The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium.

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Hematopoietic stem and progenitor cells use podosomes to transcellularly cross the bone marrow endothelium.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

The GEF Trio controls endothelial cell size and arterial remodeling downstream of Vegf signaling in both zebrafish and cell models.

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome.

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.

Inflammation-Sensitive Myosin-X Functionally Supports Leukocyte Extravasation by Cdc42-Mediated ICAM-1-Rich Endothelial Filopodia Formation.

Leukocyte transendothelial migration is key to inflammation. Leukocytes first start rolling over the inflamed endothelium, followed by firmly adhering to it. Under inflammatory conditions, endothelial cells express small finger-like protrusions that stick out into the lumen. The function and regulation of these structures are unclear. We present evidence that these ICAM-1- and F-actin-rich endothelial finger-like protrusions are filopodia and function as adhesive structures for leukocytes to transit from rolling to crawling but are dispensable for diapedesis. Mechanistically, these structures require the motor function of myosin-X, activity of the small GTPase Cdc42, and p21-activated kinase 4. Moreover, myosin-X expression is under control of TNF-α-mediated c-Jun N-terminal kinase activity and is upregulated in human atherosclerotic regions. To our knowledge, this is the first study to identify that regulation of endothelial filopodia is crucial for leukocyte extravasation, in particular for the initiation of leukocyte adhesion under flow conditions.

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution.

Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, it is not clear whether sustained and local activation of Rac1 is required for long-term flow-induced cell alignment. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 h upon long-term flow. Silencing studies show that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and, as a result, for long-term flow-induced cell alignment. Surprisingly, Trio appears to be not involved in flow-induced activation of Rac1. Our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner and that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions.

Annexin A2 Limits Neutrophil Transendothelial Migration by Organizing the Spatial Distribution of ICAM-1.

ICAM-1 is required for firm adhesion of leukocytes to the endothelium. However, how the spatial organization of endothelial ICAM-1 regulates leukocyte adhesion is not well understood. In this study, we identified the calcium-effector protein annexin A2 as a novel binding partner for ICAM-1. ICAM-1 clustering promotes the ICAM-1-annexin A2 interaction and induces translocation of ICAM-1 into caveolin-1-rich membrane domains. Depletion of endothelial annexin A2 using RNA interference enhances ICAM-1 membrane mobility and prevents the translocation of ICAM-1 into caveolin-1-rich membrane domains. Surprisingly, this results in increased neutrophil adhesion and transendothelial migration under flow conditions and reduced crawling time, velocity, and lateral migration distance of neutrophils on the endothelium. In conclusion, our data show that annexin A2 limits neutrophil transendothelial migration by organizing the spatial distribution of ICAM-1.

F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling.

During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.

Self-assembled CdSe/CdS nanorod sheets studied in the bulk suspension by magnetic alignment.

We studied spontaneously self-assembled aggregates in a suspension of CdSe/CdS core/shell nanorods (NRs). The influence of the length and concentration of the NRs and the suspension temperature on the size of the aggregates was investigated using in situ small-angle X-ray scattering (SAXS) and linear dichroism (LD) measurements under high magnetic fields (up to 30 T). The SAXS patterns reveal the existence of crystalline 2-dimensional sheets of ordered NRs with an unusually large distance between the rods. The LD measurements show that the size of the sheets depends on the free-energy driving force for NR self-assembly. More precisely, the sheets are larger if the attraction between NRs is stronger, if the temperature is lower, or if the NR concentration is higher. We show that the formation of large NR sheets is a slow process that can take days. Our in situ results of the structures that spontaneously form in the bulk suspension could further our understanding of NR self-assembly into mono- or multilayer superlattices that occurs at the suspension/air interface upon evaporation of the solvent.

Rho-GTPase signaling in leukocyte extravasation: an endothelial point of view.

Leukocyte transendothelial migration (TEM) is one of the crucial steps during inflammation. A better understanding of the key molecules that regulate leukocyte extravasation aids to the development of novel therapeutics for treatment of inflammation-based diseases, such as atherosclerosis and rheumatoid arthritis. The adhesion molecules ICAM-1 and VCAM-1 are known as central mediators of TEM. Clustering of these molecules by their leukocytic integrins initiates the activation of several signaling pathways within the endothelium, including a rise in intracellular Ca (2+), activation of several kinase cascades, and the activation of Rho-GTPases. Activation of Rho-GTPases has been shown to control adhesion molecule clustering and the formation of apical membrane protrusions that embrace adherent leukocytes during TEM. Here, we discuss the potential regulatory mechanisms of leukocyte extravasation from an endothelial point of view, with specific focus on the role of the Rho-GTPases.

The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.

Spatial distribution of nanocrystals imaged at the liquid-air interface.

The 3D distribution of nanocrystals at the liquid-air interface is imaged for the first time on a single-particle level by cryogenic electron tomography, revealing the equilibrium concentration profile from the interface to the bulk of the liquid. When the surface tension of the liquid is decreased, the interaction of the nanocrystals with the liquid-air interface shifts from adsorption to desorption. Macroscopic surface tension measurements do not detect this transition, due to the presence of surface-active molecular species.

The many faces of the guanine-nucleotide exchange factor trio.

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.