The Rho-mDia1 Pathway Regulates Cell Polarity and Focal Adhesion Turnover in Migrating Cells through Mobilizing Apc and c-Src

Abstract

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.Cell migration is indispensable in biological processes such as development, inflammation, wound healing, and tumor metastasis. Migrating cells polarize by extending protrusions at the front and retracting the tail at the rear, and make adhesions to extracellular matrix (ECM) to stabilize the forward protrusion (36). Adhesions to ECM are then used as sites to pull the cell body forward and are subsequently disassembled as the cell moves over them. This cycle of events enables cells to migrate to their destination. The actin cytoskeleton and microtubules (MTs) work critically in these events. Actin polymerization at the leading edge drives membrane protrusion, the association of the actin cytoskeleton with integrins regulates binding of the integrins to ECM, and the actin bundles within the body generate tension to pull the cell body forward and retract the tail. MTs are also polarized in migrating cells and are essential for the directed migration of many cell types (36, 37). However, how these cytoskeletons are regulated in migrating cells and work for cell polarization and adhesion turnover remains largely unknown.The Rho GTPases, including Rho, Rac, and Cdc42, work as molecular switches in cell morphogenesis by inducing specific types of actin cytoskeleton and by locally regulating MT dynamics. Accumulating evidence suggests that Cdc42 regulates cell polarity and Rac works in membrane protrusion of migrating cells. Indeed, Cdc42 is active at the cell front (17, 30), and disruption of Cdc42 function impairs directionality of migration in many cell types (1, 32). One well-characterized action of Cdc42 in cell polarity is to orient the MT organizing center (MTOC) in the front of the nucl...

Lack of Adenomatous Polyposis Coli Protein Correlates with a Decrease in Cell Migration and Overall Changes in Microtubule Stability

“…html). A histogram summarizing this data in a different format is shown in Supplemental Figure 5. contribute to changes in F-actin (Wen et al, 2004;Yamana et al, 2006). Importantly, we found that the isolated N-terminal domain of APC that contains the full complement of binding sites for IQGAP, Asef, and Kap3 is unable to cluster in cellular protrusions (Zumbrunn et al, 2001) and does not induce protrusions when overexpressed ( Figure 5B), suggesting that these protein interactions on their own are not responsible for this effect of APC.…”

T Cell Responses in Mammalian Diaphanous-related Formin mDia1 Knock-out Mice

“…Although p140mDia1 is a known RhoA effector, it is plausible that the disruption in adhesion and chemotactic migration is due to disruption of RhoA-mediated p140mDia1 activation and dysregulation of forminmediated actin dynamics. Indeed, a role for mDia1 and the related budding yeast formin Bni1 have been extensively characterized as promoting cell polarity and mediating both directional cell migration (33)(34)(35) and spontaneous cell migration (36). Furthermore, expression of constructs interfering with mDia1 function or small hairpin RNA constructs directed against mDia1 also disrupt microtubule organizing center reorientation in activated T cells, a hallmark of T cell polarization (36,37).…”

Ubiquitin C-terminal hydrolase-L1 is a key regulator of tumor cell invasion and metastasis

“…Changes in cell morphology are induced by several events. Directed cell migration requires cell polarization and adhesion turnover, phenomena in which actin cytoskeleton and microtubules play critical roles (Yamana et al, 2006). H157 cells infected with UCH-L1 RNAi lentivirus were monitored for 72 h after seeding at low density (1 Â 10 4 cells) on 60 mm cell culture dish.…”