The alphavbeta3 integrin regulates alpha5beta1-mediated cell migration toward fibronectin.

This study examines the interactions of alphavbeta3 and alpha5beta1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express alpha5beta1 endogenously were transfected with alphavbeta3 and beta3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An alphavbeta3 blocking antibody and the alphavbeta3 ligand cyclic G-Pen-GRGDSPC-A inhibited alpha5beta1-mediated migration toward Fn, but not attachment to Fn. This function was alphavbeta3-specific since alphavbeta5 transfection and alphavbeta5 blocking antibody did not produce this effect. Mutations introduced into the beta3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation beta3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation beta3-S752P into the cytoplasmic domain or its truncation (beta3-Delta717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by alphavbeta3 blocking antibody. alphavbeta3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (beta3-Delta693). These findings demonstrate alphavbeta3 regulation of alpha5beta1-mediated cell migration and suggest that the beta3 transmembrane domain is essential for this function.

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus.

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.

Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus.

The superinfection exclusion of VSV has been studied and found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: first, a decreased rate of formation of endocytic vesicles as judged by an inhibition of fluid-phase uptake at 2 hr postinfection; second, a decreased rate of internalization of receptor-bound ligands, which was detected at 4 hr postinfection; and third, a competition with newly synthesized virus for occupancy of coated pits, as indicated by electron microscopy of infected cells. At the same time that fluid-phase uptake decreased, numerous uncoated invaginations were observed at the cell surface.