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 bi-directional host-pathogen interactions. Previous studies by numerous laboratories including our own have identified overlapping pathological mechanisms that include 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. In cancer metastasis and other disease processes, normal cell behavior is subverted by processes that modify the formation of cell adhesions, migration, and invasion. We are investigating the mechanisms of invasion to identify potential targets for therapeutic intervention in the pathogenic process, e.g., key steps and target molecules such as interactions of components of integrin adhesion and signaling complexes. We are addressing the following questions: 1. How do invadopodia, which are tiny cell surface structures mediating proteolysis, initiate and function? What regulates their formation? 2. How are integrins involved in tumor cell invasion and metastasis? 3. What aspects of cell-extracellular molecule interactions can be used for potential translational applications? We have focused on developing new tools and approaches to address these questions. An approach has been to develop cutting-edge microscopy and novel invasion substrates, as well as adapting TIRF (total internal reflection fluorescence) microscopy to study invadopodia. We previously published a description of essential steps in the formation and function of invadopodia: the structural actin cores of invadopodia are formed first and then the protease MT1-MMP accumulates to mediate ECM degradation. We have recently described the first live-cell imaging of dynamic invadopodial filamentous protrusions extending outward from cortactin cores. Rapid high-resolution TIRF microscopy permitted imaging of the ventral cell membrane and cytoplasm in closest proximity to the matrix substrate as invadopodia form. Plasma membrane dynamics near the cortactin core of the invadopodia of human carcinoma cells were visualized using two different membrane markers, an IL2R subunit lacking a cytoplasmic domain fused to mCherry and myristoylated-GFP;both membrane markers yielded similar results. The actin-cortactin cores of invadopodia were visualized using GFP- or mCherry-cortactin. Invadopodium assembly was found to be initiated by docking of the cytoplasmic protein cortactin to the cell membrane adherent to extracellular matrix, associated with the formation of a distinctive invadopodial membrane process that extended from a ventral cell membrane lacuna towards the extracellular matrix. The tip of this invadopodial process flattened as it interacted with a 2D matrix, and it began to undergo rapid ruffling and dynamic formation of filament-like protrusions as the invadopodium matured. Cortactin did not extend into these protrusions, but instead stabilized the whole invasive complex. Three distinct stages of invadopodium formation could be identified: the 1) invadopodial process stage, 2) invadopodial ruffle stage, and 3) mature invadopodia stage. This invadopodial complex consisting of a cortactin-actin core and distinctive filamentous processes was also found to exist in cells invading a 3D matrix by examining invasion of breast carcinoma cells transiently expressing GFP-cortactin and IL2R-Cherry into a 3D collagen matrix. The tumor cells inserted an initial invadopodial process into the 3D matrix, and then dynamic filament-like invadopodia extended from the tip to interact with the collagen matrix. Thus, the invadopodium is a highly dynamic, filament-like extension of a complex invasive structure. This structure is distinct from podosomes, which can also mediate matrix degradation but lack these dynamic, probing filamentous invasive structures. These studies will be extended to determine whether sites of proteolytic degradation correspond to the ruffle, filament-like invadopodia, or both, as well as the roles of integrin activation and signaling in invadopodia formation and function in matrix degradation. We will continue to characterize the mechanisms and regulation of invadopodia formation and function. Interactions at the cell surface are likely to play important roles in many diseases. We have been involved in a long-term collaboration with the laboratory of Subhash Dhawan in CBER, FDA to apply basic research approaches to characterize extracellular interactions involved in the pathogenesis of infectious diseases. Previous studies have explored the roles of adhesion molecules, secreted HIV-Tat protein, and signal transduction in HIV disease, novel approaches to generate a therapeutic vaccine, and roles of extracellular protein signaling in disease. Preliminary studies have identified novel roles for induction of endogenous cellular heme oxygenase-1 as a surprisingly general inhibitor of infections by pathogens that included West Nile virus, dengue, and influenza. These findings were filed as Invention Report E-276-2009: """"""""Infections by West Nile virus, dengue, poxvirus, leishmania, and influenza are inhibited by heme oxygenase-1 induction.""""""""
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