A glioma produces some of the most rapidly growing, angiogenic, and invasive primary brain tumor cells known. A lack of understanding about the intricately coupled molecular mechanisms that result in cell transformation is responsible, in part, for the minimal progress made in treating this disease over the past century. To begin dissecting molecular interrelationships in time and space within living normal and transformed cells, a morphological assay is being developed wherein patient-derived tumor cells are allowed to attach to a substrate, spread, change shape, and locomote. During this process (approximately 6.5 h), low magnification phase contrast images of the cells are recorded at 1 min intervals. Quantitative image analyses of these time-lapse images are used to measure dynamic parameter such as projected cell area, shape, and displacement for each cell. Although there is considerable cellular heterogeneity, patterns of patient-specific tumor cell behavior are beginning to emerge. In addition, the assay is being used to test the effects of drugs that alter specific intracellular processes (e.g., cytochalasin, 2-deoxyglucose, and chemotherapeutic drugs). To dissect tumor cell physiology into its molecular components, I have focused on the actin-cytoskeleton because it is involved in the temporal and spatial orchestration of ions, metabolites, macromolecules, and organelles that underlies the interconnected processes of tumor cell growth, motility, and differentiation. I have used fluorescent analogs of actin and its associated proteins in conjunction with multimode-based light microscopy of living cells to measure the complex interrelationships responsible for motility in patient-derived normal and transformed glia. I have simultaneously measured the dynamics of actin assembly and focal contact formation using new fluorescent analogs of actin and vinculin in single migrating human glioblastoma cells and used this information to begin developing a molecular model of tumor cell migration. By engineering new protein-based reagents and fluorescence spectroscopic methodologies we will be able to measure and manipulate a greater number of molecular processes and therefore further refine the model. The ultimate goal of this work is to use living cells to diagnose and design treatment for primary brain tumors in a more patient-specific manner.