Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration

P120RasGAP competes with Rab21 for binding to the cytoplasmic domain of integrin α-subunits, thereby promoting receptor escape from early endosomes and recycling to the plasma membrane

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.Moreno-Layseca et al. identify Swip1 as an integrin-specific endocytic adaptor controlling the dynamics of integrin adhesion complexes as well as the migration and invasion of breast cancer cells

Reinigung von nativen Enzymen aus Makroalgen

Enzyme sind als biologische Katalysatoren essentiell in vielen industriellen Prozessen und diagnostischen Anwendungen. Eine bislang wenig genutzte, vielversprechende Quelle für die Entdeckung von innovativen, biotechnologisch-relevanten Enzymen sind Makroalgen. Doch die biochemische Zusammensetzung der Algen erschwert die Reinigung der Enzyme. Um besonders aktive Redoxenzyme aus Algen zu extrahieren und zu identifizieren, wurde eine Prozesskaskade entwickelt, mit der Zielsetzung die Enzyme im Anschluss rekombinant herzustellen.28301-

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex

Arl2 and Arl3, members of the Arf subfamily of smallG proteins, are believed to be involved in ciliary and microtubule-dependent processes. Recently, we could identify RP2, responsi-ble for a variant of X-linked retinitis pigmentosa, as the Arl3-specific GAP. Here, we have characterized Arl2/3 interactions.We show the formation of a ternary complex between Arl3, itscognate GAP RP2 and its retinal effector HRG4. This complexseems to be important for photoreceptor function

Distinct recycling of active and inactive β1 integrins

Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6- and early endosome antigen 1-positive compartment in an Rab 4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic

Cytokinesis failure due to derailed integrin traffic induces aneuploidy and oncogenic transformation <i>in vitro</i> and <i>in vivo</i>

Aneuploidy is frequently detected in solid tumors but the mechanisms regulating the generation of aneuploidy and their relevance in cancer initiation remain under debate and are incompletely characterized. Spatial and temporal regulation of integrin traffic is critical for cell migration and cytokinesis. Impaired integrin endocytosis, because of the loss of Rab21 small GTPase or mutations in the integrin β-subunit cytoplasmic tail, induces failure of cytokinesis in vitro. Here, we describe that repeatedly failed cytokinesis, because of impaired traffic, is sufficient to trigger the generation of aneuploid cells, which display characteristics of oncogenic transformation in vitro and are tumorigenic in vivo. Furthermore, in an in vivo mouse xenograft model, non-transformed cells with impaired integrin traffic formed tumors with a long latency. More detailed investigation of these tumors revealed that the tumor cells were aneuploid. Therefore, abnormal integrin traffic was linked with generation of aneuploidy and cell transformation also in vivo. In human prostate and ovarian cancer samples, downregulation of Rab21 correlates with increased malignancy. Loss-of-function experiments demonstrate that long-term depletion of Rab21 is sufficient to induce chromosome number aberrations in normal human epithelial cells. These data are the first to demonstrate that impaired integrin traffic is sufficient to induce conversion of non-transformed cells to tumorigenic cells in vitro and in vivo

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1

GTP-Specific Fab Fragment-Based GTPase Activity Assay

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2′/3′-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4^GTP) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations