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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.

Chromophore-assisted laser inactivation (CALI) is a technique whereby engineered proteins and dye molecules that produce substantial amounts of reactive oxygen species upon absorption of light are used to perturb biological systems in a spatially and temporally defined manner. CALI is an important complement to conventional genetic and pharmacological manipulations. In this review, we examine the applications of CALI to cell biology and discuss the underlying photochemical mechanisms that mediate this powerful technique.

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments.