Abstract. Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serumcontaining medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation.

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Abstract. We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogenous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAIl, we stained the cells live at 0°C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10 -6 M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PAmediated focal proteolysis.

Abstract.We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PAcontaining focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5 % FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion. rIESIVE interactions of cells with the extracellular matrix and their reversal are critical events for morphogenetic movements during embryonic development and for cancer cell invasion during metastasis (30, 49). The molecular structures at the sites where cultured cells contact each other and the growth substratum are thus of great interest. Focal contacts, the sites of closest cell-substratum apposition, can be visualized either by electron microscopy (1) or by interference reflection microscopy (23). Focal contacts are localized at the termini of actin-containing microfilament bundles, and contain vinculin (7, 19) and talin (6) on the cytoplasmic side, but apparently do not have fibronectin on the extracellular side (9). The so-called close contacts exhibit a greater substrate separation distance, do not contain vinculin, and probably represent a more labile type of contact (23). A third kind of substrate adhesion site termed the fibronexus (42) or extracellular matrix contact site (9) is characteristic of well-spread and stationary fibroblasts. It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15). Fibronexuses show very close transmembrane associations of actin microfilaments and fibronectin fibrils (42,43).

Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in. an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of prou-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which It depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.The process of malignant invasion and metastasis by tumor cells requires a coordinated sequence of events involving both detachment from existing contacts and the formation of new adhesive interactions with the extracellular matrix at new sites. Focal adhesions are specialized structures at the sites of closest cell-substratum contact, providing a transmembrane linkage between the extracellular matrix and the cytoskeleton (1). Focal adhesions contain several structural components, such as vinculin (2, 3), talin (4), integrins (5-7), heparan sulfate proteoglycan (8), and the urokinase-type plasminogen activator (u-PA) (9-11). Several regulatory factors have also been located at the focal adhesions, such as the tyrosine kinase oncogene products p60src, pl205ag-abl, p9(-Yes, and p8Va9-Yes (see ref.