Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains ix, 127 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 108-123).

Thesis (Ph. D. in Cellular and Molecular Pathology)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references

Abstract Anion exchanger 2 (AE2) is a member of the anion exchanger gene family, which includes three additional members, AE1, AE3, and AE4. They are also known as Na+-independent Cl-/HCO3- exchangers, and their major function is to regulate intracellular pH and chloride concentration. All four isoforms have several N-terminally truncated variants that are often expressed cell type specifically. Red blood cells express the full-length AE1 isoform that interacts with ankyrin, an adapter protein linking plasma membrane to the spectrin-based membrane skeleton. This membrane skeleton association is essential for maintaining the membrane integrity of red blood cells. AE3 variants are mainly found in the brain and heart, whereas AE4 is localized in the kidney. Anion exchanger 2 is expressed in every cell line and tissue studied thus far, and it has been mainly localized to the plasma membrane. However, we found two types of localization/targeting of the AE2 protein in several of the cell lines studied. The protein was localized to either the plasma membrane or the Golgi complex, depending on the cell type. The AE2 variant expressed in these cells was identified as the full-length AE2 protein. The determinants of differential intracellular targeting were assessed. We hypothesized that Golgi-AE2 is anchored to the Golgi membranes via its association with the Golgi membrane skeleton. We were able to show that the Golgi localization of AE2 correlated with the cell type specific expression of Ank195, a Golgi membrane skeletal protein. In cells where AE2 was targeted to the plasma membrane, Ank195 was not expressed. In addition, the detergent insolubility and co-redistribution properties of AE2 and Ank195 strongly suggested that these proteins interact with each other. The Golgi membrane skeleton has been shown to be necessary for maintaining the Golgi structure. Our studies were consistent with these findings, showing that in cells in which AE2 expression was reduced by using AE2-specific antisense oligonucleotides, the Golgi complex was dispersed. The spectrin-based membrane skeleton was probably partially detached from the Golgi membranes leading to breakdown of the Golgi structure and disorganization of the microtubules associated with it. The present findings suggest that the targeting of AE2 is cell type specific, and that Golgi-localized AE2 serves as a membrane association site for the spectrin-based Golgi membrane skeleton, thereby participating in the maintenance of the Golgi structure.

L1-type cell adhesion molecule (L1CAM) plays an essential role in the development of nervous system and is also highly relevant for the progression of diseases such as Alzheimer’s disease, stroke and cancers, some of the leading causes of human mortality. In addition to its canonical role as a plasma membrane protein organizing the cytoskeleton, recent in vitro studies have revealed that transmembrane as well as cytosolic fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus and regulate expression of genes involved in DNA post-replication repair, cell cycle control, migration and differentiation. However, little is known about the in vivo function of L1CAM in the adult nervous system. This dissertation research focuses on studying in vivo nuclear translocation and function of L1CAM. Using the Drosophila model system, we first show that the sole Drosophila L1CAM homolog, Neuroglian (Nrg), is proteolytically cleaved by Alzheimer’s associated secretases, similar to L1CAM, and is also translocated to the nucleus in the adult nervous system. Subsequently, we have shown that the deletion of highly conserved Ankyrin binding domain or FIGQY motif disrupts nuclear import. Further experiments have revealed that the nuclear translocation of Nrg is in fact regulated by the phosphorylation of the FIGQY motif. Importantly, our studies also show transgenic expression of full-length Nrg or the intracellular domain of Nrg resulted in increased myc expression, which is associated with increased sensitivity to oxidative stress and reduced life span. On the other hand, deletion of the FIGQY motif or mutations preventing its phosphorylation led to decrease in myc expression. In summary, we have identified a novel role for the highly conserved Ankyrin binding domain in nuclear translocation and transcriptional regulation of the Drosophila myc oncogene, which is of high relevance to neurodegenerative diseases and cancer associated with oxidative stress. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Le segment initial de l'axone (SIA) joue un rôle dans la maintenance de la polarité neuronale et dans l'initiation du potentiel d'action. Il se construit autour de l'ankyrine G (ankG) qui relie les protéines membranaires au cytosquelette d'actine et de microtubules. Cette structure est dynamiquement régulée par des kinases. S'il a été clairement établi que l'ankG est cruciale à la formation et à la maintenance du SIA, les mécanismes responsables de sa concentration dans la partie proximale de l'axone restent encore inconnus. Il en est de même pour la protéine kinase CK2 qui régule l'interaction entre l'ankG et les canaux sodiques (Nav1). Dans un premier temps, nous avons montré, par des approches de mutagénèse, que le domaine serine-rich (SR), notamment ses 73 premiers acides aminés, porte l'information nécessaire à l'entrée de l'ankG dans l'axone. Mais ce domaine n'est pas suffisant pour le confiner dans la partie proximale de l'axone, cette propriété est portée par le domaine Tail. En plus de la coopération de ces domaines, nous avons aussi observé que l'adressage de l'ankG est régulé par les kinases Cdk5 et PKC. Dans un second temps, nous avons montré que l'accumulation de la CK2 au SIA dépend de l'expression des Nav1. L'existence d'un complexe formé par les Nav1 et la CK2 serait donc importante au recrutement de la protéine kinase CK2 au SIA. En outre, le développement des anticorps phosphospécifiques nous a permis de monter que les Nav1 sont phosphorylés in vivo au niveau de leur motif de liaison à l'ankG. L'ensemble de nos résultats ouvre de nouvelles perspectives dans la compréhension de la formation du SIA et des mécanismes de régulation qui peuvent être associés. / The axon initial segment (AIS) is responsible for both the maintenance of neuronal polarity and the generation of action potentials. The scaffolding protein ankyrin G (ankG) is specifically expressed in the AIS where it links transmembrane proteins to the subjacent actin and microtubule cytosqueletons. Moreover, the AIS is dynamically regulated by kinases. Although, it has been clearly established that ankG directs AIS assembly and maintenance, the mechanisms regulating ankG proper transport and tethering remain unclear. Another AIS component, the protein kinase CK2 is also playing an important role via the phosphorylation of the ankG-binding motif (ABM) on sodium channels (Nav1) to strengthen their interaction with ankG. But, the mechanism regulating its targeting and anchoring to the AIS remain still unknown. Here, we report that the first 73 residues of the serine-rich domain are necessary for the targeting of ankG to the axon and the tail domain for the proper positioning along the proximal axon. We also observed that ankG axonal localization is modulated by post-translational modifications. Using phosphospecific antibodies and inhibition/depletion approaches, we also provide evidence that the ABM of Nav1 are phosphorylated in vivo and that CK2 accumulation at the AIS depends on Nav1 expression, with which they form tight complexes. This suggests that CK2-mediated phosphorylation participates in Nav1 clustering in vivo and that its specific localization at the AIS is dependent on Nav1 expression. Altogether, our results open new perspectives in understanding the formation of AIS and regulatory mechanisms that may be involved.

This body of work details the exploitation of an incredibly powerful neural culture system, which enables the in vitrostudy of events involved in peripheral nervous system (PNS) development. Using a myelinating dorsal root ganglion (DRG) explant culture system, node of Ranvier formation and maintenance and the associated generation and maturation of myelin segments was examined. In addition, Schwann cell (SC) development, dynamics, and migration were extensively studied. First, in characterizing these cultures, the discrete axonal localization of specific ankyrin isoforms was revealed. Ankyrins are peripheral membrane proteins that immobilize classes of integral membrane proteins to the spectrin based-membrane skeleton. Ankyrins interact with proteins such as the voltage-dependent/gated sodium channel (vgsc) and members of the L1 family of cell adhesion molecules. These interactions are physiologically relevant to the formation of membrane specializations involved in axon guidance and the initiation and propagation of action potentials. We examined ankyrinB and ankyrinG expression in cultured DRG explants, which allowed visualization of individual axons. AnkyrinB and ankyrinG exhibited differential localizations to specific axonal populations. This was evident as early as one day in vitro and persisted over time. In mature pre-myelinated cultures, axons having an apparent diameter of less than 1 µm predominantly expressed ankyrinB, whereas axons having a diameter greater than or equal to 1 µm predominantly expressed ankyrinG (based on immunocytochemical reactivity). When myelination was induced, ankyrinGwas appropriately localized to sites of nodal development flanked by myelinating glial processes in the large caliber axons. These observations suggest that axons destined for myelination may express a distinct complement of peripheral, and perhaps integral, membrane proteins as compared to those observed in non-myelinated axons. These distinguishing features may play a role in the selection of axons for myelination. This work was followed with defining the role axo-glial interactions play in organizing domains along the axon being myelinated. Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the PNS, axon-glial cell contacts have been implicated in SC differentiation and the formation of nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using the myelinating explant culture system, we observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50kDa (EBP50)/regulatory co-factor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin (ERM) family of proteins, concomitant with myelination in response to inductive signals. These components were targeted to the SC distal tips where live cell imaging revealed novel, dynamic growth cone-like behavior. Further, localized activation of the Rho signaling pathway at SC tips gave rise to these microvillar component-enriched “caps” and influenced the efficiency of node formation. Extending these findings, a more profound examination of SC dynamics was undertaken. This was a particularly important experimental transition, as SC motility is crucial in the development and regeneration of the PNS. The seemingly equivalent bipolarity of mature SCs represents a conundrum in terms of directed motility. Fluorescence time-lapse microscopy of transfected SCs within the myelinating DRG explants revealed a novel cycling of these cells between static, bipolar and motile, unipolar morphologies via asymmetric process retraction and extension. Concentrations of PIP2 (phosphatidylinositol (4,5)-bisphosphate), activated ERMs, and EBP50 delineated the transitory asymmetry associated with the generation and neuron-like migration of the unipolar cell. EBP50 over-expression enhanced unipolar SC migration, suggesting a new role for this adaptor protein in cell motility. Further, the ERMs themselves were found to be essential to both motility and process dynamics with ERM disruption yielding a dysfunctional, multipolar SC phenotype. We propose this novel form of motility may be associated with the correct alignment and spacing of SCs along axons prior to elaboration of the myelin sheath. These compiled studies present significant advances in understanding and examining axo-glial interactions in the PNS. This work establishes the foundation for further, novel exploration of normal PNS development and the regeneration and repair mechanisms involved in PNS injury and disease states.