin and F-actin in an unusual beaded pattern at lateral membranes. In polarized cells, intracellular aggregates formed with Rac1V12, F-actin, and an apical membrane protein (GP135). At low cell density, RhoAV14 and RhoAN19 were localized in the cytoplasm, and cells were generally flattened and more fibroblastic than epithelial in morphology. In polarized RhoAV14 cells, F-actin was diffuse at lateral membranes and prominent in stress fibers on the basal membrane. GP135 was abnormally localized to the lateral membrane and in intracellular aggregates, but E-cadherin distribution appeared normal. In RhoAN19 cells, F-actin, E-cadherin, and GP135 distributions were similar to those in controls. Expression of either RhoAV14 or RhoAN19 in Rac1V12 cells disrupted Rac1V12 distribution and caused cells to adopt the more fibroblastic, RhoA mutant phenotype. We suggest that Rac1 and RhoA are involved in the transition of epithelial cells from a fibroblastic to a polarized structure and function by direct and indirect regulation of actin and actin-associated membrane protein organizations.

Key Words: GTPases • RhoA • cell polarity • cell adhesion • membrane domains

Abbreviations used in this paper: DC, doxycycline; ERM, ezrin-radixin-moesin; PIP2, phosphatidylinositol (4,5)-bisphosphate.

We are very grateful to the following colleagues who made various reagents available to us: Dr. Rong-Guo Qiu (Onyx Pharmaceutical) for the RhoA and Rac1 mutant plasmids; Dr. Marc Symons and Dr. Bonnee Rubinfeld (Onyx Pharmaceutical) for Glu-Glu tagged RhoAV14, RhoAN19, Rac1V12, and Rac1N17 cDNA; Dr. George Cann (Stanford University) for mouse anti-myc hybridoma, 9E10; Dr. George Ojakian (SUNY Brooklyn) for the anti-GP135 hybridoma; and Dr. James Goldenring (Medical College of Georgia) for advice on rab11 staining and distribution. We thank members of the Nelson laboratory and Marc Symons (Onyx Pharmaceutical) for their criticisms and help during the course of this work.

Address all correspondence to W. James Nelson, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Beckman Center, B121, Stanford, CA 94305-5345. Tel.: (650) 725-7596. Fax: (650) 498-5286. E-mail: wjnelson{at}leland.stanford.edu


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