Die in der vorliegenden Habilitationsschrift zusammengefaßten Publikationen stellen Untersuchungen zu zwei Themenschwerpunkten dar: 1. Verankerungsmechanismen von Membranproteinen der basolateralen und der apikalen Plasmamembrandomäne der Parietalzellen mit dem Membranzytoskelett und 2. die regulierte Fusion von zytoplasmatischen Vesikeln mit der apikalen Plasmamembran dieser Zellen. Die strukturell und molekular sehr unterschiedlich gestaltete apikale und basolaterale Membrandomäne der Parietalzellen sollte funktionell charakterisiert und die Mechanismen der Membranumbauvorgänge aufgeklärt werden, die nach Aktivierung der Zellen im apikalen Membrankompartiment ablaufen. Für die strukturelle Stabilität der basolateralen Domäne spielt wahrscheinlich die Verankerung von AE2 über das Verknüpfungsprotein Ankyrin mit dem Membranzytoskelett eine wichtige Rolle. Die apikale Membrandomäne der Parietalzellen kann in drei Kompartimente unterteilt werden. Die freie apikale Membran, die canalikuläre Membran und die Membranen der tubulären Vesikel. Entlang der freien apikalen und der canaliculären Plasmamembran kommen wie auf der basolateralen Seite die Zytoskelett-Proteine Actin und Spectrin vor. Nach unseren Untersuchungen könnte es während der Sekretionsphase zu einer temporären Verbindung von H+,K+-ATPase Molekülen mit dem Membranzytoskelett kommen. Diese Verbindung wird wahrscheinlich durch das Verknüpfungsprotein Ezrin vermittelt. Der Mechanismus des Fusionsvorgangs der tubulären Vesikel mit der canaliculären Membran war bisher nicht bekannt. In Parietalzellen konnten die neuronalen SNARE-Proteine Synaptobrevin 2, Syntaxin 1 und SNAP25 sowie das zur Familie der kleinen G-Proteine gehörende Protein Rab3A und die Regulatorproteine NSF und alpha/beta SNAP nachgewiesen werden. Das in Parietalzellen gefundene Verteilungsmuster der SNARE-Proteine entspricht nicht der klassischen Vorstellung einer heterotypischen Membranfusion, vielmehr entspricht diese Verteilung einer homotypischen Fusion, wie sie für Vakuolen in Hefezellen beschrieben wurde. Die Bedeutung der SNARE-Proteine für die Fusion der tubulären Vesikel mit der canaliculären Membran und damit für die Steigerung der HCl-Sekretion konnte durch Inkubation der Zellen mit Tetanus Neurotoxin (TeNt) gezeigt werden. Die Behandlung der Parietalzellen mit TeNt führte zum vollständigen Ausbleiben der, nach Stimulation mit cAMP bei Kontrollzellen beobachteten Erhöhung, der Säuresekretion / The publications summarized here cover two topics: 1. the anchorage mechanism of membrane proteins of the basolateral and the apical plasma membrane with the membrane cytoskeleton of parietal cells and 2. the regulated fusion of cytoplasmic vesicles with the apical plasma membrane of these cells. It was the aim of these studies to characterize the structural and molecular differences between the apical and basolateral membrane domains in parietal cells. Moreover the mechanisms involved in membrane traffic within the apical membrane compartment following stimulation were investigated. We found that anchorage of AE2 with the membrane cytoskeleton through the linkage protein ankyrin seems to be important for the stability of the basolateral membrane. The apical membrane domain of parietal cells can be subdivided into three compartments. The free apical membrane, the canalicular membrane and the tubulovesicular membrane. The cytoskeletal proteins spectrin and actin can be found at the basolateral, the free apical and the canalicular membrane. We have shown that the H+K+-ATPase molecules appear to be temporary linked to the membrane cytoskeleton during acid-secretion. This contact is most likely mediated by the linker-protein ezrin. Until now the mechanism of fusion of the tubulovesicles with the canalicular membrane was unknown. In parietal cells the neuronal SNARE-proteins synaptobrevin 2, Syntaxin 1, SNAP25, the small G-protein rab3A, and the regulatory proteins NSF and alpha/beta-SNAP were detected. The subcellular distribution of these proteins does not support the notion of a neuron-like heterotypic fusion. Instead it shows similarity with the homotypic fusion process of vacuoles in yeast. The importance of SNARE-proteins for the fusion of tubulovesicles with the canalicular membrane and, by consequence also for the increase of acid-secretion was shown by incubation of the cells with tetanus neurotoxin (TeNt). The measurable increase of acid secretion by parietal cells after stimulation with c-AMP was inhibited completely through an incubation with TeNt.

No description available.

Le dépliement thermique et chimique est raisonnablement compris, mais pas les effets déstabilisants de la pression. Dans une tentative de caractérisation des facteurs à la base des effets de la pression sur les protéines, nous avons étudié le dépliement sous pression d'une protéine modulaire, le domaine ankyrine du récepteur Notch (Nank1-7*), ainsi que plusieurs mutants en nombre de motifs. Nos expériences montrent un dépliement à deux états sous pression. La dépendance à la température des sauts de pression a montré qu'à faibles températures l'ensemble d'état de transition(TSE) est proche en volume de l'état replié alors que son énergie est proche de celle de l'état déplié,ce qui est significatif d'une déshydratation importante de la barrière énergétique, et a montré que l'augmentation de la température conduit à un TSE plus grand en volume que l'état replié. Ce comportement révèle une grande plasticité du TSE de Nank1-7*. L'étude des mutants (délétion demotifs) nous a montré que le ΔV n'est déterminé ni par l'hydratation des liaisons peptidiques ni par l'hydratation différentielle des acides aminés, mais par l'existence de vides internes exclus du solvant. Seule une petite fraction des cavités est déterminante pour le ΔV, celles qui ne sont pas significativement solvatées dans les coeurs hydrophobes. Finalement, nous avons déterminé que le TSE possède même des cavités plus grandes, et nous émettons l'hypothèse que l'énergétique et la dynamique contribuent aux effets de la pression sur les protéines. / Thermal and chemical unfolding of proteins are reasonably well understood, but the destabilizingeffects of pressure are not. In an attempt to characterize the factors at the basis of pressure effects,we investigated the pressure unfolding of a modular protein, the ankyrin domain of the Notch receptor(Nank17*) and several of its deletion constructs. All our experiments were consistent with a simpletwo-state folding/unfolding transition under pressure. The temperature dependence of pressure-jumpsdemonstrated that at low temperature, the transition state ensemble (TSE) lies close in volume to thefolded state despite its unfolded-like energetics, consistent with significant dehydration at the barrier,and that increasing temperature leads to a volume of the TSE larger than that of the folded state. Thisbehavior reveals a high degree of plasticity of the TSE of Nank17*. Studies of the deletion mutantsshowed us that ΔV is determined not by hydration of peptide bonds or by differential hydration ofresidues, but by the existence of internal solvent-excluded void volumes. Only a small fraction of theinternal cavities are relevant of the ΔV, those that are not significantly solvated in the hydrophobiccore. Finally, we determined that the transition state ensemble show even larger cavities, and wehypothesize both energetics and dynamics contribute to pressure effects on proteins.

Les canaux potassiques à deux domaines P (K2P) contrôlent l’excitabilité cellulaire et jouent un rôle central dans l’établissement et le maintien du potentiel de repos membranaire dans la majorité des cellules animales. Depuis leur identification dans les années 90, ces canaux ont été impliqués dans de nombreuses fonctions comme la modulation de l’activité neuronale, l’activité du muscle cardiaque ou encore la physiologie rénale. Malgré l’importance de ces canaux, peu de données existent sur les processus cellulaires qui contrôlent leur fonction in vivo. Au cours de ma thèse, j’ai utilisé des approches génétiques, d’imagerie et d’électrophysiologie pour comprendre comment l’expression, la distribution et l’activité du canal K2P UNC-58 sont contrôlés chez le nématode modèle C. elegans.Pour cela, j’ai effectué un crible suppresseur du phénotype locomoteur du mutant gain de fonction unc-58(e665). J’ai ainsi obtenu 133 mutants présentant une large gamme de niveaux de suppression, suggérant l’implication de plusieurs gènes dans les processus de régulation du canal. En utilisant les technologies de reséquençage complet de génome, j’ai pu cloner six nouveaux gènes requis pour la fonction d’unc 58.J’ai ensuite caractérisé en détail le rôle d’unc-44/ankyrine dans le contrôle du trafic d’unc 58. Ce travail a conduit à 4 conclusions majeures : (1) UNC-58, malgré sa structure de canal potassique, possède en réalité une sélectivité ionique altérée favorisant le passage des ions sodium, (2) l’addition à UNC 58 de protéine fluorescente par approche CRISPR/Cas9 nous a permis pour la première fois d’observer directement la distribution du canal UNC-58 in vivo, (3) l’ankyrine est nécessaire à l’adressage du canal UNC-58 à la surface des muscles et dans les axones des neurones mécanosenseurs ALM. Cette fonction fait intervenir une poche d’interaction lipidique localisée au sein du module Zu5N-Zu5C-UPA d’UNC-44, (4) ce mécanisme est hautement sélectif puisqu’il n’est pas requis pour l’adressage de 6 autres canaux potassiques musculaires. Mon crible a également identifié une interaction génétique entre unc-70/ß-spectrine et unc-44/ankyrine. Toutefois, la nature moléculaire de cette interaction reste encore à préciser / Two-pore potassium channels (K2P) control cell excitability and play a central role in the establishment and the maintenance of the resting membrane potential of almost all animal cells. Since their identification in the late 90s, these channels have been implicated in a large number of functions ranging from neuronal and cardiac activity to kidney physiology. Despite the crucial functions of these channels, comparatively little is known about the cellular processes controlling their function in vivo. During my PhD, I used a wide range of strategies including genetics, microscopy and electrophysiology to understand how the expression, the distribution and the activity of the K2P channel UNC-58 are controlled in the model nematode C. elegans. I have first performed a genetic suppressor screen targeting the locomotion phenotype of the gain of function mutant unc-58(e665). The screen yielded 133 mutants, displaying a wide range of suppression level, suggesting that several genes may be implicated in the channel regulation process. By using whole genome sequencing technologies, I’ve been able to clone six new genes required for the function of UNC-58.Then, I’ve characterized in detail the role of unc-44/ankyrin in the trafficking of UNC 58. This project led to 4 main conclusions : (1) UNC-58, despite its potassium channel structure, has an altered ionic selectivity, allowing preferably sodium ions to pass through the channel (2) the addition of a fluorescent protein to UNC-58 by CRISPR/Cas9 approaches allowed us for the first time to directly observe the addressing of the UNC-58 channel to the muscle surface and axons of ALM mechanosensory neurons. This function involves a lipid binding pocket located within the Zu5N-Zu5C-UPA module of UNC-44, (4) this mechanism is highly selective since it is not required for the addressing of 6 other muscular channels.My screen also identified a genetic interaction between unc-70/ß-spectrin and unc-44/ankyrin. However, the exact molecular nature of this interaction remains to be elucidated

Actually, there is a strong incidence of psychiatric diseases, representing a 13% of total burden diseases and 450 million of people affected. The etiology of psychiatric diseases remains unknown. However, scientific evidences suggest a maldevelopment of nervous system (NS). The diagnosis is inaccurate, and international manuals (ICD-10 and DSM-IV) identify pathologies according to a list of symptoms but no underlying biological cause of disease. The aim of the thesis is to identify potential biomarkers -related to the development of NS- in peripheral blood of psychiatric patients diagnosed as different mental diseases, such as autism spectrum disorders (ASD), schizophrenia and bipolar disorders. It is intended to contribute with the improvement of diagnostic, prognostic and treatment of subjects. The thesis is divided into 4 chapters: 1) study of neurotrophins in ASD, where the results show the relationship of this family of molecules with the disease, 2) study of Latrophilin-3 (LPHN3) in the TEA, which was obtained in association with lower cognitive level of ASD, 3) study of the Eph-receptor A4 in the pathology of schizophrenia and bipolar disorder, results of which show no association, and finally 4) study of Ankyrin-3 (ANK3) in schizophrenia and bipolar disorder, which shown a relationship with bipolar disorder but not with schizophrenia. / Actualment, hi ha una forta incidència de les patologies psiquiàtriques, representant un 13% del total de les malalties i 450 milions de persones afectades. L’etiologia de les patologies psiquiàtriques és desconeguda. Tot i així, evidències científiques suggereixen un mal desenvolupament del sistema nerviós (SN). El diagnòstic és poc precís, i els manuals internacionals (ICD-10 i DSM-IV) identifiquen les patologies d’acord a un llistat de símptomes, però sense cap causa biològica subjacent de la patologia. L’objectiu de la tesi és la identificació de biomarcadors potencials –relacionats en el desenvolupament del SN- en sang perifèrica de pacients diagnosticats amb diferents patologies mentals, com ara els trastorns de l’espectre autista (TEA), esquizofrènia i desordres bipolars. És pretén contribuir amb la millora del diagnòstic, el pronòstic i el tractament de les persones que les pateixen. La tesi s’estructura en 4 capítols: 1) estudi de les neurotrofines en els TEA, on els resultats evidencien la relació d’aquesta família de molècules amb la patologia, 2) estudi de la Latrofilina-3 (LPHN3) en els TEA, on s’ha obtingut associació amb el nivell cognitiu més baix dels TEA, 3) estudi del receptor EPH A4 en les patologies d’esquizofrènia i desordres bipolars, resultats del qual no mostren associació i, per últim 4) estudi de la Ankirina-3 (ANK3) en l’esquizofrènia i els desordres bipolars, en el qual si que es troba una relació amb els desordres bipolars, però no amb l’esquizofrènia. / Actualmente, hay una fuerte incidencia de las patologías psiquiátricas, representando un 13% del total de las enfermedades y 450 millones de personas afectadas. La etiología de las patologías psiquiátricas es desconocida. Aún así, evidencias científicas sugieren un mal desarrollo del sistema nervioso (NS). El diagnóstico es poco preciso, y los manuales internacionales (ICD-10 y DSM-IV) identifican las patologías de acuerdo a un listado de síntomas, pero sin ninguna causa biológica subyacente de la patología. El objetivo de la tesis es la identificación de biomarcadores potenciales –relacionados con el desarrollo del SN- en sangre periférica de pacientes diagnosticados con diferentes patologías mentales, como son los trastornos del espectro autista (TEA), esquizofrenia y desordenes bipolares. Se pretende contribuir en la mejora del diagnóstico, el pronóstico i el tratamiento de las personas que las padecen. La tesis se estructura en 4 capítulos: 1) estudio de las neurotrofinas en los trastornos del espectro autista (TEA), en el cual los resultados evidencian la relación de esta familia de moléculas con la patología, 2) estudio de la Latrofilina-3 (LPHN3) en los TEA, donde se ha obtenido una asociación con el nivel cognitivo más bajo de los TEA, 3) estudio del receptor EPH A4 en las patologías de la esquizofrenia y los desordenes bipolares, resultados del cual no muestran asociación y, por último 4) estudio de la Ankirina-3 (ANK3) en la esquizofrenia y los desordenes bipolares, en el cual si que se ha encontrado una relación con los desordenes bipolares, pero no con la esquizofrenia.

<p>Studies in the ankyrin-B+/- mouse reveal that ankyrin-B deficiency is associated with both the benefits of enhanced cardiac contractility and the costs of arrhythmia, early senescence, reduced lifespan, and impaired glucose tolerance. This constellation of traits is known as ankyrin-B syndrome, which may have important implications for humans possessing functional ankyrin-B mutations. We found that ankyrin-B variants are surprisingly common, ranging from 2 percent of European individuals to 8 percent in individuals from West Africa. Furthermore, by studying of the metabolic phenotype associated with ankyrin-B mouse, we have uncovered a major new dimension to ankyrin-B syndrome, a link between ankyrin-B and parasympathetic control of insulin secretion. Stimulation of pancreatic beta cells by acetylcholine augments glucose-stimulated insulin secretion by inducing inositol-trisphosphate receptor (InsP3R)-mediated Ca2+ release. We report that ankyrin-B is also enriched in pancreatic beta cells. Ankyrin-B-deficient islets display impaired potentiation of insulin secretion by the muscarinic agonist carbachol, blunted carbachol-mediated intracellular Ca2+- release, and reduced InsP3R stability. Ankyrin-B(+/-) mice also display postprandial hyperglycemia, consistent with impaired parasympathetic potentiation of glucose-stimulated insulin secretion. R1788W mutation of ankyrin-B impairs its function in pancreatic islets and associates with type 2 diabetes in Caucasians and Hispanics. Finally, we have generated knockin mice corresponding to the R1788W and L1622I mutations. Functional characterization of these animals will allow us to better understand the relationship between human ankyrin-B variants and ankyrin-B syndrome.</p> / Dissertation

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.

In recent years it has become apparent that neuronal development and function relies not just on the regulation of transcription but also on post-transcriptional events. Two prevalent mRNA-based regulatory mechanisms in neurons are asymmetric mRNA localisation and the generation of different 3’UTR isoforms by alternative polyadenylation (APA). While experiments in mammalian systems indicate that subcellular mRNA localisation plays an important role in regulating local expression of proteins in neuronal processes, little is known about how mRNAs reach their destinations. It has been proposed that APA allows the production of mRNA isoforms with different roles. However, the importance of 3’UTR extensions has not been addressed in detail, particularly at the organismal level. In my PhD, I investigated the mechanisms of mRNA localisation and functional consequences of APA using the Drosophila embryonic nervous system as a genetically tractable model. I screened for mRNAs that localise in embryonic axons using an available transgenic library of 3’UTR sequences, as well as publically available in situ hybridisation data. I found that Ankyrin2 (Ank2) mRNA localises in Drosophila embryonic sensory neurons, and showed that this is dependent on the kinesin-1 motor and microtubules. These data reveal an active mRNA transport system in embryonic neurons. I also showed that the Ank2 mRNA has an extended 3’UTR that is found in axons, suggesting that APA could be relevant to axonal functions of Ank2. I demonstrated that while mRNA molecules could still localise to axons upon CRISPR-Cas9-mediated deletion of the Ank2 3’UTR extension, a fraction of the mutant embryos had a disrupted nervous system. Interestingly, embryos that lack the ability to make Ank2 protein have an overtly normal embryonic nervous system. This observation reveals that the extension does not simply promote Ank2 protein function. Further experiments revealed that the extended 3’UTR is required for efficient locomotion of adult flies. While the exact function of the Ank2 3’UTR extension requires future investigation, I show that it is unlikely to be associated with the trafficking of associated proteins into axons. RNA affinity purifications from embryonic extracts provide evidence that the 3’UTR extension selectively binds conserved RNA-binding proteins. I speculate that the extension plays a role in regulating axonal morphogenesis by regulating the relative expression level of different Ank2 protein isoforms.

The cell differentiation process is associated with activation and repression of different genes, whereby the formation of heterochromatin is mediated by spreading of repressor proteins along large chromatin domains. Some of these proteins are methyltransferases, including GLP and G9a that are implicated in the addition of mono- and dimethyl groups to lysine 9 at Histone 3. Despite extensive research the exact mechanism of binding and spreading of G9a and GLP is unclear. To better understand the molecular mechanisms through which G9a and GLP bind to chromatin we tested the in vivo binding of a mutant G9a that is unable to bind to H3K9me2 histone marks via its Ankyrin domain. Murine erythroleukemia (MEL) cell line with expression of mutant G9a was generated using recombinant DNA technologies; G9a binding targets genome-wide were detected by the analysis of ChIP-sequencing data. We validated ChIP-sequencing data providing a reliable tool to visualize G9a targets in MEL cells. We also found that G9a Ankyrin mutant bound to all tested regions suggesting that the Ankyrin domain is not the only factor that contributes to the binding of G9a on chromatin in vivo.

<p>The formation and retention of distinct membrane domains in the fluidic membrane bilayer is the key process in establishing spatial organization for mediating physiological functions in metazoans. The spectrin-ankyrin network organizes diverse membrane domains including T-tubule and intercalated disc of cardiomyocytes, basolateral membrane of epithelial cells, costameres of striatal muscle, and axon initial segments and nodes of Ranvier in nervous system. This thesis identifies a novel function of 480 kDa ankyrin-G, an alternatively spliced isoform of the ankyrin family, in promoting somatodendritic GABAA receptor synaptogenesis both in vitro and in vivo. In the nervous system, an insertion of a neuronal specific exon (exon 37) occurs in ankyrin-G polypeptide which results in a 480 kDa isoform. 480 kDa ankyrin-G (giant ankyrin-G) has been shown to coordinate formation and maintenance of the axon initial segment (AIS) and nodes of Ranvier. This thesis research began with the discovery that giant ankyrin-G, previously thought to be confined to the axon initial segment, forms developmentally-regulated and cell-type specific somatodendritic "outposts" on the plasma membrane of pyramidal neurons. This somatodendritic 480 kDa ankyrin-G outpost forms micron-scale membrane domains where it associates with canonical AIS binding partners including voltage-gated sodium channel and neurofascin. This thesis further discovered that the giant insert of 480 kDa ankyrin-G interacts with GABARAP, a GABAA receptor-associated protein. Both the interaction with GABARAP and the membrane association through palmitoylation of giant ankyrin-G are required for the formation of somatodendritic GABAergic synapses. This work further found that ankyrin-G associates with extrasynaptic GABAA receptors and stabilizes receptors on the extrasynaptic membrane through opposing endocytosis. This story demonstrates for the first time the existence of giant ankyrin-G somatodendritic outpost as well as its function in directing the formation of GABAergic synapses that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and neurodevelopmental disorders.</p><p>Additional work presented in the Appendix characterized novel ankyrin-G full length transcripts in the heart and kidney with unique domain compositions though alternative splicing. The preliminary work further identified biochemical properties and potential role of an insert C in the C-terminus of ankyrin-G in mediating cytokinesis and cellular migration in mouse fibroblasts. Together, this thesis work expands the knowledge of giant ankyrin-G functions in the nervous system and offers insights into the diversified roles of distinct ankyrin-G peptides acquired from alternative splicing in organizing specific membrane domains and interacting with defined intracellular pathways in different tissues.</p> / Dissertation