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Regulation of Wnt Receptor Activation by the Tumor Suppressor APCSaito-Diaz, Vicente Kenyi 27 March 2017 (has links)
The Wnt pathway is a highly-conserved pathway that controls many developmental processes and is mutated in many human diseases (e.g., cancer). The tumor suppressor adenomatous polyposis coli (APC) is a critical negative regulator of Wnt signal transduction. Mutations in the APC gene resulting in constitutive activation of the Wnt pathway occur in over 80% of human colorectal cancers (CRC). Despite its critical role in the Wnt pathway, the exact mechanism of APC function in Wnt signal transduction is not clear. The lab developed a monoclonal antibody (mAb7E5) that targets the co-receptor LRP6 and inhibits Wnt signaling in APC-mutant CRC cells. Using the antibody mAb7E5, I found that APC regulates Wnt receptor activation. Furthermore, I found that, in APC-depleted cells, the co-receptor LRP6 is constitutively active in a manner independent of Wnt ligands and that LRP6 is internalized by the clathrin-dependent endocytic machinery. Finally, I demonstrate that APC, clathrin, and the AP-2 adaptor protein interact as a complex. Thus, my studies reveal a new role for APC function in Wnt signal transduction and provide insight into the development of therapeutic agents targeting APC-mutant tumors.
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Dissecting Pancreatic β-cell Stress Using Whole Transcriptome SequencingStancill, Jennifer Susan 29 March 2017 (has links)
Type 2 diabetes is characterized by failure of pancreatic β-cells to secrete adequate insulin to meet the needs of the body. This β-cell failure is thought to be caused by increased metabolic load due to mounting insulin resistance, but the molecular mechanisms by which this dysfunction occurs are not fully understood. To better understand how β-cells fail, we took a whole-transcriptome approach, collecting RNA-sequencing datasets from purified β-cell populations from several mouse models of β-cell stress. First, we used mice lacking Abcc8, a key component of the β-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on β-cell identity and gene expression. We found that chronically elevated β-cell [Ca2+]i results in the dysregulation of over 4,200 genes, as well as modest loss of β-cell identity, characterized by decreased expression of key functional genes, increased expression of genes associated with β-cell dedifferentiation, increased β-cell transdifferentiation to PP-expressing cells, and decreased β-cell function. These studies prompted us to propose a model by which chronically elevated β-cell [Ca2+]i acts through a putative Ca2+-regulated transcription factor, ASCL1, to disrupt a network of genes, contributing to β-cell failure.
In addition to exploring the effects of chronically elevated [Ca2+]i on β-cell gene expression, we analyzed β-cells from mice ectopically expressing human growth hormone (hGH), mice made insulin resistant by feeding a high-fat diet (HFD), and mice of different sexes. We found that both ectopic hGH and HFD have beneficial effects (induction of β-cell proliferation genes) as well as deleterious effects (increased expression of ER stress genes) on β-cell function. Ultimately, the collection of 17 RNA-sequencing datasets allowed us to perform weighted gene correlation network analysis (WGCNA) to generate modules of similarly-expressed genes. Several of these initial modules have meaningful correlations to specific β-cell stresses. Overall, these studies highlight the power of using whole transcriptome datasets from highly-pure cell populations and have allowed us to elucidate how stress alters the β-cell gene regulatory network.
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Actin and Microtubule Cross-talk during CytokinesisLandino, Jennifer Elaine 30 March 2017 (has links)
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.
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Synaptotagmin IV and Myt factors promote β-cell functional maturation and maintenanceHuang, Chen 31 March 2017 (has links)
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.
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Identification of a role for integrin alpha 5 in colonic epithelial morphogenesisStarchenko, Alina 04 April 2017 (has links)
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.
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Dcbld2/esdn Is Essential For Proper Optic Tract Formation And Retinal LaminationJoy, Ryan Mears 01 January 2016 (has links)
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.
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Structural Analysis of the Helicobacter pylori Toxin VacAPyburn, Tasia Marie 21 March 2017 (has links)
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.
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Variables that influence transcription factor-mediated acinar to beta cell reprogrammingClayton, Hannah Worchel 24 February 2017 (has links)
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.
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Uncovering mechanisms that control myosin-1a membrane binding and targeting dynamicsMazerik, Jessica Nicole 09 May 2013 (has links)
Epithelial cells called enterocytes line the lumen of the small intestine and are responsible for nutrient processing and barrier maintenance. Enterocytes have highly ordered actin arrays, or brush borders, on their apical surfaces. The brush border is composed of microvilli, membrane based protrusions of parallel actin bundles. Within microvilli, myosin-1a laterally links the actin cytoskeleton to the overlying membrane, and contributes to membrane tension regulation and vesicle shedding. These physiological functions require proper localization of this motor, a process that depends on the membrane binding tail homology 1 (TH1) domain. The goal of this thesis is to provide mechanistic details as to how myosin-1a targets to microvillar membrane and how its cellular dynamics are controlled. We find that in vitro and in cells myosin-1a interacts electrostatically with phosphatidylserine through basic residues in two independent bona fide membrane binding motifs. Because membrane binding controls myosin-1a targeting and previously published solution kinetic studies show the motor/actin interaction is short lived, we hypothesized that TH1 is the master regulator of dynamics for this molecule. We used live cell single molecule total internal reflection fluorescence microscopy in combination with single particle tracking and mean squared displacement analysis to measure membrane bound lateral mobility for myosin-1a and TH1. Many myosin-1a molecules display long-lived low mobility dynamics. Similar events are absent from TH1 analysis, indicating the motor domain makes an unexpected contribution to limiting mobility of myosin-1a at the membrane/cytoskeletal interface. Structure/function analysis confirmed this result and revealed the neck region is also important to controlling myosin-1a dynamics. In the context of full length myosin-1a, the neck region also plays a role in regulating localization, perhaps through a conformational change that involves calmodulin/calcium interactions. This is the first study to examine live cell dynamics for any class I myosin at single molecule resolution. The results presented within this thesis provide novel insight as to how myosin-1a cellular targeting and dynamics are controlled, and how biochemical and biophysical properties of myosin-1a manifest in cells to help this molecule carry out physiological roles in the brush border.
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Novel Regulators of Epithelial-to-Mesenchymal Transformation in Cardiogenesis are Identified Through Next-Generation SequencingDeLaughter, Daniel Morris 10 June 2013 (has links)
Epithelial-to-Mesenchymal Transformation (EMT) is an important process in development, and occurs during key steps in both valvular and coronary vessel development. This dissertation uses transcriptional profiling strategies to identify novel regulators of EMT during cardiogenesis.
An early step in valvulogenesis occurs when endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo EMT to yield valvular interstitial cells. We developed an unbiased strategy to identify genes important in endocardial EMT using a spatial transcriptional profile. RNA-seq analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick (HH18) and mouse (E11.0) embryos at comparable stages of development was performed. EMT occurs in the AVC and OFT cushions but not the VEN at this time. We identified genes >2-fold enriched in chick and mouse cushions compared to VEN. Gene ontology and Gene Regulatory Network (GRN) analysis of cushion-enriched genes were consistent with cells undergoing EMT. Further analysis accurately identified and validated previously unrecognized novel candidate genes (Meis2, Id1, Hapln1, Foxp2) and the NF-κB pathway as regulators of endocardial cell EMT in vitro.
Epicardial EMT is a critical step in coronary vessel formation which is dysregulated in mice lacking TGFβR3. To elucidate the role of TGFβR3 in EMT we developed a strategy to identify genes downstream of TGFβR3 in cultured epicardial cells. Tgfbr3+/+ and Tgfbr3-/- immortalized epicardial cells were incubated with vehicle or ligands known to promote TGFβR3-dependent invasion (TGFβ1, TGFβ2, BMP2) and harvested for RNA-seq analysis. GRN analysis of genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells in each ligand incubation group revealed dysregulated NF-ĸB signaling. TGFβ2 or BMP2 incubation stimulated NF-ĸB activity in Tgfbr3+/+ but not Tgfbr3-/- epicardial cells. Inhibiting NF-ĸB signaling reduced TGFβ2- or BMP2-promoted invasion of Tgfbr3+/+ cell, further supporting a role for NF-ĸB signaling in promoting invasion downstream of TGFβR3. The genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.
These transcriptional profiling strategies identified and validated novel regulators of endocardial and epicardial EMT.
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