<p><italic>Cryptococcus neoformans</italic> pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of <italic>C. neoformans</italic> yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.</p> / Dissertation

<p>Adherens junctions are calcium-dependent cell-cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl family of tyrosine kinases have been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins.</p><p>Here we use pharmacological inhibition and RNA interference to identify the Abl family kinases as critical mediators of cadherin-mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl-dependent regulation of the Rho-ROCK-myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto-myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain-of-function promotes adherens junction formation through a Crk-dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation.</p><p>Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl-Crk-Rac.</p><p>Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover.</p> / Dissertation

Foot and Mouth Disease (FMD) could have a significant impact on the U.S. agriculture industry and the welfare of U.S. producers and U.S. consumers. In order to address the potential impact from animal disease outbreaks, this project is designed to utilize a combined epidemic and economic modeling framework to evaluate animal disease management strategies which can be used to reduce the potential losses in an unusual event such as FMD outbreaks. In this study, we compare the welfare changes among three different parties with different strategies using, 1) ANOVA analysis; 2) cost benefit analysis; and 3) Risk Aversion Coefficient (RAC) analysis. Four types of index feedlots are selected in the study including, Feedlot Type 1 (> 50,000 heads of animals), Feedlot Type 4 (backgrounder feedlot), Large Beef Grazing (>100 heads of animals), and Backyard (<10 heads of animals). Results suggest that early detection of FMD events has the advantage in reducing risk as shown in the epidemiological impacts. Enhanced surveillance is found to be a preferred mitigation strategy for U.S. consumers in the scenario of smaller feedlot disease introductions (e.g. Large Beef Grazing and Backyard) and for U.S. producers in the larger feedlot disease introduction scenarios (e.g. Feedlot Type 1 and Feedlot Type 4). Adequate vaccination is not cost effective when seeking to minimize average loss but becomes a preferred strategy when the risk aversion rises. Risk modeling with stochastic programming adopted in this study also confirms the importance of incorporating risk evaluation into decision making process. It offers another option for us to evaluate the mitigation strategies. Two portfolio models are adopted in this study including, E-V model (mean variance portfolio choice model) and Unified model. The results show that the preference for control strategies depends on risk attitude. Early detection proves to be preferable for U.S. consumers and is also preferred by U.S. processors and producers as Risk Aversion Parameters (RAP) rises. Adequate vaccination strategy can benefit U.S. consumers but does not give U.S. processors a better outcome. Adequate vaccination provides a better choice for U.S. producers when the RAP rises. Enhanced surveillance is preferred for U.S. consumers. For U.S. processors, enhanced surveillance does not give a better risk/return outcome. U.S. producers are likely to switch their preferences from regular surveillance to enhanced surveillance as their RAP rises.

<p>Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.</p><p>This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.</p> / Dissertation

Cell polarity is fundamental to numerous cellular processes including migration, molecular transport, and cell division. The establishment and organization of polarity is crucial to the maintenance of cellular homeostasis in mammalian systems. Deregulation of cell polarity is observed in disease states, including cancer. Numb is an adaptor protein that functions in regulating endocytic trafficking events. Numb was originally identified in Drosophila as an asymmetrically localized cell fate determinant, and was subsequently found to be conserved in vertebrates. In mammalian polarized epithelial cells, Numb is distributed asymmetrically along the basolateral membrane domain. The work herein describes phosphorylation of Numb by the Par complex protein, atypical Protein Kinase C (aPKC), as a means of regulating membrane localization and asymmetric distribution of Numb. A mutant of Numb that cannot be phosphorylated by aPKC accumulates on the plasma membrane and localizes to both apical and basolateral membranes. In aPKC-depleted cells, endogenous Numb is unable to achieve polarized distribution and localizes around the entire cell cortex. We demonstrate that this mechanism is conserved in Drosophila as mutation of the corresponding phosphorylation sites disrupts Numb asymmetric localization in dividing sensory organ precursor cells. In polarized epithelial cells, one function of Numb is to promote epithelial morphology when cells are challenged with external stimuli that disrupt cell-cell adhesion. For example, depletion of Numb results in enhanced sensitivity of cells to lose cell-cell contacts when treated with calcium chelating agents. Loss of Numb potentiates hepatocyte growth factor (HGF)-induced lamellipodia formation and cell dispersal – early steps in epithelial-mesenchymal transition (EMT). In Numb-depleted cells, Rac1-GTP loading is enhanced, which corresponds with increased rate in loss of cell-cell adhesion and increased lamellipodia formation, following depletion of extracellular calcium and HGF stimulation, respectively. Together, this work identifies a mechanism that regulates polarized distribution of Numb and provides insight into its function in polarized epithelial cells.

Cell polarity is fundamental to numerous cellular processes including migration, molecular transport, and cell division. The establishment and organization of polarity is crucial to the maintenance of cellular homeostasis in mammalian systems. Deregulation of cell polarity is observed in disease states, including cancer. Numb is an adaptor protein that functions in regulating endocytic trafficking events. Numb was originally identified in Drosophila as an asymmetrically localized cell fate determinant, and was subsequently found to be conserved in vertebrates. In mammalian polarized epithelial cells, Numb is distributed asymmetrically along the basolateral membrane domain. The work herein describes phosphorylation of Numb by the Par complex protein, atypical Protein Kinase C (aPKC), as a means of regulating membrane localization and asymmetric distribution of Numb. A mutant of Numb that cannot be phosphorylated by aPKC accumulates on the plasma membrane and localizes to both apical and basolateral membranes. In aPKC-depleted cells, endogenous Numb is unable to achieve polarized distribution and localizes around the entire cell cortex. We demonstrate that this mechanism is conserved in Drosophila as mutation of the corresponding phosphorylation sites disrupts Numb asymmetric localization in dividing sensory organ precursor cells. In polarized epithelial cells, one function of Numb is to promote epithelial morphology when cells are challenged with external stimuli that disrupt cell-cell adhesion. For example, depletion of Numb results in enhanced sensitivity of cells to lose cell-cell contacts when treated with calcium chelating agents. Loss of Numb potentiates hepatocyte growth factor (HGF)-induced lamellipodia formation and cell dispersal – early steps in epithelial-mesenchymal transition (EMT). In Numb-depleted cells, Rac1-GTP loading is enhanced, which corresponds with increased rate in loss of cell-cell adhesion and increased lamellipodia formation, following depletion of extracellular calcium and HGF stimulation, respectively. Together, this work identifies a mechanism that regulates polarized distribution of Numb and provides insight into its function in polarized epithelial cells.

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

No description available.

Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF to study the effect of different levels of endogenous CTGF on osteoblast cytoskeleton reorganization and motility. Our assays showed enhanced cell adhesion, spreading and Rac expression in CTGF OE osteoblasts, while in CTGF KO osteoblasts, cell adhesion, spreading and Rac expression were significantly decreased. In contrast, CTGF OE osteoblasts that showed high adhesion and spreading, exhibited diminished cell motility and low levels of RhoA expression, while KO cells migrated quickly and expressed high levels of RhoA. Together, these experiments establish CTGF as an adhesion protein for osteoblasts; they demonstrate that the alpha-v beta1 integrin is a functional signaling receptor for CTGF; they confirm that osteoblast differentiation is enhanced when cultured on CTGF matrix through activation of regulatory signaling molecules; and finally, these experiments establish a role for CTGF in the regulation of small RhoGTPases expression, which in turn implies a significant role for CTGF in cell cytoskeleton reorganization and motility. / Cell Biology