The final steps of cell division are tightly coordinated in space and time but whether mechanisms exist to couple the actin and microtubule cytoskeletons during anaphase and cytokinesis (C phase) is largely unknown. We show here that spindle midzone microtubules are stabilized as cells initiate cleavage furrow ingression. This stabilization is dependent on actomyosin contraction, suggesting that there is active coordination between furrow ingression and microtubule dynamics during C phase. Midzone microtubule stabilization also depends on the kinase activity of Aurora B, the catalytic subunit of the Chromosomal Passenger Complex (CPC), uncovering a feedback mechanism that couples furrowing with microtubule dynamics. We further show that the CPC scaffolding protein INCENP binds actin. Interaction between actin and INCENP is important for cytokinesis, and for midzone microtubule stabilization following furrow ingression. Pharmacological stabilization of midzone microtubules rescues cytokinesis in INCENP actin-binding mutant expressing cells, demonstrating that the CPC is integral for coupling furrow ingression with midzone microtubule stabilization. We also found that actin binding is required to localize the CPC to the midzone and equatorial cortex during C phase. As the sub-cellular localization of the CPC is tied to its mitotic functions we investigated the role of actin-binding in recruiting the CPC to the equatorial cortex. The transport of the CPC from the centromeres to the cell middle is thought to depend on microtubule plus-end directed transport by the kinesin Mklp2 (Kif20a), however, we observed that in the absence of motor-based transport on MTs, the CPC can still target the cortex, in manner that depends on INCNEP-actin binding. This demonstrates that the mechanisms involving both the actin and MT cytoskeletons cooperate to precisely position CPC during C phase. We also observed that Mklp2 and the CPC remain associated throughout C phase and that the Mklp2-CPC complex diffuses once it reaches midzone MT plus ends, or the cell cortex, suggesting that motor activity does not define the bulk of the dynamic behavior we observe during anaphase. We find that cortical diffusion of Mklp2-CPC relies on F-actin, suggesting INCENP-actin binding promotes cortical recruitment and diffusion. Finally, cortically-localized CPC is sufficient to rescue furrow closure in Mklp2-depleted cells, indicating that this population is functional to promote cleavage furrow ingression. Collectively, our work demonstrates that the activities of the actin and microtubule cytoskeletons are coordinated during C phase, through both cytoskeletal cross-talk and cooperative CPC positioning, in order to ensure successful cell division.

Both type I and type II diabetes are related to β-cell defects in the pancreatic islet of Langerhans. Deriving β-cells from stem cells and other mature cell types provides an important cell source for transplantation-based therapy to treat diabetes. Mechanistic studies of β-cell maturation and functional maintenance are crucial in providing novel insights for the generation of glucose-responsive and long-term sustainable β-cells. In this study, I found that two gene/gene families, synaptotagmin IV (Syt4) and Myt factors are essential to promote β-cell maturation and functional maintenance. Mouse studies provided evidence that Syt4 modulates insulin Ca2+ vesicle sensitivity to facilitate β-cell maturation the neonatal stage. Loss of Syt4 in mice resulted in Ca2+ hypersensitivity of insulin vesicles in β-cells and compromised glucose-stimulated insulin secretion. Conversely, Syt4 overexpression reduced insulin Ca2+ vesicle sensitivity and established the mature insulin secretion profile in the newborn β-cells. Moreover, Myt factors are essential to generate functional β-cells. Postnatal β-cells in a Myt knockout mouse model were characterized by functional failure, cell apoptosis and loss of mature β-cell identity. Loss of Myt factors in β-cells disrupted the expression of genes involved in insulin secretion, β-cell survival and identity maintenance. These combined results suggest that Syt4 and Myt factors can be exploited as molecular targets to promote β-cell maturity and long-term functional maintenance for better clinical β-cell regeneration.

Apico-basolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of colorectal cancer (CRC). Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We observed an increase in collagen disorganization and higher prevalence of an integrin β1/EGF receptor-containing complex in human CRC. To better understand the contribution of integrin signaling to epithelial cell morphogenesis and receptor tyrosine kinase (RTK) signal transduction, we used an approach combining 3D type-1 collagen culture and integrin β1 function-altering antibodies. We found that induction of integrin α5β1 clustering at lateral, intercellular surfaces contributes to apico-basolateral polarization in a fibronectin-dependent manner. Preliminary work suggests a role for integrin α5β1 in regulating CRC-derived cell response to RTK ligands EGF, NRG1 and HGF. All together, these data show a novel role for integrin α5β1 in regulating colonic epithelial morphogenesis.

ABSTRACT The Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2/ESDN/CLCP1) is a type-I, transmembrane receptor that mediates diverse cellular processes such as angiogenesis, vascular remodeling, cellular migration and proliferation. Identification of DCBLD2 in a proteomics screen to identify substrates of Src family tyrosine kinases that bind the Src homology 2 domain of CT10 regulator of kinase-Like (CrkL), a critical scaffolding protein for neuronal development, led to a need for further characterization of the protein. To elucidate the role of this interaction and potential novel function of DCBLD2, an in vivo approach utilizing Danio rerio (zebrafish) was conducted. dcbld2 was found localized in neuronal tissues during development, with strong expression in the retina. Knockdown of the protein led to a deficiency of retinal ganglion cells and the optic tracts, or nerve bundles, they project to innervate the brain. Serial sections revealed malformation of the normally discrete layering of retinal cell types, and smaller eye area overall. These findings suggest a role for dcbld2 in developing nervous tissue, specifically neuronal migration during interkinetic nuclear migration. While it is has been shown that dcbld2 has a role in the developmental patterning of intersegmental vessels in the tail of zebrafish, the protein has not been investigated in the context of neurogenesis. The loss of RGCs and lamination defects observed in the eye, along with its association with the CrkL-SH2 domain, implicate it in processes that allow for the proper differentiation of neurons. This study has brought us further down the path to understanding the multiple functions of the receptor; however, further studies are required to delineate the exact mechanistic function of the dcbld2 receptor.

CELL AND DEVELOPMENTAL BIOLOGY Structural Analysis of the Helicobacter pylori toxin VacA Tasia Marie Pyburn Dissertation under the direction of Associate Professor Melanie Ohi Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. One of the most important H. pylori virulence determinants is a secreted pore-forming toxin known as vacuolating cytotoxin A (VacA). Secreted as an 88 kDa protein, VacA is composed of an N-terminal p33 domain and a C-terminal p55 domain which assemble into multiple types of water-soluble oligomers including hexamers, heptamer, dodecamers, and tetradecamers. We have determined three-dimensional (3D) structures of VacA s1/i1/m1 oligomeric conformations at ~15 Ã resolution as well as three mutant forms of VacA. At this resolution, differences between the mutants and VacA s1/i1/m1 could not be discerned. Therefore, cryo-EM has been performed on VacA s1/i1/m1 and a structure has been determined of a VacA dodecamer to the highest resolution to date, ~10Ã resolution. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. Our goal is to understand how VacA transitions from a soluble protein to a membrane inserted protein and how it organizes on membrane. Using a combination of in vitro liposome binding, biochemical assays, and single particle electron microscopy (EM), we show membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle EM reveals structural differences within the central pore-forming region of the oligomers indicating that membrane interactions induce a structural change within the p33 domain. Analyses of VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels. Approved _______________________________________________ Date __________________ Melanie D. Ohi, Ph.D.

Reprogramming of pancreatic cells into new beta-like cells is a potential therapy for Type 1 diabetes. Pancreatic acinar cells are an appealing target for cellular reprogramming since they are abundant, derived from a common progenitor cell during pancreatic organogenesis, and exhibit significant transcriptional plasticity. Towards this end, it has been reported that adenoviral-mediated expression of three pancreas-specific transcription factors MafA, Pdx1 and Neurog3 (3TF) in immunocompromised Rag1-/- mice resulted in the conversion of pancreatic acinar cells into new, insulin-secreting, beta-like cells. Using a transgenic mouse model to express 3TF in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to beta-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new beta-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new beta-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.

The regulation of protein biosynthesis at fertilisation was studied in North Sea Plaice (Pleuronectus platessa). RNA and protein synthesis were studied in fertilised and unfertilised plaice eggs. There was no detectable RNA synthesis in the egg in early embryonic stages, but protein synthesis at all stages of development. However, using RITA synthesis inhibitors (actinomycin D and ethidium bromide), it was demonstrated that certain groups of proteins were synthesised independently of any newly synthesised messenger RNA. Different rates of protein synthesis in various subcellular fractions from both fertilised and unfertilised eggs were demonstrated. Proteins precipitated by vinblastine were shown to exhibit the most changes between the egg and fertilised egg. These proteins were further characterized on several polyacrylamide-gel electrophoresis systems. RNA was isolated from the polysomes and the supernatant fractions of eggs and fertilised eggs. This RITA was tested for messenger RITA activity in a rabbit reticulocyte cell-free system and also in a wheat germ in vitro system. Messenger RNA activity was demonstrated in both the polysomal and supernatant fractions from the egg and also from the fertilised egg, and was purified by affinity chromatography and sucrose gradient centrifugation. Regulatory mechanisms have been demonstrated at the translational level of protein synthesis in early plaice embryogenesis.

<p> Na⁺/Ca²⁺ exchangers are low affinity/high capacity transporters that mediate Ca²⁺ extrusion by coupling Ca²⁺ efflux to the influx of Na⁺ ions. Their primary function is to maintain Ca²⁺ homeostasis in cells of all organisms and they play a particularly important role in excitable cells that experience transient Ca²⁺ fluxes. While their functions have been studied extensively in muscle cells, much is still unknown about their contributions to the nervous system. Data suggests that Na⁺/Ca²⁺ exchangers play a key role in neuronal processes such as memory formation, learning, oligodendrocyte differentiation and axon guidance. They are also implicated in pathologies such as Alzheimer&rsquo;s Disease, Parkinson&rsquo;s Disease, Multiple Sclerosis and Epilepsy. While they are implicated in critical neuronal processes, a clear understanding of their mechanism remains unknown. This dissertation examines the role of Na⁺/Ca²⁺ exchangers in the invertebrate model organism <i>Caenorhabditis elegans </i>. There are ten identified Na⁺/Ca²⁺ exchanger genes in <i>C. elegans</i> (labeled <i>ncx-1</i> to <i>ncx</i>-10). Data presented here is the first comprehensive description of their genetics and function in <i>C. elegans</i>. The expression pattern of all 10 Na⁺/Ca²⁺ exchanger genes is described and their phylogeny is examined comparatively across humans and flies. Analysis of <i>ncx-2</i> and <i>ncx-8</i> mutants shows important roles for Na⁺/Ca²⁺ exchanger genes in egg-laying, lipid storage and longevity, suggesting a role in diverse biological functions for Na⁺/Ca²⁺ exchangers in <i>C. elegans</i>. The function of an NCLX type Na⁺/Ca²⁺ exchanger NCX-9 is also detailed comprehensively. Analysis of <i> ncx-9</i> mutants shows that NCX-9 is required for asymmetrical axon guidance choices made by the DD and VD GABAergic motor neuron circuit. Pathway analysis shows that NCX-9 regulates asymmetric circuit patterning through RAC-dependent UNC-6/Netrin signaling and LON-2/Glypican Heparan Sulfate signaling. <i> In vitro</i> analysis of NCX-9 physiology in HEK cells shows that NCX-9 is a mitochondrial Na⁺/Ca²⁺ exchanger, similar to NCLX, which is its homolog in humans.</p>

<p>Abstract</p><p>During embryonic development many structures crucial for breathing and eating arise from the pharyngeal and anterior foregut epithelium (FGE), which contains the oral ectoderm and the foregut endoderm. Proper differentiation and signaling within and from this epithelial tissue is necessary for the development of the mandible, the esophagus, and the trachea. Many birth defects occur in these structures that greatly disrupt the ability of affected infants to breathe and eat. This dissertation investigates the importance of the pharyngeal and anterior FGE in mandible and foregut development. </p><p>The most rostral portion of the pharyngeal epithelium contributes to the development of the mandible. At embryonic day 10.5 the mandible is a bud structure, composed of neural crest-derived mesenchyme and core mesoderm surrounded by pharyngeal epithelium. The mesenchyme needs to receive Hedgehog signaling for mandible development, but the epithelial tissue that signals to the mesenchyme has not been identified in mammals. Data presented in Chapter 2 show that Sonic Hedgehog is necessary at two distinct stages of mandible development by using a tissue specific genetic ablation to remove Sonic Hedgehog from the pharyngeal endoderm. First, we show that Sonic Hedgehog promotes cell survival prior to cartilage differentiation through immunostaining for Caspase-9, an apoptosis marker. Second, a rescue of early cell death with the p53 inhibitor pifithrin-&#945; shows that Sonic Hedgehog is necessary for cartilage condensation and differentiation later in development. Without cartilage differentiation the mandible is unable to elongate properly and hypoplasia occurs.</p><p>Caudal to the pharyngeal epithelium is the anterior FGE, which develops into the larynx, esophagus and trachea. The anterior FGE is a single endodermal tube at E9.5 and by E11.5 compartmentalizes into two distinct tubes: the esophagus and trachea. While the signaling pathways involved in proper compartmentalization of the foregut are well studied, nothing is known about the cellular behaviors that drive this complex event. One important event during foregut compartmentalization is the establishment of dorso-ventral patterning, which is necessary for separation to occur. To elucidate the importance of dorso-ventral patterning, we take advantage of two genetic mouse models with disrupted patterning, an activation of and a removal of &#946;-catenin in the ventral foregut endoderm. Data presented in Chapter 3 show that &#946;-catenin is important for epithelial pseudostratification and the establishment of a region of double-positive cells at the dorso-ventral midline through close examination of epithelial morphogenesis at E10.5 prior to compartmentalization. This data has established two mouse models for studying changes in epithelial morphology during foregut compartmentalization. In total, this body of work details how signals originating in the pharyngeal and anterior foregut epithelium regulate both mesenchymal and epithelial behaviors during mandible and foregut development.</p> / Dissertation

<p> Sudden infant death syndrome (SIDS) occurs when a sleeping infant experiences a challenge to cardiorespiratory homeostasis which it fails to overcome. Analyses of brain tissue from SIDS cases from around the world consistently show abnormalities in the brainstem serotonin systems. These include increased numbers of neurons that test positive for serotonergic markers, but have an immature phenotype, reduced brain tissue serotonin concentrations and decreased serotonin receptor binding in projection sites important to cardiorespiratory homeostasis, including the nucleus of the solitary tract (NTS). The NTS is of particular interest in the pathophysiology of SIDS because it is the integration center for afferent projections involved in eliciting several apnea-inducing reflexes long suspected of contributing to SIDS. The laryngeal chemoreflex (LCR), an airway protective reflex which is initiated when water, acidic solutions, or low [Cl-] solutions activate chemoreceptors in the larynx, is one such reflex. In infants, inhibitory reflex responses to hypoxia (apnea, bradycardia, decreased metabolic activity) that are adaptive for a fetal environment that precludes the possibility of the fetus acquiring more oxygen by increasing breathing, persist for some time into the postnatal period. Therefore, hypoxia resulting from apnea caused by the LCR can result in a cataclysmic downward spiral of apnea, followed by increasing hypoxic inhibition of respiration, which ultimately leads to SIDS. We hypothesized that increasing serotonin signaling in the brainstems of rat pups would shorten the apnea and respiratory disruption caused by eliciting the LCR. We have shown that both intracisternal injections of serotonin, and microinjections of serotonin into the caudal NTS, dramatically shorten the LCR. This effect is also seen after microinjection into the NTS of the 5-HT3 specific agonist CPG. Chemical stimulation by microinjection of AMPA of neurons in the raphe obscurus, some of which send serotonergic projections to the NTS, also shortens the LCR, but this effect is blocked by prior injection of a 5-HT3 antagonist in the NTS. Our work suggests that serotonergic projections to the NTS from the caudal raphe may play an important role in limiting the duration of apnea following inhibitory reflexes like the LCR and in the subsequent restoration of eupnea.</p>