We hypothesize that related biological processes are involved not only in normal morphogenesis and tissue repair, but also in pathological processes such as tumor cell invasion and HIV pathogenesis. Previous studies by numerous laboratories including our own have identified overlapping mechanisms that include choreographed changes in cell-matrix and cell-cell adhesion, migration, and associated signal transduction pathways. Understanding these processes should help to clarify mechanisms of pathogenesis and to identify novel potential targets for therapeutic intervention. ? ? Both tumor cells and certain HIV-infected cells can migrate and invade tissues. The role of proteases in these processes is currently uncertain. We are focusing on three facets of this question: (1) What initiates and regulates the formation and function of invadopodia, the small cell surface structures postulated to mediate proteolysis? (2) Where on the cell surface do cells degrade the matrix during cell motility, and is proteolysis essential for migration? (3) In comparison, how do normal cells such as fibroblasts migrate through 3D matrices, and do they require proteases?? ? We have been developing new tools in collaborations to address these questions. One approach has been to develop new microscopy methods and to adapt TIRF (total internal reflection microscopy) to study proteolysis in 2D regular tissue culture and in 3D matrices. A second approach is to try to develop sensors for local proteolysis. To study the dynamics of invadopodial function, we developed a high-sensitivity fluorescent gelatin substrate to detect invadopodia-degraded holes in the matrix and 3-channel live-cell imaging for parallel analysis of the dynamics of multiple proteins. We published the first analysis of essential steps in formation of invadopodia, identifying four stages with stage-specific functional roles for cortactin and MT1-MMP. We are extending these studies to 3D matrices to determine whether the mechanisms are similar and to identify early steps in invadopodial formation.? ? We have been involved in a long-term collaboration for a dozen years with the Dhawan laboratory in CBER, FDA to characterize cell-surface and extracellular interactions involved in AIDS pathogenesis. Ongoing studies have explored the roles of adhesion molecules, secreted HIV-Tat protein, and signal transduction in HIV disease, and we have attempted to develop a novel approach to generate a therapeutic vaccine, which we feel is more practical at the current time in the field than a preventive vaccine. HIV-Tat peptides that we had previously identified as being important for its function as an extracellular effector were tested for ability to vaccinate mice, using peptide immunogens with certain amino acid residues modified to suppress the potent cell biological activity of these peptides. We recently demonstrated that this novel vaccine candidate generates antibodies capable of blocking HIV-1 viral infectivity and cytopathic effects in human cells against multiple HIV-1 isolates.
