The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin–based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1–Rac signaling. Tiam1 interacts with Par3 and aPKCζ, which are two components of the conserved Par3–Par6–aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCζ, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3–Par6–aPKC polarity complex.

Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by Arp2/3 complex. Although regulation of Arp2/3-complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an EGF-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments activating the Arp2/3 complex and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium/ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.

Formin proteins are key regulators of the cytoskeleton involved in developmental and homeostatic programs, and human disease. For these reasons, small molecules interfering with Formins’ activity have gained increasing attention. Among them, small molecule inhibitor of Formin Homology 2 domains (SMIFH2) is often used as a pharmacological Formin blocker. Although SMIFH2 inhibits actin polymerization by Formins and affects the actin cytoskeleton, its cellular mechanism of action and target specificity remain unclear.Here we show that SMIFH2 induces remodelling of actin filaments, microtubules and the Golgi complex as a result of its effects on Formins and p53.We found that SMIFH2 triggers alternated depolymerization-repolymerization cycles of actin and tubulin, increases cell migration, causes scattering of the Golgi complex, and also cytotoxicity at high dose. Moreover, SMIFH2 reduces expression and activity of p53 through a post-transcriptional, proteasome-independent mechanism that influences remodelling of the cytoskeleton.As the action of SMIFH2 may go beyond Formin inhibition, only short-term and low-dose SMIFH2 treatments minimize confounding effects induced by loss of p53 and cytotoxicity.

Tiam1 is a ubiquitously expressed activator of the small GTPase Rac. Previously, we found that Tiam1 knockout (KO) mice are resistant to DMBA-induced skin tumorigenicity, which correlated with increased apoptosis in keratinocytes of the skin epidermis. Here, we have studied the mechanisms by which Tiam1 protects against apoptosis. We found that Tiam1-KO keratinocytes show increased apoptosis in response to apoptotic stimuli, including growth factor deprivation and heat-shock treatment. Expression of catalytically active Tiam1, but not inactive Tiam1, rescues the apoptosis susceptibility of Tiam1-KO keratinocytes, indicating that this defect is caused by impaired Tiam1-mediated Rac activation. Apoptosis induced by growth factor starvation correlates with impaired ERK phosphorylation in Tiam1-KO keratinocytes. Moreover, Tiam1-KO keratinocytes contain lower levels of intracellular reactive oxygen species (ROS) when compared with wild-type cells. The ROS content of keratinocytes is dependent on both Tiam1 and the activity of NADPH oxidase (Nox), and is required for ERKmediated survival signaling. Indeed, Tiam1 deficiency or the inhibition of intracellular ROS production blocks ERK phosphorylation and sensitizes wild-type keratinocytes to apoptotic stimuli. Our results indicate that the Rac activator Tiam1 controls the intracellular redox balance by Nox-mediated ROS production, which regulates ERK phosphorylation and the susceptibility of keratinocytes to apoptotic signaling.