SPATIOTEMPORAL DYNAMICS AND REGULATION OF CYCLIC NUCLEOTIDES AND THEIR EFFECTORS IN LIVING CELLS

Cellular signaling is a tightly regulated process which defines how cells interact with their environment and maintain homeostasis. It is also the mechanism by which they change. Neuronal development is one such process that depends on the concerted activities of many molecular signaling pathways to proceed through highly typified stages. A neuron’s morphology is a perfect example of spatially distinct compartments which change over time. The spatial and temporal regulation of signaling pathways within the dendrites, soma, and axon during development require second messengers and kinases for proper establishment of neuronal morphology. The second messenger 3'-5'-cyclic adenosine monophosphate (cAMP) and kinase cAMP-dependent protein kinase (PKA) are considered critical axon determinants; meaning without them the axon does not form. At the second messenger level, cAMP is generated by adenylyl cyclase and degraded by phosphodiesterases in the cellular milieu to balance the cAMP concentration in specific signaling microdomains. When activated by cAMP, PKA phosphorylates its substrates which can be both upstream and downstream of itself. But the cAMP/PKA signaling pathway components are not randomly strewn throughout the cell. Instead, they are bound and tightly regulated within a signaling platform called the A-kinase anchoring protein (AKAP) signalosome. AKAPs bring PKA into close proximity to its substrates, source of cAMP, and scaffold other signaling partners to produce profound effects on a neuron’s fate. To understand how cAMP and PKA control neuronal polarization in living neurons, we utilized fluorescence resonance energy transfer (FRET)-based biosensors shed light on their activities in different regions and at different stages of development. This dissertation is composed of four chapters: an introduction, the major project, a minor project, and concluding remarks. The first chapter introduces major concepts and information regarding cyclic nucleotide signaling, cyclic nucleotide effectors, signal compartmentalization and methods used to visualize molecular activity in living cells. The second chapter contains the bulk of the thesis work in which we investigated the role of spatiotemporally compartmentalized cAMP and PKA signaling in developing neurons. Here, we used the FRET-based cAMP reporter ICUE3 and PKA reporter AKAR4 to study how their molecular activities differ between the spatially distinct soma, dendrites, and axons of developing hippocampal neurons. In this study we have demonstrated how gradients of cAMP and PKA in the axon are regulated by phosphodiesterase activity and scaffolding by AKAPs. We also show the functional significance of AKAP anchoring in neuronal development by analyzing axon outgrowth. Lastly, we link the functional outcomes to the gradients of molecular activity and developed a conceptual model for our observations. In the third chapter, we describe and characterize a new suite of targeted cGMP biosensors to help further the understanding of cGMP compartmentalization. In the fourth and last chapter, we provide a review of the thesis work and provide some perspective on future directions. As a whole, the studies presented here demonstrate how cAMP, PKA, and cGMP signaling within the spatially distinct compartments of living cells is regulated. The gradients alluded to in the literature over the years are now revealed using the spotlight of genetically encoded FRET-based biosensors. Furthermore, we present the field of neurodevelopment with a new target of study, the AKAPs, which we show have significant implication in the growth and differentiation of hippocampal neurons