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Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1[DELTA]wsc1[DELTA] synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1[DELTA] cells. Inactivation of RHQ3 or RHQ4 was able to rescue the rgd1[DELTA]wsc1A synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC-mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC-MAP kinase pathway.

Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive.We carried out a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells shows that MTs are essential during a specific time frame of tracheal cell elongation while the branch extends. In the absence of MTs, one tracheal cell per branch over-elongates, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs are crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs.Maintaining E-Cadherin/Par-3 levels at the apical domain requires de novo synthesis rather than internalisation and recycling from and to the apical plasmamembrane. However, apical targeting of E-Cadherin and Par-3 requires functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. We demonstrate that apical enrichment of recycling endosomes is dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, we establish that E-Cadherin dynamics and MT requirement differ in remodelling tracheal cells versus planar epithelial cells. Altogether, our results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis. [ABSTRACT FROM AUTHOR]

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae. [ABSTRACT FROM AUTHOR]

The article proposes that Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae, participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the protein kinase C-mitogen-activated protein kinase pathway. It details the yeast strains and genetic procedures. The article also discusses the production of Rho3 and Rho4 GTPases.