ConFERMing the role of talin in integrin activation and mechanosignaling.

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.

Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation.

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.

Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation.

Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate integrin activity.

Comparison of two methods of neutralization and wet air oxidation for treating wastewater spent caustic produced by oil refineries.

Today, problems of treating wastewater spent caustic produced by refinery units with high toxic compounds and chemical oxygen demand (COD) become concerns of many managers of these industries and environmental experts. Hence, emission without application of treatment methods will have adverse environmental impacts. In this study, two direct acid neutralization (DAN) and wet air oxidation (WAO) processes were selected to treatment the wastewater of the Bandar Abbas oil refinery in southern Iran. The aim was to reduce COD and harmful substances, compare the two methods, optimize processes, and evaluate their performance. Experimental experiments were performed in a reactor system with different input variables related to two different methods. Parameter optimization was performed based on Box-Behnken (BBD) method and Design Expert software. The analysis of the results based on statistical methods and response procedure diagrams was used to evaluate the status of COD changes to the parameters. The best operating conditions of temperature, pressure, residence time, and stoichiometric coefficient of air were 148.269 °C, 15.716 bar, 3.563 h, and 8.415 l/h respectively in WAO with 68% reduction in COD, and for DAN process, temperature, pH, and agitation speed were 30.082 °C, 2.008, and 203.672 rpm, respectively, with 43% reduction in COD. Results of rapid impact assessment matrix (RIAM) showed that WAO process with a higher score is a more environmentally friendly method and DAN process has been considered by experts due to its popularity, ease of testing, less equipment requirements, and lower cost.

Environmental impact assessment studies for mining area in Goa, India, using the new approach.

The mining industry is a fundamental source for building infrastructures and an enabler for a country's growth. Over the last decade, the act of mining has been among the top in the list of human activities which has the most disturbing and catastrophic impacts on environment, therein extensively affecting the ecological, economic, and social elements in the vicinity. There is an exigency for a pragmatic balance to exist between the global demand satisfaction of metal and environmental sustenance. In this paper, a comprehensive case study on Environmental Impact Assessment (EIA) of a mining site has been presented using the new approach. This new approach is an improved version of the traditional matrix method, incorporating a modified version of Rapid Impact Assessment Matrix (RIAM) integrated with analytical hierarchy process (AHP), thereby knocking out the limitations in the existing EIA techniques. The data used in this study is an outcome of a broad survey conducted among the people associated in both direct and indirect ways to the project actions related to the mining industry and, hence, minimizing issues such as assessors' reproducibility, subjectivity, and non-inclusivity of all stakeholders' opinion, which can contribute to misleading outcomes. This new approach delivers more precise and practical results for the assessment of environmental impact data.

Inhibition of talin-induced integrin activation by a double-hit stapled peptide.

Integrins are ubiquitously expressed cell-adhesion proteins. Activation of integrins is triggered by talin through an inside-out signaling pathway, which can be driven by RAP1-interacting adaptor molecule (RIAM) through its interaction with talin at two distinct sites. A helical talin-binding segment (TBS) in RIAM interacts with both sites in talin, leading to integrin activation. The bispecificity inspires a "double-hit" strategy for inhibiting talin-induced integrin activation. We designed an experimental peptidomimetic inhibitor, S-TBS, derived from TBS and containing a molecular staple, which leads to stronger binding to talin and inhibition of talin:integrin interaction. The crystallographic study validates that S-TBS binds to the talin rod through the same interface as TBS. Moreover, the helical S-TBS exhibits excellent cell permeability and effectively suppresses integrin activation in cells in a talin-dependent manner. Our results shed light on a new class of integrin inhibitors and a novel approach to design multi-specific peptidomimetic inhibitors.

Structural, biochemical, and functional properties of the Rap1-Interacting Adaptor Molecule (RIAM).

Leukocytes, the leading players of immune system, are involved in innate and adaptive immune responses. Leukocyte adhesion to endothelial cells during transmigration or to antigen presenting cells during T cell activation, requires integrin activation through a process termed inside-out integrin signaling. In hematopoietic cells, Rap1 and its downstream effector RIAM (Rap1-interacting adaptor molecule) form a cornerstone for inside-out integrin activation. The Rap1/RIAM pathway is involved in signal integration for activation, actin remodeling and cytoskeletal reorganization in T cells, as well as in myeloid cell differentiation and function. RIAM is instrumental for phagocytosis, a process requiring particle recognition, cytoskeletal remodeling and membrane protrusion for engulfment and digestion. In the present review, we discuss the structural and molecular properties of RIAM and the recent discoveries regarding the functional role of the Rap1/RIAM module in hematopoietic cells.

TLR4 inhibition ameliorated glucolipotoxicity-induced differentiation suppression in osteoblasts via RIAM regulation of NF-κB nuclear translocation.

TLR4 is a key innate immune signal that mediates glucolipid toxicity through yet unclear mechanisms. Here, TLR4 truncation ameliorated bone metabolism disorders in diabetic rats, and the underlying mechanisms were explored by proteomics. Our study showed that TLR4 truncation inhibited bone loss induced by diabetes in rats. In addition, a proteomic analysis screen exposed the differential proteins associated with immune reactivity and T cell activation (RIAM and Class II histocompatibility antigen, M ß1 chain). Further cellular experiments showed that TLR4 mediated the inhibition of osteoblast differentiation induced by glucolipotoxicity and promoted an increase in the nuclear level of RIAM-NF-κB. Mechanistic studies showed that TLR4 mediated glucolipotoxicity induced damage in bone metabolism primarily by regulating RIAM-NF-κB interactions, which promoted RIAM-NF-κB nuclear translocation. In conclusion, we confirmed that TLR4 inhibition could delay bone metabolism disorders induced by glycolipid toxicity via RIAM regulation of NF-κB nuclear translocation.

Integrin Regulators in Neutrophils.

Neutrophils are the most abundant leukocytes in humans and are critical for innate immunity and inflammation. Integrins are critical for neutrophil functions, especially for their recruitment to sites of inflammation or infections. Integrin conformational changes during activation have been heavily investigated but are still not fully understood. Many regulators, such as talin, Rap1-interacting adaptor molecule (RIAM), Rap1, and kindlin, are critical for integrin activation and might be potential targets for integrin-regulating drugs in treating inflammatory diseases. In this review, we outline integrin activation regulators in neutrophils with a focus on the above critical regulators, as well as newly discovered modulators that are involved in integrin activation.

Expression of the phagocytic receptors αMß2 and αXß2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMß2, CD11b/CD18) and CR4 (αXß2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to ß2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (ß2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMß2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMß2 and αXß2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Phosphorylation of RIAM Activates Its Adaptor Function in Mediating Integrin Signaling.

Integrins are cellular receptors that regulate cell adhesion and many other cellular functions. Integrins can be activated via an "inside-out pathway" that is promoted by RAP1 GTPase. RAP1-GTP-Interacting Adaptor Molecular (RIAM) mediates integrin activation by linking RAP1 GTPase to talin, an integrin activator. RIAM's function in integrin signaling is tightly regulated. In this commentary, we review recent studies of the molecular mechanisms underlying RIAM autoinhibition via both intramolecular interaction and oligomer assembly, and the phosphorylation-dependent activation of RIAM.

Environmental impact assessment of a steel industry development plan using combined method involving Leopold matrix and RIAM.

The present study was conducted to determine positive and negative impacts of Sepid-Farab Kavir steel (SKS) complex development plan and to propose suitable managerial strategies by a combined method involving Leopold matrix and Rapid Impact Assessment matrix (RIAM). The SKS complex is located in Aran-Bidgol city, Isfahan, Iran. Two scenarios of project implementation and project cancellation were formulated for SKS complex development plan, which has two sub-phases: construction and operation Using Leopold and RIAM matrices, the direct and indirect impacts of the project on the study area was investigated. The impact analysis for project cancellation scenario showed that the obtained scores of construction and operation phases were -119 and -52, respectively. Also, for project implementation scenario, the obtained scores of construction and operation phases were + 302 and + 382, respectively. The number of positive impacts in the implementation and cancellation scenarios were 354 and 48, respectively, and the number of negative impacts in implementation and cancellation scenarios were 270 and 127, respectively. Also, comparison of positive and negative impacts frequency in the two scenarios, and in the two sub-phases, in RIAM indicated the project implementation will have positive impacts in social-cultural and economic-operational aspects compared to option of prevention, especially in operation phase. The results of environmental impact assessment of the mentioned project indicated the superiority of positive impacts over the negative ones.

Binding of Rap1 and Riam to Talin1 Fine-Tune ß2 Integrin Activity During Leukocyte Trafficking.

ß2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent ß2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the ß2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that ß2 integrin regulation primarily occurs via these two pathways.

Phosphorylation of RIAM by src promotes integrin activation by unmasking the PH domain of RIAM.

Integrin activation controls cell adhesion, migration, invasion, and extracellular matrix remodeling. RIAM (RAP1-GTP-interacting adaptor molecule) is recruited by activated RAP1 to the plasma membrane (PM) to mediate integrin activation via an inside-out signaling pathway. This process requires the association of the pleckstrin homology (PH) domain of RIAM with the membrane PIP2. We identify a conserved intermolecular interface that masks the PIP2-binding site in the PH domains of RIAM. Our data indicate that phosphorylation of RIAM by Src family kinases disrupts this PH-mediated interface, unmasks the membrane PIP2-binding site, and promotes integrin activation. We further demonstrate that this process requires phosphorylation of Tyr267 and Tyr427 in the RIAM PH domain by Src. Our data reveal an unorthodox regulatory mechanism of small GTPase effector proteins by phosphorylation-dependent PM association of the PH domain and provide new insights into the link between Src kinases and integrin signaling.

RIAM-VASP Module Relays Integrin Complement Receptors in Outside-In Signaling Driving Particle Engulfment.

The phagocytic integrins and complement receptors αMß2/CR3 and αXß2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of ß2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

Adhesions Assemble!-Autoinhibition as a Major Regulatory Mechanism of Integrin-Mediated Adhesion.

The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of tissues, organs and complex multicellular life forms. The development of such organisms required tight regulation of dynamic adhesive structures by signaling pathways that coordinate cell attachment. Integrin-mediated adhesion to the extracellular matrix provides cells with support, survival signals and context-dependent cues that enable cells to run different cellular programs. One mysterious aspect of the process is how hundreds of proteins assemble seemingly spontaneously onto the activated integrin. An emerging concept is that adhesion assembly is regulated by autoinhibition of key proteins, a highly dynamic event that is modulated by a variety of signaling events. By enabling precise control of the activation state of proteins, autoinhibition enables localization of inactive proteins and the formation of pre-complexes. In response to the correct signals, these proteins become active and interact with other proteins, ultimately leading to development of cell-matrix junctions. Autoinhibition of key components of such adhesion complexes-including core components integrin, talin, vinculin, and FAK and important peripheral regulators such as RIAM, Src, and DLC1-leads to a view that the majority of proteins involved in complex assembly might be regulated by intramolecular interactions. Autoinhibition is relieved via multiple different signals including post-translation modification and proteolysis. More recently, mechanical forces have been shown to stabilize and increase the lifetimes of active conformations, identifying autoinhibition as a means of encoding mechanosensitivity. The complexity and scope for nuanced adhesion dynamics facilitated via autoinhibition provides numerous points of regulation. In this review, we discuss what is known about this mode of regulation and how it leads to rapid and tightly controlled assembly and disassembly of cell-matrix adhesion.

Leukocyte arrest: Biomechanics and molecular mechanisms of ß2 integrin activation.

Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell-cell and cell-matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called activation. Here we review the molecular biomechanics during conformational changes of integrins, integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in integrin activation.

The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism.

The small GTPase Rap1 induces integrin activation via an inside-out signaling pathway mediated by the Rap1-interacting adaptor molecule (RIAM). Blocking this pathway may suppress tumor metastasis and other diseases that are related to hyperactive integrins. However, the molecular basis for the specific recognition of RIAM by Rap1 remains largely unknown. Herein we present the crystal structure of an active, GTP-bound GTPase domain of Rap1 in complex with the Ras association (RA)-pleckstrin homology (PH) structural module of RIAM at 1.65 Å. The structure reveals that the recognition of RIAM by Rap1 is governed by side-chain interactions. Several side chains are critical in determining specificity of this recognition, particularly the Lys31 residue in Rap1 that is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases. Lys31 forms a salt bridge with RIAM residue Glu212, making it the key specificity determinant of the interaction. We also show that disruption of these interactions results in reduction of Rap1:RIAM association, leading to a loss of co-clustering and cell adhesion. Our findings elucidate the molecular mechanism by which RIAM mediates Rap1-induced integrin activation. The crystal structure also offers new insight into the structural basis for the specific recruitment of RA-PH module-containing effector proteins by their small GTPase partners.