Browsing by Subject "Semaphorins"
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Item Characterizing the Molecular Mechanisms of Axon Guidance: Activation and Regulation of the Axon Guidance Receptor Plexin A(2012-07-17) Yang, Taehong; Terman, Jonathan R.Neuronal connectivity is precisely determined by axonal pathfinding during development. The navigating axons detect attractive and repulsive environmental cues by axon guidance receptors. However, the biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are the largest family of axon guidance cues and utilize Plexin receptors to exert repulsive effects on axon extension. The intracellular region of Plexins contains a Ras GTPase activating protein (GAP) domain, which is necessary for repulsive guidance effects. Previous studies suggest that activation of Plexin RasGAP requires interactions with both Semaphorin at the extracellular region and a Rho-family GTPase at the Rho family GTPase-binding domain (RBD). Interestingly, Semaphorin repulsion can be rapidly "turned-off" by other distinct cues and signaling cascades. However, the molecular mechanisms to activate or modulate Plexin RasGAP remain unclear. First, to further understand how the Plexin RasGAP is activated, I collaborated with the Zhang lab, and following determination of the crystal structure of the intracellular region of Plexin, I examined the roles of residues interfacing with the RasGAP domain using functional mutagenesis in the Drosophila model system. Our results demonstrate that Plexin exhibits an auto-inhibited conformation, and suggest that interaction among the previously uncharacterized juxtamembrane segment, the RBD, and the RasGAP domain is critical for Plexin RasGAP activation. Second, to better understand how Semaphorin/Plexin signaling is modulated, I characterized the results of a large-scale screen to look for proteins interacting with the cytoplasmic portion of Plexin and identified the phosphoserine binding protein 14-3-3epsilon as a specific Plexin-interacting protein. My results reveal that 14-3-3epsilon is specifically required for axon guidance during development. Moreover, Protein kinase A is found to phosphorylate Plexin in the RasGAP domain and mediates the 14-3-3epsilon interaction. Plexin-14-3-3epsilon interactions prevent Plexin from interacting with its Ras-family GTPase substrate, which effectively switches Plexin-mediated axonal repulsion to Integrin-mediated adhesion. These findings uncover both a new molecular integration point between important axon guidance signaling pathways and a biochemical logic by which this guidance information is coalesced to steer the growing axon. Therefore, these new observations on activating and silencing specific signals that are repulsive to axon growth also illuminate new approaches to neutralize axonal growth inhibition and encourage axon regeneration.Item Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling(2017-04-14) Rich, Shannon Kay Good; Johnson, Jane E.; Terman, Jonathan R.; Krämer, Helmut; Alto, NealDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology.