No abstract

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

Microtubule (MT) stabilization is regulated by the small guanosine triphosphate (GTP)-binding protein Rho and its effector, mammalian homolog of Diaphanous (mDia), in migrating cells, but factors responsible for localized stabilization at the leading edge are unknown. We report that integrin-mediated activation of focal adhesion kinase (FAK) at the leading edge is required for MT stabilization by the Rho-mDia signaling pathway in mouse fibroblasts. MT stabilization also involved FAK-regulated localization of a lipid raft marker, ganglioside GM1, to the leading edge. The integrin-FAK signaling pathway may facilitate Rho-mDia signaling through GM1, or through a specialized membrane domain containing GM1, to stabilize MTs in the leading edge of migrating cells.

Autophagy is a tightly regulated catabolic process that plays key roles in normal cellular homeostasis and survival during periods of extracellular nutrient limitation and stress. The environmental signals that regulate autophagic activity are only partially understood. Here, we report a direct link between glutamine (Gln) metabolism and autophagic activity in both transformed and nontransformed human cells. Cells cultured for more than 2 days in Gln-containing medium showed increases in autophagy that were not attributable to nutrient depletion or to inhibition of mammalian target of rapamycin. Conditioned medium from these cells contained a volatile factor that triggered autophagy in secondary cell cultures. We identified this factor as ammonia derived from the deamination of Gln by glutaminolysis. Gln-dependent ammonia production supported basal autophagy and protected cells from tumor necrosis factor-alpha (TNF-alpha)-induced cell death. Thus, Gln metabolism not only fuels cell growth but also generates an autocrine- and paracrine-acting regulator of autophagic flux in proliferating cells.