| Signaling events in cells are often localized and transient. Understanding how these pathways are regulated in space and time requires the development of new tools that reveal spatiotemporal transduction. Subcellular events can be visualized in real-time by high-resolution light microscopy. Technological advances in live-cell imaging have offered the means to not only observe the phenotypic consequences of signaling events, but to visualize and manipulate the activity of their components. This dissertation describes four studies in which microscopy is implemented to manipulate or visualize signal transduction in living cells. The first study demonstrates contributions to Chromophore Assisted Laser Inactivation, a light-mediated loss of function tool. The second study describes the generation of a new probe to visualize the activation of Src-family kinases. The third study utilizes a biosensor for the GTPase RhoA to reveal novel information about how this signaling component is spatiotemporally regulated in neurons. Finally, the fourth study describes a new computational method for the automated identification and tracking of protein structures called focal adhesions. Together these studies demonstrate the power of using microscopy to gain key insights to the spatiotemporal details of signal transduction. |
