Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.
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In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions. (+info)
Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.
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Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins. (+info)
Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.
(43/20287)
The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection. However, mRNA injection has several disadvantages. The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages. To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively. In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS). Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators. This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes. (+info)
Analysis of macrophage scavenger receptor (SR-A) expression in human aortic atherosclerotic lesions.
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The class A scavenger receptors (SR-As) are trimeric, integral membrane glycoproteins that exhibit unusually broad ligand-binding properties. A number of studies have suggested that these receptors may play an important role in host defense and in many macrophage-associated pathological processes, including atherosclerosis and Alzheimer's disease. The study of the expression and function of these receptors in human disease has been hampered by the lack of suitable antibodies recognizing human SR-A. This has generated questions regarding the nature of receptors responsible for scavenger receptor activity detected in a variety of cell types, including monocytes, macrophages, smooth muscle cells, and endothelial cells. To address these questions, we have produced high-titer antisera recognizing human SR-A by using mice deficient for SR-A (SR-A -/-). We show that SR-A -/- mice produce a significantly higher-titer immune response than do wild-type (SR-A +/+) littermates, with antisera of the former having a broad species reactivity and recognizing SR-A from humans, mice, and rabbits. The antisera recognize both type I and II SR-A in a wide range of immunological techniques. Using these antisera we show that the expression of SR-A protein is induced during monocyte to macrophage differentiation and that SR-A mediates 80% of the uptake of acetylated low density lipoprotein by human monocyte-derived macrophages. We also establish that human SR-A is expressed by tissue macrophages in liver and lung and by macrophage-derived foam cells within aortic atherosclerotic lesions, with little detectable expression by smooth muscle cells or aortic endothelium. (+info)
Reduced in vitro repair in endothelial cells harvested from the intercostal ostia of porcine thoracic aorta.
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The ability of large-vessel endothelium to repair itself rapidly after injury is important in the maintenance of its barrier function and in limiting the development and progression of atherosclerosis. Because dysfunctional repair may be involved in the pathogenesis of some atherosclerotic plaques, including those at the ostia of aortic branches, linear mechanical denuding wounds were made in confluent monolayers of endothelial cells harvested by scraping from the flow divider, the upstream wall of the intercostal branch and unbranched regions in the thoracic aorta. The extent of wound closure was significantly lower in cells derived from either side of the intercostal branches, compared with cells from unbranched areas. The wound edge of cells harvested from the flow divider and its opposite wall closed by 22+/-0.084 microm and 22+/-1.3 microm, respectively, versus control, unbranched endothelial cells (30+/-2.2 microm) at 24 hours and by 48 hours, 48+/-3.4 microm and 47+/-3.6 microm compared with control (61+/-3.4 microm). Extent of wound closure in cells harvested by scraping from unbranched regions was comparable with collagenase-harvested endothelial cells at 24 and 48 hours. Distribution of F-actin microfilaments, tubulin and centrosomes have been shown to be disrupted at the wound edge in poorly migrating cells. In our study, however, no differences were observed in cytoskeletal distribution between cells from branched, unbranched and control areas. Thus, aortic endothelial cells from the intercostal branch region show a reduced ability to repair wounds compared with cells harvested from unbranched aorta. The mechanism for this difference is currently unknown. (+info)
Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation.
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The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the DeltaB5R and DeltaF13L deletion strains, but not DeltaA34R, were still able to induce actin tails. The DeltaA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from DeltaA34R- and DeltaA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation. (+info)
Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin.
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The nucleocapsid (NC) domain of the retrovirus Gag protein plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of human immunodeficiency virus type 1 (HIV-1) Gag was found to drastically reduce virus particle production in CD4(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type HIV-1 Gag, unmyristylated Gag, and p6(Gag) domain-truncated Gag molecules copurified with the host cell cytoskeleton, most of the mutant Gag molecules lacking both the NC and p6(Gag) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against actin coimmunoprecipitated full-length Gag and p6(Gag) domain-truncated Gag molecules from cell lysates but failed to precipitate the truncated mutant Gag molecules lacking NC plus p6(Gag). Purified recombinant NCp7, but not CAp24, was able to bind F-actin in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular actin-binding protein (the villin headpiece), bound F-actin in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind F-actin directly and that interaction between HIV-1 Gag and the actin cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle. (+info)
Self-regulated polymerization of the actin-related protein Arp1.
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The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments. (+info)