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

Abstract

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.

Importance of spatial activation of Cdc42 and Rac small G proteins by frabin for microspike formation in MDCK cells

“…2A and B). Consistent with earlier observations (Hordijk et al 1997;Takaishi et al 1997;Jou & Nelson 1998;Kodama et al 1999), both proteins increased the accumulation of F-actin (data not shown) and E-cadherin at cell-cell AJs in MDCK cells ( Fig. 2C and D).…”

“…A recent series of experiments on the roles of the Rho small G protein family, including the Rho, Rac and Cdc42 subfamilies, in cultured MDCK cells have revealed that the Rho subfamily (Rho) regulates the formation of stress ®bres and focal adhesions, whereas the Cdc42 and Rac subfamilies (Cdc42 and Rac) regulate the accumulation of actin ®laments (F-actin) and E-cadherin at cadherin-based cell-cell adherens junctions (AJs) (Hordijk et al 1997;Takaishi et al 1997;Jou & Nelson 1998;Kodama et al 1999). Rho does not directly affect the accumulation of F-actin or E-cadherin at cell-cell AJs, although it indirectly affects it .…”

Turn‐off, drop‐out: Functional state switching of cadherins

“…p120 ctn also alters the relative activities of the small G-proteins Rho A, Rac, and Cdc42 Grosheva et al, 2000;Noren et al, 2000). This may well be related to changes in the activities of these proteins initiated by adhesion Noren et al, 2001) and involved in actin-driven cadherin clustering (Braga et al, 1997(Braga et al, , 1999Jou and Nelson, 1998;Takaishi et al, 1997).…”

Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix

“…In addition, scribble has 16 canonical leucine-rich repeats (LRRs), another protein -protein interaction motif. Related repeats to the LRRs are known to bind small GTPases of the Ras superfamily (Jou and Nelson, 1998;Kaibuchi et al, 1999). Recently, proteins with both PDZ domains and LRRs have been reported to have common functions in the control of the cell shape and polarity.…”