No abstract

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.Human immunodeficiency virus (HIV) therapies have been highly successful in slowing disease progression, increasing health and well-being, and prolonging life. However, viral resistance is now becoming common, and since most existing drugs target only two viral proteins, reverse transcriptase and protease, cross-resistance is a significant problem. One solution to the issue of resistance is development of new complementary therapies based on novel mechanisms of action. The discovery that the chemokine receptors CCR5 and CXCR4, in addition to CD4, are required for viral entry not only furthered understanding of the fusion and infection process but provided two new targets for therapeutic intervention (3,12,14,17,18,22,44).The entry mechanism as currently understood is an ordered process in which the viral envelope protein, gp120, following interaction with CD4, undergoes a conformational change allowing binding to the appropriate chemokine receptor, CCR5 for macrophagetropic or R5 strains, and CXCR4 for T-celltropic or X4 s...

The fibronexus (FNX), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substratebinding surface of fibroblasts in tissue culture (Singer, I . I ., 1979, Cell, 16 :675-685) . To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds . At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy . These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy . We observed three kinds of FNXs: (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs, in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils . Coniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial . Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin . The finding that these FNXs appear to connect Collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation .The in vitro enhancement of fibroblast adhesion and spreading by fibronectin has been well documented (20,29,55,56), whereas the importance of the cytoskeleton in these processes has only recently been realized (23,26,27,42,43,46). The observation that actin microfilament bundles (stress fibers) are highly developed in well-spread stationary fibroblasts in vitro (23,27,35,43,46) transformed cells, strongly suggests a dominant role for the cytoskeleton in the fibroblast-to-substrate adhesion mechanism. A fascinating aspect ofthis phenomenon is the coincident distribution of fibronectin-containing extracellular matrix fibers and bundles of actin microfilaments on a global scale at the substrate adhesive surface (22,26,27). Electron microscopic studies of this substrate-binding plasma mem-

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin- coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate- bound vitronectin.

Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

IntroductionThe destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. (21,(26)(27)(28), and induction of the cytokines IFNy, TNFa, and IL-1IB (20, 29-1. Abbreviations used in this paper:

Mevinolin is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), an enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. We have been studying the hepatic distribution of reductase with immunofluorescence microscopy and liver ultrastructure with electron microscopy in normal and drug-treated rats. In control animals, only about 20% of the hepatocytes were reductase positive. These cells were localized in the periportal lobular zones. The numbers of positive hepatocytes in animals given mevinolin or cholestyramine (or both) were directly proportional to the activities of the HMG-CoA reductase determined biochemically. This induction of HMG-CoA reductase immunofluorescence was centered periportally. Rats given 0.075% mevinolin alone had a homogeneous distribution of reductase staining in their hepatocyte cytoplasm, whereas a combination of 0.25% mevinolin and 3% cholestyramine caused a 150-fold increase in enzyme activity and induced prominent juxtanuclear immunofluorescent globules of HMG-CoA reductase in all hepatocytes. With electron microscopy, these bodies were composed of tightly packed stacks of smooth endoplasmic reticulum cysternae and aggregates of branched smooth endoplasmic reticulum tubules. Our data suggest that a subpopulation of periportal rat hepatocytes may be uniquely specialized for cholesterol synthesis.Mevinolin, a fungal metabolite isolated from Aspergillus terreus, is a potent competitive inhibitor of the rate-limiting enzyme of cholesterol biosynthesis, microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34) (1). A similar but somewhat less active inhibitor, compactin (ML-236B), has been purified from Penicillium citrium (2). These compounds are effective cholesterol-lowering agents in several species, including dogs (1, 3, 4) and humans (5-10) but not rats (11,12). Furthermore, 50-70% reductions in the plasma cholesterol levels of dogs and humans have been obtained with combinations of bile acid sequestrants and mevinolin or compactin (4, 8-10).The synthesis of HMG-CoA reductase in rat liver is exquisitely sensitive to a number of factors, including diurnal variation (13,14). Treatment of rats with either an HMGCoA reductase inhibitor or a bile acid sequestrant (or both) results in a marked elevation of enzyme level (14-17). This induction is the result of a dramatic increase in reductase mRNA levels and an enhancement of enzyme stability (15-17). Similarly, in UT-1 cells, a compactin-resistant line, reductase levels are greatly elevated but may be repressed abruptly by low density lipoprotein administration (18). Microscopic studies of these cells reveal a dramatic development of the smooth endoplasmic reticulum (SER) associated with high concentrations of HMG-CoA reductase (19-21).The purpose of this study was to determine the hepatic distribution of HMG-CoA reductase and to correlate putative alterations in reductase patterns and ultrastructure with the extent of enzyme ...

Abstract. We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CDllb/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CDI8, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six-to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that upregulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.XTRAVASATION of polymorphonuclear leukocytes (PMNs) m and monocytes and their entrance into inflammatory tissue sites requires that they adhere to and penetrate the capillary endothelium, the basement membrane, and the stromal connective tissue (14,42,60). The hematogenous spread of metastatic cells occurs via a similar mechanism (3, 32). Certain cell surface receptors for extracellular l igands play central roles in these adhesion-dependent processes, and the molecular mechanisms underlying these interactions are being elucidated (27,36,48,49).The dominant adhesive ligand of basement membranes is laminin (LN) (36), whereas fibronectin (FN) and vitronectin (VN) are attachment ligands of the connective tissue stroma (23,48,49); other important adhesive molecules such as ICAM-1 and ELAM-1 are expressed on the endothelial cell surface (8,9,13,35). Several types of laminin receptors (LNRs) have been described. A LNR with a molecular mass of 67 kD has been identified in neoplastic cells, PMNs, mac-1. Abbreviations used in this paper: ECMR, extracellular matrix receptor; fMLP, f-met-leu-phe; FN, fibronectin; FNR, fibronectin receptor; IEM, immunoeleetron microscopy; LN, laminin; LNR, laminin receptor; LPS, lipopolysaccharide; PMN, polymorphonuclear; TNF, tumor necrosis factor; VN, vitronectin; VNR, vitronectin receptor. rophages, and myocytes (25,31,37,46,60,61,68,78). This receptor consists of a si...