<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

<p>The spectrin-ankyrin network assembles diverse plasma membrane domains including axon initial segments and nodes of Ranvier, cardiomyocyte T-tubules and intercalated discs, epithelial lateral membranes, costameres and photoreceptor inner and outer segments. However the mechanism that targets the spectrin-ankyrin network to those plasma membrane domains is unknown. This thesis identifies two lipid inputs from protein palmitoylation and phosphoinositides that together control the precise localization of the spectrin-ankyrin network. In Chapter 2, we identify a linker peptide encoded by a single divergent exon that distinguishes the subcellular localization of ankyrin-B and -G by selectively suppressing protein binding through autoinhibition. In Chapter 3, we demonstrate that ankyrin-G is S-palmitoylated at a conserved C70 residue which is required to assemble epithelial lateral membranes and neuronal axon initial segments. We continue to interrogate how palmitoylation regulates ankyrin-G activities in Chapter 4, and identify DHHC5 and DHHC8 as the palmitoyltransferases in MDCK cells. We showed that palmitoylated ankyrin-G, in concert with phosphoinositide lipids, determines the polarized localization of beta II spectrin though a coincidence detection mechanism. This palmitoyltransferases/ ankyrin-G/beta II spectrin pathway determines the cell height of columnar epithelial cells. In Chapter 5, we elucidated the molecular mechanism through which the spectrin-ankyrin network assembles epithelial lateral membranes. We demonstrated that ankyrin-G and beta II spectrin function by opposing clathrin-mediated endocytosis to build the lateral membrane in MDCK cells. Together, this thesis dissects the mechanisms of how the spectrin-ankyrin network achieves precise membrane targeting and how it assembles lateral membranes to determine the morphogenesis of columnar epithelial cells, and provides the first molecular insight to understand how cells control the assembly of diverse plasma membrane domains.</p> / Dissertation