Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linkerassociated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.cell motility | receptor tyrosine kinase | Memo protein | glycogen synthase kinase-3 | adenomatous polyposis coli M icrotubules (MTs) have several important functions during cell motility. They participate in cell detachment at the cell back, regulate the turnover of focal adhesions at the cell front, and contribute to the establishment and maintenance of cell orientation (1). MTs are polarized polymers, which in interphasic cells are assembled from the MT organizing center and elongate toward the cell periphery. MT plus-ends explore the cytoplasmic area in search of cortical targets, where they will be stabilized. MT assembly and stability are under the control of MT-associated proteins. Plus-end tracking proteins (+TIPs) are specialized MTassociated proteins that concentrate at the plus-end of MTs. They include several families of structurally unrelated proteins (2). Although some proteins, such as kinesin-13 family members (3), bind to depolymerizing MTs, most of them, including EB1, cytoplasmic linker protein-170, cytoplasmic linker-associated proteins (CLASPs), or the tumor suppressor APC (adenomatous polyposis coli), decorate only growing MTs. +TIPs interact with MT ends and with each other, generating a network of proteins held together by selective and transient interactions (2). Plus-end associated complexes interact in turn with membrane-associated proteins, such as RhoGTPases, via intermediate proteins, such as IQGAP1 or the mDia formins (4). +TIPs can also associate directly with cortical factors (5) or actin, as illustrated by ...

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.