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<p> The urgency for deeper knowledge about nervous system function and dysfunction has never been greater. With increasing rates of mental disorders and expanding healthcare costs, deciphering details of axonal development is essential to meeting this imperative. Models of neuronal growth are improving as roles of microtubules and motor proteins surface. However, traditional motor protein studies focus on intracellular cargo transport, leaving deficits in knowledge about how these proteins organize cytoskeletal filaments in the axon and growth cone during neuronal development. Inconsistent findings on microtubule activity in growing axons also leave gaps in quantitative assessments of microtubule translocation and assembly, limiting the ability to construct a comprehensive model of axonal elongation.</p> The goal of this study was to provide a more complete neuronal growth cone model by determining how individual microtubule translocation and assembly, mass microtubule movements, and motor protein activity contribute to axonal elongation. The underlying mechanisms of these processes were investigated by testing the roles of dynein and microtubule dynamics in axonal elongation of <i>Aplysia </i><i>californica </i>neurons using transillumination, fluorescent speckle, and super-resolution microscopy. Pharmacologically inhibiting either dynein activity or microtubule assembly reduced both bulk and individual microtubule anterograde translocation and neurite elongation rates. Suppressing both processes simultaneously had compensatory rather than additive effects. Super-resolution imaging also revealed fewer dynein motors co-localized with microtubules when microtubule assembly was inhibited. These results strongly suggest that disrupting microtubule assembly blocks neurite outgrowth partly because it inhibits dynein-mediated bulk microtubule translocation.

Chemical senses play a critical role in predator-prey and social interactions of many animals. Predators often evoke adaptive escape responses by prey, one of which is the release of chemicals that induce adaptive avoidance behaviors from both predators and conspecifics. I explore the use of chemicals in predator-prey and social interactions, using a crustacean model system, the spiny lobster. As predators, spiny lobsters are opportunistic, polyphagous feeders, and they rely heavily on their chemical senses during feeding. Some of their potential prey deter attacks through chemical defenses that act through the spiny lobsters’ chemical senses. An example of this is sea hares, Aplysia californica, which secrete an ink when vigorously attacked by sympatric spiny lobsters, Panulirus interruptus. I show that that this ink defends sea hares from spiny lobsters through several mechanisms that include phagomimicry, sensory disruption, and deterrence, and that the ink’s efficacy is enhanced by its naturally high acidity. As prey, spiny lobsters rely heavily on their chemical senses to assess risk from predators. One way to assess risk of predation is through ‘alarm cues’, which are injury-related chemicals. I show that injured Caribbean spiny lobsters, Panulirus argus, release alarm cues in their hemolymph, and that nearby conspecifics detect these cues using olfaction. Hemolymph from conspecifics induces primarily alarm behavior in the form of retreat, sheltering, and suppression of appetitive responses. In contrast, hemolymph from heterospecifics, depending on phylogenetic relatedness, induces either mixed alarm and appetitive behaviors or primarily appetitive behaviors. Spiny lobsters also use chemical cues to assess risk during social interactions with conspecific. I show that spiny lobsters use urine-borne chemical signals and agonistic behaviors to communicate social status and that these chemical signals are detected exclusively by the olfactory pathway. Dominant animals increase urine release during social interactions, whereas subordinates do not. Experimental prevention of urine release during interactions causes an increase in agonism, but this increase is abolished when urine of dominants is reintroduced. My findings lay the foundation for neuroethological studies of risk-assessment systems mediated by intraspecific chemical cues.

<p dir="ltr">This dissertation delves into two research projects that aim to characterize post-translational modifications in two distinct proteins, each originating from a different species – one from the eukaryotic sea slug Aplysia californica and the other from the bacterial pathogen Bordetella bronchiseptica.</p><p dir="ltr">Aplysia have an unusually large neuron and therefore serve as an excellent model for studying cell signaling regulating neuronal chemotaxis. Cortactin is an actin binding protein that is regulated by post-translational modifications, including acetylation and phosphorylation. Studies have shown that Src2 tyrosine kinase phosphorylates cortactin to regulate lamellipodia protrusion and filopodia formation in Aplysia bag cell neurons. However, these in vivo phenotypes have not been tested mechanistically in vitro. To this end, the goal of my thesis work was to validate in vivo observations. The following work describes the methodology we developed to purify homogenous non-phosphorylated proteins. Our collaborative results show that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro.</p><p dir="ltr"> Filamentation induced by cAMP (Fic) proteins constitute a recently characterized family of enzymes that are being recognized to regulate diverse cellular processes in bacteria and metazoans. While Fic proteins predominantly utilize adenosine triphosphate (ATP) to post-translationally modify target proteins via a covalent addition of AMP, two Fic proteins have been reported that utilize uridine triphosphate (UTP) and cytidine diphosphate-choline (CDP-choline) to alter the activity of their target. In this dissertation, we report the discovery of the first guanosine triphosphate (GTP) specific Fic protein – BB0907 (BbFic) from Bordetella bronchiseptica. BbFic displays weak to no binding to ATP; instead has a 10-fold increased preferential usage for GTP. We identify key residues involved in GTP recognition. Additionally, sequence similarity network (SSN) analyses reveal that BbFic represents a distinct clade of Fic proteins, highlighting BbFic as a representative new class of guanylyltransferase. Our discovery adds to the functional diversity of the growing Fic protein family and frames the groundwork for understanding Fic-mediated GMPylation as a novel signaling paradigm. </p><p dir="ltr">Taken together, my thesis work provides novel insights into biological consequences of Fic-mediated GMPylation in bacteria and Src-mediated phosphorylation in filopodia formation.</p><p><br></p>