The long-term goal is to develop nanobiotechnology that allows the understanding of the initial signal transduction process in time and in space, and with that knowledge to devise strategies to alter the behavior of cells in general, with focus on cancer cells in specific. The idea is based on the observation that clustering of integrin and other focal proteins such as tensin are correlated to the downstream cell signaling. By varying the geometric parameters of nanostructures of integrin-binding sites, such as size, shape, and separation, the localization of tensin at focal adhesion would change accordingly, thus altering the corresponding cell signaling pathways. The two co-PIs have complementary expertise in nanotechnology (Liu) and cell biology of focal adhesion involving tensin (Lo), respectively. Preliminary studies from the co-PI (Lo) laboratory reveal that tensin localization at focal adhesion impacts on corresponding cell functions such as motility and migration. Preliminary study from the PI (Liu) group has demonstrated that scanning probe microscopy based nanofabrication enables precise positioning of simple molecular ligands, proteins and DNA on surfaces. The first goal is to develop a new methodology, combined atomic force and confocal microscopy for single cell imaging and for visualization of protein complexes at focal adhesion. Confocal microscopy will be utilized to (a) reproduce previously known immunofluorescence studies in order to validate this new method; and (b) to guide atomic force microscopy (AFM) probes to penetrate into cells to reach focal adhesion sites for high resolution imaging of protein complexes. Arrays of nanostructures consisting of integrin-binding ligands such as fibronectin will be produced with designed size and geometry using Liu's nanofabrication methodologies. Finally, NIH 3T3 cells will be seeded onto these nanostructure arrays where integrin/tensin clusters will form, and be characterized using this combined AFM/confocal microscopy to reveal how the tensin localization varies as a function of the ligand nanostructures underneath. Once this concept of regulating tensin translocation via nanotechnology is proven, future work can be planned to investigate how cell functions can be regulated by forming designed integrin clusters and tensin location using ligand nanostructures underneath the cells. Formation of other protein complexes such as tyrosine kinases at focal adhesion may be regulated following the same concept. This nanoengineering approach should provide a new paradigm for controlling cell signaling and behavior, which enables new methods be developed for cancer therapy. ? ? ?
