SnapShot: talin and the modular nature of the integrin adhesome.

Integrin αß transmembrane heterodimers play central roles in metazoan development and physiology by mediating adhesion and by transmitting forces and biochemical signals across the plasma membrane. In this SnapShot, we present a simplified "modular" view of the integrin adhesome, centered on the talin-integrin interaction, and provide examples of how this view can help to unravel the adhesome's remarkable functional diversity and plasticity.

Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

Regulation of integrin activation.

Regulation of cell-cell and cell-matrix interaction is essential for the normal physiology of metazoans and is important in many diseases. Integrin adhesion receptors can rapidly increase their affinity (integrin activation) in response to intracellular signaling events in a process termed inside-out signaling. The transmembrane domains of integrins and their interactions with the membrane are important in inside-out signaling. Moreover, integrin activation is tightly regulated by a complex network of signaling pathways. Here, we review recent progress in understanding how the membrane environment can, in cooperation with integrin-binding proteins, regulate integrin activation.

Transcriptomic signatures of individual cell types in cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets. Video Abstract.

Kindlin-3 recruitment to the plasma membrane precedes high-affinity ß2-integrin and neutrophil arrest from rolling.

Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the ß2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates ß2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and ß2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ ß2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity ß2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ ß2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity ß2-integrin.

ß1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

ß1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active ß1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of ß1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting ß1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

Cerebral Cavernous Malformation: From Mechanism to Therapy.

Cerebral cavernous malformations are acquired vascular anomalies that constitute a common cause of central nervous system hemorrhage and stroke. The past 2 decades have seen a remarkable increase in our understanding of the pathogenesis of this vascular disease. This new knowledge spans genetic causes of sporadic and familial forms of the disease, molecular signaling changes in vascular endothelial cells that underlie the disease, unexpectedly strong environmental effects on disease pathogenesis, and drivers of disease end points such as hemorrhage. These novel insights are the integrated product of human clinical studies, human genetic studies, studies in mouse and zebrafish genetic models, and basic molecular and cellular studies. This review addresses the genetic and molecular underpinnings of cerebral cavernous malformation disease, the mechanisms that lead to lesion hemorrhage, and emerging biomarkers and therapies for clinical treatment of cerebral cavernous malformation disease. It may also serve as an example for how focused basic and clinical investigation and emerging technologies can rapidly unravel a complex disease mechanism.

Bioprotection of Transportation and Facilities from SARS-CoV-2 (COVID-19).

The recent COVID-19 pandemic has led to a nearly world-wide shelter-in-place strategy. This raises several natural concerns about the safe relaxing of current restrictions. This article focuses on the design and operation of heating ventilation and air conditioning (HVAC) systems in the context of transportation. Do HVAC systems have a role in limiting viral spread? During shelter-in-place, can the HVAC system in a dwelling or a vehicle help limit spread of the virus? After the shelter-in-place strategy ends, can typical workplace and transportation HVAC systems limit spread of the virus? This article directly addresses these and other questions. In addition, it also summarizes simplifying assumptions needed to make meaningful predictions. This article derives new results using transform methods first given in Ginsberg and Bui. These new results describe viral spread through an HVAC system and estimate the aggregate dose of virus inhaled by an uninfected building or vehicle occupant when an infected occupant is present within the same building or vehicle. Central to these results is the derivation of a quantity called the "protection factor"-a term-of-art borrowed from the design of gas masks. Older results that rely on numerical approximations to these differential equations have long been lab validated. This article gives the exact solutions in fixed infrastructure for the first time. These solutions, therefore, retain the same lab validation of the older methods of approximation. Further, these exact solutions yield valuable insights into HVAC systems used in transportation.

Optogenetics-based localization of talin to the plasma membrane promotes activation of ß3 integrins.

Interaction of talin with the cytoplasmic tails of integrin ß triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbß3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVß3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbß3 or αVß3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in ß3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated ß3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in ß3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.

Talin-1 is the principal platelet Rap1 effector of integrin activation.

Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin ß cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin-1 F1 interaction in platelets markedly decreases talin-1-mediated activation of platelet ß1- and ß3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1-talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.

The final steps of integrin activation: the end game.

Cell-directed changes in the ligand-binding affinity ('activation') of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin's affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.

MARCH Proteins Mediate Responses to Antitumor Antibodies.

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice.

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1 ECKO ) or Pdcd10 (Pdcd10 ECKO ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10 ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10 ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Phenotypic characterization of murine models of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.

Cutting Edge: Loss of T Cell RIAM Precludes Conjugate Formation with APC and Prevents Immune-Mediated Diabetes.

Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity.

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

The Rap1-RIAM-talin axis of integrin activation and blood cell function.

Integrin adhesion receptors mediate the adhesion of blood cells, such as leukocytes, to other cells, such as endothelial cells. Integrins also are critical for anchorage of hematopoietic precursors to the extracellular matrix. Blood cells can dynamically regulate the affinities of integrins for their ligands ("activation"), an event central to their functions. Here we review recent progress in understanding the mechanisms of integrin activation with a focus on the functions of blood cells. We discuss how talin binding to the integrin ß cytoplasmic domain, in conjunction with the plasma membrane, induces long-range allosteric rearrangements that lead to integrin activation. Second, we review our understanding of how signaling events, particularly those involving Rap1 small guanosine triphosphate (GTP)hydrolases, can regulate the talin-integrin interaction and resulting activation. Third, we review recent findings that highlight the role of the Rap1-GTP-interacting adapter molecule (RIAM), encoded by the APBB1IP gene, in leukocyte integrin activation and consequently in leukocyte trafficking.