Epithelial to mesenchymal transition (EMT) is a fundamental biological process that occurs during development and tumorigenesis. The Rho family of GTPases (Rho‐family) is a well‐characterized regulator of actin cytoskeleton that gives rise to EMT‐associated cell activities. Meanwhile, there are in total at least 66 different Rho‐GTPase‐activating proteins (Rho‐GAPs), which, as an upstream regulator, inactivate specific members of the Rho‐family in a cell context‐dependent manner. However, molecular roles of individual Rho‐GAPs are poorly understood, particularly regarding their involvements in EMT. Here, based on comprehensive screening on the whole Rho‐GAP family, we identified specific Rho‐GAPs that are responsible for the maintenance of epithelial cell phenotypes, suppressing EMT in human mammary epithelial cells. Specifically, we revealed that at least two Rho‐GAPs, that is, ARHGAP4 and SH3BP1, critically regulate the cell morphology. Among them, we focused on ARHGAP4 and demonstrated with multidisciplinary approaches that this specific Rho‐GAP regulates epithelial/mesenchymal‐selective marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber‐driven physical force generation in a manner reminiscent of the EMT process. Furthermore, we identified Septin9 with proteomic analyses as a negative regulator of ARHGAP4, which promotes the occurrence of EMT by activation of the FAK/Src signaling pathway. These findings shed light on the novel Rho‐GAP‐associated pathway in the EMT process under development and tumorigenesis.

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

The Rho family of GTPases are inactivated in a cell context-dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 as well as overexpressing its two mutually independent upstream regulators SEPT2 and SEPT9 all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion-associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the upregulated Integrin Beta 1. These results provide novel insights to cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.