Browsing by Author "Bennett, Vann"

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.

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.

Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration.

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.

Organelle transport in eukaryotic cells is regulated by a precisely coordinated activity of phosphoinositide lipids, small GTPases, and molecular motors. Despite the extensive study of functional activities of individual regulators, how these activities promote precise deliveries of particular membrane proteins to specific cellular locations remained unclear. Ankyrin-B, which is previously well recognized as a plasma membrane adaptor that assembles diverse specialized plasma membrane domains, exhibited an unexpected role in intracellular transport. This thesis establishes ankyrin-B as a master integrator of the polarized long range organelle transport via direct interactions with Rab GTPase Activating Protein 1 Like (RabGAP1L), phosphatidylinositol 3-phosphate (PI3P) and dynactin 4. In Chapter 2, I identified an ankyrin-B death domain binding partner, RabGAP1L, that specifically interacts with ankyrin-B on intracellular organelles and requires ankyrin-B for its proper localization. In Chapter 3, I demonstrated that ankyrin-B is a PI3P-effector in mouse embryonic fibroblasts (MEFs) and promotes the polarized transport of associated organelles in migrating cells in a RabGAP1L-dependent manner. I continued to investigate what membranes/membrane-associated proteins utilize the ankyrin-B/RabGAP1L pathway in Chapter 4 and identified α5β1-integrin as a cargo whose polarized transport and recycling are ankyrin-B-dependent. I further presented that ankyrin-B, through recruiting RabGAP1L to PI3P-positive/Rab22A-associated endosomes containing α5β1-integrin, promotes polarized recycling of α5β1-integrin in migrating mouse embryonic fibroblasts. In collaboration with James Bear (UNC Chapel Hill), we further demonstrated that this ankyrin-B/RabGAP1L-mediated pathway is required for haptotaxis along fibronectin gradients. In Chapter 5, I elucidated the in vivo interaction between ankyrin-B and RabGAP1L. I demonstrated that ankyrin-B specifically interacts with RabGAP1L at long axon tracts in the brain and at costameres in the skeletal muscle. I also show the phenotypic analysis of ankyrin-B floxDD mice as an initial attempt to determine the physiological function of the ankyrin-B death domain in vivo. Together, this thesis dissects an ankyrin-B-mediated molecular mechanism for polarized endosomal trafficking and α5β1-integrin recycling during directional cell migration, and provides new insights into how phosphoinositide lipids, Rab GTPases, and molecular motor activities are coordinated to control the directional transport of specialized membrane cargos.

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.

The ankyrin family of proteins form specialized membrane domains in various cell types, including neurons and cardiomyocytes but little is known about how this process occurs. The majority of ankyrins localize to the plasma membrane in these cells while ankyrins in fibroblasts are largely intracellular. This property of fibroblasts allows us to study intracellular ankyrin and potentially how ankyrin forms membrane domains in other cell types. In this thesis, we use western blots, immunofluorescence, RT-PCR to characterize the expression pattern of ankyrin in fibroblasts and find that both ankyrin-B and -G localize to both nuclear and intracellular compartments. The size of the compartments of both ankyrin-B and -G is affected by the genetic deletion of the large isoforms of ankyrin-B and -G. The ankyrin-B compartment has a weak association with recycling endosomes suggesting that ankyrin-B may be involved in membrane protein trafficking.

Organization of membrane domains in axons is key for the normal transmission of information from one neuron to another. Giant ankyrin proteins are two such members of axonal membrane domains, where giant ankyrin-B (440kDa) is localized to the plasma membrane of axons and giant ankyrin-G (480kDa) is localized to excitable membrane domains of axons as well as somatodendritic regions later in development. Mutations in these proteins are found in autistic individuals (giant ankyrin-B) and bipolar and schizophrenia patients (giant ankyrin-G). However, the function of giant ankyrin-B and the regulation of giant ankyrin-G remain elusive. Here we developed two transgenic mouse lines lacking giant ankyrin-B: one that completely eliminates giant ankyrin-B by Cre-dependent removal of the inserted sequence, and another CRISPR induced frameshift inside of the inserted sequence that mimics the variant in one autistic individual, to decipher its role in the central nervous system. Using analyses of cellular, brain-wide connectivity, and behavioral assays of these transgenic mice, we show the L1CAM-giant ankyrin-B complex is essential for normal axon branching and brain connectivity. Perturbations to brain connectivity by giant ankyrin-B mutation leads to selective deficits in pup ultrasonic vocalizations, male territory marking, and increased plasticity for reversal learning in a binary water T-maze. Secondly, I developed a novel solubilization technique for the denatured isolation of previously biochemically inaccessible giant ankyrin proteins. With this technique, we determined that phosphorylation was the major post-translational modification on giant ankyrin-G, and also on beta-4 spectrin sigma six isoforms. The stoichiometry of the phosphorylation was increased in these two excitable membrane proteins in comparison to the other isoforms of these proteins. By comparing developmental stages, there is a trend of decreased phosphorylation of giant ankyrin proteins within the giant inserted region during maturation of excitable membrane domains. Finally, we show a developmental change in ankyrin-G polypeptide levels and pinceau synapse formation on Purkinje cells in a mouse model with a disrupted ankyrin-G/GABARAP binding site. Together, these findings demonstrate that giant ankyrin proteins are critical proteins for the normal connectivity and conductance of neurons and that they are dynamically regulated through post-translational phosphorylation.

During development, oriented cell divisions are crucial for correctly organizing and shaping a tissue. Mitotic spindle orientation can be coupled with cell fate decisions to provide cellular diversity through asymmetric cell divisions (ACDs), in which the division of a progenitor cell results in two daughters with different cell fates. Proper tissue morphogenesis relies on the coupling of these two phenomena being highly regulated. The development of the mouse epidermis provides a powerful system in which to study the many levels that regulate ACDs. Within the basal layer of the epidermis, both symmetric and asymmetric cell divisions occur. While symmetric divisions allow for an increase in surface area and progenitor cell number, asymmetric divisions drive the stratification of the epidermis, directly contributing additional cell layers (Lechler and Fuchs 2005; Poulson and Lechler 2010; Williams, Beronja et al. 2011).

Utilizing genetic lineage tracing to label individual basal cells I show that individual basal cells can undergo both symmetric and asymmetric divisions. Therefore, the balance of symmetric:asymmetric divisions is provided by the sum of individual cells' choices. In addition, I define two control points for determining a cell's mode of division. First is the expression of the mInscuteable gene, which is sufficient to drive ACDs. However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, I show that p63, a transcriptional regulator of stratification, does not control either of these processes, rather it controls ACD indirectly by promoting cell polarity.

Given the robust control on NuMA localization to prevent excess ACDs, I sought to determine how targeting of NuMA to the cortex is regulated. First, I determined which regions within the protein were necessary and sufficient for cortical localization. NuMA is a large coiled- coil protein that binds many factors important for ACDs, which include but are not limited to: microtubules, 4.1, and LGN. Interestingly, while the LGN binding domain was necessary, it was not sufficient for proper NuMA localization at the cortex. However, a fragment of NuMA containing both the 4.1 and LGN binding domains was able to localize to the cortex. Additionally, the NuMA-binding domain of 4.1 was able to specifically disrupt NuMA localization at the cortex. These data suggested an important role for a NuMA-4.1 interaction at the cortex. While the 4.1 binding domain was not necessary for the cortical localization of NuMA, it was important for the overall stability of NuMA at the cortex. I hypothesize that 4.1 acts to anchor/stabilize NuMA at the cortex to provide resistance against pulling forces on the mitotic spindle to ensure proper spindle orientation.

Finally, to determine if post-translational modifications of NuMA could regulate its localization I tested the importance of a conserved Cdk-1 phosphorylation site. Interestingly, a non-phosphorylatable form of NuMA localized predominately to the cortex while the phosphomimetic protein localized strongly to spindle poles. In agreement with these data, use of a CDK-1 inhibitor was able to enhance the cortical localization of NuMA. Unexpectedly, the non-phosphorylatable form of NuMA did not require LGN to localize to the cortex. Additionally, restoration of cortical localization of the phosphomimetic form of NuMA was accomplished by the overexpression of either LGN or 4.1. Thus, phosphorylation of NuMA may alter its overall affinity for the cortex.

Overall, my studies highlight two important regulatory mechanisms controlling asymmetric cell division in the epidermis. Additionally, I show a novel role for the interaction between NuMA and 4.1 in providing stability at the cortex. This will ultimately provide a framework for analysis of how external cues control the important choice between asymmetric and symmetric cell divisions.