Two processes influence the success of mitosis, the process by which eukaryotic cells divide their replicated genome into two new daughter cells. First, the cell must build a bipolar array of microtubules called the mitotic spindle, which is accomplished by microtubule sliding by the Kinesin-5 Eg5; second, a subset of those microtubules, called kinetochore-microtubules (K-MTs), must attach correctly to each chromosome. The stability of these K-MT attachments to the chromosome influences whether chromosomes will segregate correctly in anaphase, but an influence of K-MT stability on the ability of cells to build or maintain bipolar spindles had not been recognized. Here we show that human cell lines with high K-MT stability are better able to maintain bipolar spindles when Eg5 is inhibited compared to cell lines with low K-MT stability. Artificially stabilizing K-MTs promoted bipolarity maintenance, while destabilizing K-MTs undermined the maintenance of bipolarity. In addition, inhibition of the mitotic master-regulator cyclin-dependent kinase-1 (CDK-1) in the G2 phase of the cell cycle stabilized K-MTs after CDK-1 inhibition was relieved, which promoted bipolar spindle maintenance without Eg5 but undermined mitotic fidelity. These results indicate that the dynamics of K-MTs play a previously unappreciated role in determining the overall geometry of the mitotic spindle.

Myocardial infarction (MI) causes irreversible tissue damage, leading to heart failure. Our laboratory found canonical Wnt signaling and the Wnt10b ligand are strongly induced in mouse and human hearts after MI. Wnt10b regulates cell fate in various organs, yet its role in the heart is unknown. To investigate the effects of Wnt10b gain-of-function on cardiac repair mechanisms and functional outcomes after injury, we generated αMHC-Wnt10b transgenic (TG) mouse line that overexpresses Wnt10b in adult cardiomyocytes. Following acute myocardial injury, the TG mice displayed improved recovery of cardiac function, accompanied by enhanced neovascularization and attenuated scar fibrosis. Wnt10b stimulated expression of vascular endothelial growth factor receptor 2 in endothelial cells and angiopoietin-1 in vascular smooth muscle cells through nuclear factor-κB activation to promote stabilized blood vessel formation. Wnt10b also reduced the number of myofibroblasts to mitigate fibrosis. My findings may lead to novel strategies to optimize the inherent repair capacity of the heart and prevent the onset of heart failure.

Diabetic Retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is an early pathologic
feature of DR. Though it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was identified to play a significant role in human retinal pericyte apoptosis. Inhibition of the GAPDH/Siah1 pro-apoptotic complex blocks diabetes-induced pericyte apoptosis, widely considered a hallmark feature of DR.

The Wnt signaling pathway plays essential roles in a wide variety of biological processes including early animal development, cell fate determination, cell proliferation, organogenesis, and stem-cell renewal. Deregulation of the Wnt pathway can lead to human disease (e.g. developmental defects and cancers). Our lab had previously demonstrated that X-linked Inhibitor of Apoptosis (XIAP) is required for Wnt signaling via a mechanism that involves XIAP-facilitated dissociation of Gro/TLE from TCF/Lef. We had shown that XIAP is recruited onto TCF/Lef complexes upon Wnt pathway activation and ubiquitinates Gro/TLE-bound TCF/Lef. Ubiquitinated Gro/TLE has decreased affinity for TCF/Lef, allowing beta-catenin to bind. Inhibition of GSK3 by lithium causes XIAP to be recruited to TCF/Lef, although the precise mechanism was unknown. We found that XIAP is phosphorylated by GSK3, a known component of the Wnt pathway. Using mass spectrometry analysis with purified proteins, we identified two GSK3 phosphorylation sites on XIAP that are strongly phosphorylated. Mutational analysis of these two sites indicate that they are required by XIAP to fully activate Wnt signaling, as assessed by reporter assays in cultured mammalian cells and axis duplication assays in Xenopus embryos. Using purified proteins, we found that the XIAP phosphomutants have similar ubiquitination activity as wild-type XIAP. In cultured mammalian cells, however, the XIAP phosphomutants have a markedly decreased capacity to ubiquitinate Gro/TLE. We also showed that the phosphomutants also have a decreased affinity for Gro/TLE. We propose a model in which phosphorylation is necessary for the interaction between XIAP and Gro/TLE to activate Wnt signaling. GSK3 phosphorylates XIAP at T180 and S239 to facilitate its binding to Gro/TLE. XIAP can then ubiquitinate and remove Gro/TLE for subsequent binding of beta-catenin to form the transcriptionally active complex.

The spliceosome is a dynamic macromolecular machine composed of five different small nuclear ribonucleoproteins (snRNPs), the Nineteen Complex (NTC), RNA helicases, and other proteins that catalyze the removal of introns from pre-mRNA. Although the spliceosome has been extensively studied, there are still many unanswered questions regarding the molecular mechanisms of pre-mRNA splicing. In this work we used the Schizosaccharomyces pombe model system to characterize the structure and function of the spliceosome. By examining the in vitro RNA ability of S. pombe Cdc5, a core component of the NTC, we were able to propose a mechanism where protein components of the NTC can bind and stabilize RNA within the active site of the spliceosome. We also used an RNA helicase mutant, S. pombe prp16-1, to isolate a second step splicing complex. This work has provided insight into the general mechanisms on how the spliceosome functions to process pre-mRNA.

A developing vertebrate embryo can only subsist for a finite time without a vasculature. The vascular system is one of the earliest organ systems to develop and allows continued rapid maturation of complex multicellular organisms. Failure of the cardiovascular system to develop results in early termination of the fetus. Together, my thesis research demonstrates the important implications that development and cell biology have on vascular function and repair. First, a thorough evaluation of characteristics of vascular smooth muscle cells from differing regions of the adult and embryonic aorta revealed that differences between the ascending and descending embryonic aortae converge in the adult. These data have important implications for vascular development and disease. The second story examines the cell biological implications of the loss of centromere protein F (CENP-F). A cardiac specific deletion of CENP-F results in dilated cardiomyopathy and by understanding how CENP-F alters cell biology in a model cell line, we now know how loss of CENP-F modifies cardiac biology. Ultimately, these seemingly disparate studies further our understanding of vascular biology and disease.

The ubiquitinâproteasome system (UPS) influences gene transcription in multiple ways. One way in which the UPS impacts transcription centers on transcriptional activators, the function of which can be stimulated by components of the UPS that also trigger their destruction. Activation of transcription by the yeast activator Gcn4, for example, is attenuated by mutations in the ubiquitin-ligase that mediates Gcn4 ubiquitylation or by inhibition of the proteasome, leading to the idea that ubiquitin-mediated proteolysis of Gcn4 is required for its activity. Here, I probe the steps in Gcn4 activity that are perturbed by disruption of the UPS. I show that the ubiquitylation machinery and the proteasome control different steps in Gcn4 function, and that proteasome activity is required for the ability of Gcn4 to bind to its target genes in the context of chromatin. Curiously, the impact of proteasome inhibition on Gcn4 activity is suppressed by mutations in the ubiquitin-selective chaperone Cdc48, revealing that proteolysis per se is not required for Gcn4 activity. My data highlights the role of Cdc48 in controlling promoter occupancy by Gcn4 and support a model in which ubiquitylation of activatorsânot their destructionâis important for function.

Eukaryotic cells respond to stress by reprogramming their gene expression program to inhibit global protein synthesis and direct translationally silenced mRNAs to cytoplasmic foci known as stress granules (SGs). SGs function as sites where mRNAs are sorted for storage, decay or translation. SGs are linked with neurodegeneration, cancer and viral infections. However, the molecular mechanisms underlying SG function during stress and in diseases are poorly understood. DEAD-box proteins (Dbps) are RNA-dependent ATPases that mediate changes in mRNA-protein complex (mRNP) structure. Several Dbps, including DDX3, are recruited to SGs and regulate mRNP entry and exit. But, it is unclear how the Dbp activity is regulated in SGs. In the work presented here, we show that human (h) Gle1 is a novel factor of SGs and it regulates SGs by modulating the dynamic equilibrium between SGs and translation through its regulation of DDX3. We further show that two alternatively spliced isoforms of the GLE1 gene perform distinct and non-overlapping functions in a cell; hGle1A is required for SG function, whereas hGle1B functions in SGs. Interestingly, our results also show that an amyotrophic lateral sclerosis (ALS)-linked mutation in GLE1 disrupts functional specificity of hGle1 isoforms and the resulting protein variant is bifunctional. Overall, this study provides insight into the critical roles of hGle1 in SG biology and also gives clues as to how mutation of GLE1 contributes towards ALS pathogenesis.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. In this dissertation we show that polymeric immunoglobulin receptor-deficient (pIgRâ/â) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodeling and emphysema in pIgRâ/â mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-kappa B activation, leukocyte infiltration, and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgRâ/â mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodeling, while repetitive inhalation of bacterial products exacerbates disease. In addition, we demonstrate that p73 is required for PIGR expression in mice, and that mice lacking p73 also develop airway remodeling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodeling and emphysema. Based on this data, we propose that altered mucosal immunity due to SIgA deficiency contributes to chronic inflammation and airway remodeling in COPD.

Programmed cell death (PCD) is an important process necessary for the maintenance of tissues in adult organisms and the crafting of distinct tissues in development. The two main types of PCD, apoptosis and necroptosis (i.e. programmed necrosis), are characterized through differing morphologic presentations and outcomes. Death receptor signaling is a context in which both apoptotic or necroptotic outcomes can occur. Several recent studies implicate proteins involved in apoptotic signaling in the inhibition of necroptosis including Caspase-8, FADD, and cFlipL. Bid, a member of the BCL-2 family of proteins, is cleaved by Caspase-8 which promotes its activation and translocation to the mitochondrion, promoting apoptosis. To evaluate what role Bid might play in the necroptotic arm of death receptor signaling we developed a mouse with Bid and its apoptotic arm of function (Bax and Bak) removed in hematopoietic cells. Loss of these three proteins leads to loss of restraint of necroptosis leading to increased necroptotic death, inflammatory signaling, and perturbation of tissue homeostasis in the hematopoietic and gastrointestinal organ systems. These findings in mice have implications for Myelodysplastic Syndrome, a bone marrow failure disorder characterized by increased PCD, and Inflammatory Bowel Diseases, inflammatory diseases characterized by overwhelming inflammation in the gastrointestinal system.