A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

A pro-tumorigenic function of S100A8/A9 in carcinogen-induced hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is a heterogeneous disease, driven by different risk factors and presenting diverse clinicopathological features and outcomes. Epidemiological and experimental data indicate that the damage-associated molecular pattern molecules S100A8 and S100A9, forming a heterodimer called calprotectin, might be critically involved in HCC development. However, deletion of S100a9 in an inflammation- and cirrhosis-driven mouse model did not show any impairment in liver tumorigenesis, most likely due to functional compensation by other inflammatory cytokines. Here, we investigated the effect of calprotectin ablation in mice treated with diethylnitrosamine, a carcinogen-driven HCC model mimicking cancer development caused by acute liver damage in the absence of prominent chronic inflammation and tissue damage. We found that tumor cell proliferation was diminished in the absence of S100A8/A9, leading to significant reduction of tumor size. Our results demonstrate that calprotectin is required for the progression of non-inflammation driven liver tumor and might represent a therapeutic target for the treatment of HCC formed in non-cirrhotic liver.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction.

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation.

Chronic liver inflammation and hepatocellular carcinogenesis are independent of S100A9.

The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC). Apart from this evidence, the role of calprotectin in hepatocyte transformation and tumor microenvironment is still unknown. The aim of this study was to define the function of S100A8/A9 in inflammation-driven HCC. Mice lacking S100a9 were crossed with the Mdr2(-/-) model, a prototype of inflammation-induced HCC formation. S100a9(-/-) Mdr2(-/-) (dKO) mice displayed no significant differences in tumor incidence or multiplicity compared to Mdr2(-/-) animals. Chronic liver inflammation, fibrosis and oval cell activation were not affected upon S100a9 deletion. Our data demonstrate that, although highly upregulated, calprotectin is dispensable in the onset and development of HCC, and in the maintenance of liver inflammation.

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia.

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9(-/-) mice that lack heterodimer expression in myeloid cells. S100a9(-/-) mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild-type mice recovered. Over this time period, S100a9(-/-) mice displayed an average 6-fold reduction in circulating and lung-recruited neutrophils. Taqman analysis of S100a9(-/-) lungs revealed decreased production of a dominant subset of 5 cytokines and chemokines associated with neutrophil recruitment. The greatest differential was with the cytokine granulocyte colony-stimulating factor (G-CSF) that causes bone marrow release of neutrophils into the circulation (1900-fold difference at 48 h). Treating S100a9(-/-) mice with G-CSF reversed their increased susceptibility to infection by enhancing both circulating neutrophils and neutrophil recruitment into infected lungs, by reducing pneumococcal colony forming units, and by elevation of chemokine CXCL1, cytokine IL-6, and endogenous G-CSF proteins. Thus S100A9, potentially with its partner S100A8, makes a major contribution in the host response to pneumococcal infection by increasing circulating neutrophils principally regulation of G-CSF production.

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis.

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14⁻/⁻ mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14⁻/⁻ mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14⁻/⁻ mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.

S100A9 has a protective role in inflammation-induced skin carcinogenesis.

The S100A8/A9 heterodimer is expressed by myeloid cells where its function has been extensively investigated. Immune cell S100A8/A9 promotes proinflammatory effects, and its absence is often associated with lack of leukocyte recruitment resulting in protection in terms of disease progression. S100A8/A9 is also expressed by certain epithelia, either constitutively as in mucosal epithelia or following stimulation as in skin keratinocytes. The role of the heterodimer in this context has not been as frequently explored. In this study, the incidence of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) in S100a9(-/-) mice has been investigated. Unlike the immune disorders and certain models of cancer, absence of S100A8/A9 caused an increased incidence in skin of papillomas and, subsequently, squamous cell carcinomas. Although associated in S100a9(-/-) mice with increased recruitment of neutrophils and T cells, a bone marrow chimera experiment revealed the major defect to be primarily due to the absence of S100A8/A9 in the skin keratinocytes. S100a9(-/-) skin displayed enhanced Ki-67 expression over the time period of appearance of the papillomas suggesting an effect of S100A8/A9 in regulating proliferation in the epidermal layer. Thus, despite immune cell recruitment in S100a9(-/-) mouse skin that might have been predicted to promote tumor growth, it was the absence of S100A8/A9 in skin keratinocytes that dominated in terms of papilloma formation. The study highlights the importance of the S100A8/A9-expressing skin epidermal layer in controlling skin tumor formation and suggests that the influence of the heterodimer is dependent on the tissue context in which it is expressed.

A new leukocyte hyperadhesion syndrome of delayed cord separation, skin infection, and nephrosis.

Leukocyte adhesion deficiency (LAD) I is a well-described genetic disorder in which leukocytes are unable to migrate to sites of inflammation due to mutations in the ITGB2 gene coding for the ß subunit of ß2 (CD18) leukocyte integrins. The classic symptoms of the disease present in the newborn period as failure of separation of the umbilical cord and recurrent bacterial infections, which continue throughout life. We report on a patient with these clinical manifestations but with normal ITGB2 gene sequencing excluding LAD-I, normal carbohydrate-deficient transferrin testing excluding LAD-II, and normal platelet function excluding LAD-III. With testing for CD18 integrin function by flow cytometry, adhesion assay analysis, and time-lapse microscopy, we found the patient's T lymphocytes to express normal levels of ß1 and ß2 integrins but to be highly adhesive to integrin ligands and to display decreased migration compared with control T lymphocytes. The hyperadhesiveness of the cells suggests that they might be prevented from reaching infected tissues. Interestingly, administration of glucocorticoids, for the patient's nephrotic syndrome, alleviated the patient's chronic diarrhea and decreased the incidence of skin infections. The hyperadhesiveness rather than adhesion deficiency of the patient's leukocytes suggests that a novel lesion in a pathway regulating integrin adhesion is responsible for the patient's unique LAD-I-like symptoms.

S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3.

Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin. S100A9 is shown here to function as a chromatin component modulating C3 expression in mouse and human cells by binding to a region upstream of the C3 start site. When S100A9 was genetically deleted in mouse models of skin inflammation, the psoriasis-like skin disease and inflammation were strongly attenuated, with a mild immune infiltrate and decreased amounts of C3. In addition, inhibition of C3 in the mouse model strongly reduced the inflammatory skin disease. Thus, S100A8-S100A9 can regulate C3 at the nuclear level and present potential new therapeutic targets for psoriasis.

SHARPIN regulates uropod detachment in migrating lymphocytes.

SHARPIN-deficient mice display a multiorgan chronic inflammatory phenotype suggestive of altered leukocyte migration. We therefore studied the role of SHARPIN in lymphocyte adhesion, polarization, and migration. We found that SHARPIN localizes to the trailing edges (uropods) of both mouse and human chemokine-activated lymphocytes migrating on intercellular adhesion molecule-1 (ICAM-1), which is one of the major endothelial ligands for migrating leukocytes. SHARPIN-deficient cells adhere better to ICAM-1 and show highly elongated tails when migrating. The increased tail lifetime in SHARPIN-deficient lymphocytes decreases the migration velocity. The adhesion, migration, and uropod defects in SHARPIN-deficient lymphocytes were rescued by reintroducing SHARPIN into the cells. Mechanistically, we show that SHARPIN interacts directly with lymphocyte-function-associated antigen-1 (LFA-1), a leukocyte counterreceptor for ICAM-1, and inhibits the expression of intermediate and high-affinity forms of LFA-1. Thus, SHARPIN controls lymphocyte migration by endogenously maintaining LFA-1 inactive to allow adjustable detachment of the uropods in polarized cells.

Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images. The program also automatically tracks rolling cells in addition to those that are more firmly attached and migrating in random directions. We demonstrate its use in the analysis of lymphocyte arrest mediated by one or more active conformations of the integrin LFA-1. Activation of LFA-1 is a multistep process that depends on several proteins including kindlin-3, the protein that is mutated in leukocyte adhesion deficiency-III patients. We find that the very first stage of LFA-1-mediated attaching is unable to proceed in the absence of kindlin-3. Our evidence indicates that kindlin-3-mediated high-affinity LFA-1 controls both the early transient integrin-dependent adhesions in addition to the final stable adhesions made under flow conditions.

Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.

The kindlin 3 pleckstrin homology domain has an essential role in lymphocyte function-associated antigen 1 (LFA-1) integrin-mediated B cell adhesion and migration.

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.

T lymphocytes orient against the direction of fluid flow during LFA-1-mediated migration.

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm(-2). T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.

A role for LFA-1 in delaying T-lymphocyte egress from lymph nodes.

Lymphocytes use the integrin leukocyte function-associated antigen-1 (LFA-1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA-1 dependent. We show that LFA-1 mediates prolonged LN residence as LFA-1(-/-) CD4 T cells have significantly decreased dwell times compared with LFA-1(+/+) T cells, a distinction lost in hosts lacking the major LFA-1 ligand ICAM-1. Intra-vital two-photon microscopy revealed that LFA-1(+/+) and LFA-1(-/-) T cells reacted differently when probing the ICAM-1-expressing lymphatic network. While LFA-1(+/+) T cells returned to the LN parenchyma with greater frequency, LFA-1(-/-) T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA-1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T-cell response to antigen. Thus, we identify a novel function for LFA-1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.

Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide.

T lymphocytes have an essential role in adaptive immunity and rely on the activation of integrin lymphocyte function-associated antigen-1 (LFA-1) to mediate cell arrest and migration. In cancer, malignant cells modify the immune microenvironment to block effective host antitumor responses. We show for the first time that CD4 and CD8 T cells from patients with chronic lymphocytic leukemia (CLL) exhibit globally impaired LFA-1-mediated migration and that this defect is mediated by direct tumor cell contact. We show that following the coculture of previously healthy T cells with CLL cells, subsequent LFA-1 engagement leads to altered Rho GTPase activation signaling by downregulating RhoA and Rac1, while upregulating Cdc42. Of clinical relevance, repair of this T-cell defect was demonstrated using the immunomodulatory drug lenalidomide, which completely rescued adhesion and motility function by restoring normal Rho GTPase activation signaling. Our report identifies a novel cancer immune evasion mechanism whereby tumor cells induce Rho GTPase signaling defects in T cells that prevent appropriate LFA-1 activation and motility. We believe these findings identify important biomarkers and highlight the clinical utility of immunotherapy to rescue normal T-cell function in CLLs that are likely to have relevance in other cancers.

A role for Rap2 in recycling the extended conformation of LFA-1 during T cell migration.

T lymphocytes make use of their major integrin LFA-1 to migrate on surfaces that express ICAM-1 such as blood vessels and inflamed tissue sites. How the adhesions are turned over in order to supply traction for this migration has not been extensively investigated. By following the fate of biotinylated membrane LFA-1 on T lymphocytes, we show in this study that LFA-1 internalization and re-exposure on the plasma membrane are linked to migration. Previously we demonstrated the GTPase Rap2 to be a regulator of LFA-1-mediated migration. SiRNA knockdown of this GTPase inhibits both LFA-1 internalization and also its ability to be re-exposed, indicating that Rap2 participates in recycling of LFA-1 and influences its complete endocytosis-exocytosis cycle. Confocal microscopy images reveal that the intracellular distribution of Rap2 overlaps with endosomal recycling vesicles. Although the homologous GTPase Rap1 is also found on intracellular vesicles and associated with LFA-1 activation, these two homologous GTPases do not co-localize. Little is known about the conformation of the LFA-1 that is recycled. We show that the extended form of LFA-1 is internalized and in Rap2 siRNA-treated T lymphocytes the trafficking of this LFA-1 conformation is disrupted resulting in its intracellular accumulation. Thus LFA-1-mediated migration of T lymphocytes requires Rap2-expressing vesicles to recycle the extended form of LFA-1 that we have previously found to control migration at the leading edge.

A CXCL1 paracrine network links cancer chemoresistance and metastasis.

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

The Gαq/11 proteins contribute to T lymphocyte migration by promoting turnover of integrin LFA-1 through recycling.

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration. Gαq/11 has an impact on LFA-1 expression at plasma membrane level and also on its internalization. Additionally Gαq co-localizes with LFA-1- and EEA1-expressing intracellular vesicles and partially with Rap1- but not Rab11-expressing vesicles. However the influence of Gαq is not confined to the vesicles that express it, as its reduction alters intracellular trafficking of other vesicles involved in recycling. In summary vesicle-associated Gαq/11 is required for the turnover of LFA-1 adhesion that is necessary for migration. These G proteins participate directly in the initial phase of recycling and this has an impact on later stages of the endo-exocytic pathway.