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.