Red bar marks basal level expected from random Golgi/centrosome reorientation (33%)

Red bar marks basal level expected from random Golgi/centrosome reorientation (33%). monolayers has been used to study aspects of directed cell migration in a variety of cell types, including fibroblasts, astrocytes, endothelial cells, and epithelial cells. With most primary nontransformed cells, migration involves coordinated movement of the monolayer in a manner more similar to the morphogenetic movements seen during development, such as dorsal closure and convergent extension, than the movement of Rabbit polyclonal to POLR3B single cells such as in neutrophil chemotaxis. Disruption of the monolayer causes the loss of cellCcell contacts and a major consequence of this is to induce polarity in cells proximal to the scratch. One aspect of polarization is the formation of actin-rich protrusions, specifically at the front of the cell (Nobes and Hall, 1999). A second aspect of polarization involves the microtubule cytoskeleton ST271 and can be visualized as reorientation of the centrosome and Golgi to face the front of the cell. This involves the association of microtubule plus-end tips with plasma membrane complexes at the leading edge as well as movement of the nucleus to the back of the cell (Kupfer et al., 1982; Etienne-Manneville and Hall, 2001; Gomes et al., 2005). Numerous studies have now shown that the small GTPase Cdc42, or one of its close relatives, is required for polarization ST271 of the actin and microtubule cytoskeletons in astrocytes, primary fibroblasts, 3T3 fibroblasts, Vero epithelial cells, and endothelial cells (Nobes and Hall, 1999; Etienne-Manneville and Hall, 2001, 2003; Palazzo et al., 2001b; Tzima et al., 2003; Watanabe et al., 2004; Cau and Hall, 2005; Gomes et al., 2005). Studies of the signaling pathways controlling microtubule polarization in different adherent cell types have identified a complex consisting of the scaffold protein Par6 and an atypical PKC (aPKC) downstream of Cdc42. Localized activation of Cdc42 leads to localized activation of the Par6/aPKC complex, and ST271 this has now been described in astrocytes (Etienne-Manneville and Hall, 2001, 2003), primary rat fibroblasts (Nobes and Hall, 1999; Cau and Hall, 2005), 3T3 fibroblasts (Gomes et al., 2005), and endothelial cells (Tzima et al., 2003). The Par6/aPKC complex has at least two crucial activities in this process. First, it is required for the accumulation of the tumor suppressor protein adenomatous polyposis coli (APC) at the plus-end tips of microtubules, specifically at the leading edge. In primary astrocytes, GSK-3 is phosphorylated at Ser9 by PKC (Etienne-Manneville and Hall, 2003), and this was assumed to be the likely mechanism for inhibition of kinase activity leading to APC accumulation. A second activity of the Par6/aPKC complex is to promote the accumulation of another tumor suppressor protein Dlg (Discs Large) in the plasma membrane at the leading edge. The subsequent association of microtubule-bound APC with membrane-bound Dlg is required for microtubule polarization and centrosome reorientation (Etienne-Manneville et al., 2005). It is likely that many other cellular activities are required for reorganization of the microtubule cytoskeleton; for example IQGAP, another Cdc42 effector, is required both for protrusion polarity as well APC and microtubule polarity (Watanabe et al., 2004) and the dynein/dynactin complex is required for centrosome reorientation and mDia and EB1, regulated ST271 by Rho, also contribute to APC localization and stabilization (Palazzo et al., 2001a; Wen et al., 2004). In this report, we reexamined the significance of GSK-3 phosphorylation using fibroblasts derived from knock-in mice in which the phosphorylation sites of both GSK3 and isoforms (Ser21 and Ser9, respectively) have been replaced with Ala (McManus et al., 2005). We find.