Browsing by Subject "Cell Movement"
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Item Characterization of QSEA and QSED in the Quorum Sensing Cascade of Enterohemorrhagic Escherichia Coli(2005-08-11) Sharp, Faith Christine; Sperandio, VanessaEnterohemorrhagic E. coli O157:H7 (EHEC) is an enteric pathogen that has been implicated in many outbreaks of bloody diarrhea worldwide. EHEC senses its environment through quorum sensing, a mechanism by which bacteria use chemical signals, termed autoinducers, to regulate key genes. In the gastrointestinal tract, EHEC responds to AI-3 produced by the endogenous gastrointestinal microbial flora and epinephrine/norepinephrine produced by the host to regulate expression of virulence genes. In particular, EHEC utilizes quorum sensing to regulate virulence processes, including motility and chemotaxis and the production of attaching and effacing lesions. Motility and chemotaxis processes are controlled under the complex flagella regulon in EHEC. The expression of genes within the locus of enterocyte effacement (LEE) results in the production of the characteristic attaching and effacing lesions created as a result of production of a type III secretion apparatus. The LEE1 operon encodes for a transcriptional activator, Ler, which is responsible for the activation of other genes within the pathogenicity island. The virulence mechanisms that enable EHEC to circumvent the host defenses and compete for essential nutrients for survival are controlled by several transcriptional regulators, many of which are controlled in response to quorum sensing in EHEC. Quorum sensing E. coli regulator A, QseA, recently was described as a transcription factor that is activated via quorum sensing in EHEC. QseA, which belongs to the family of LysR transcription factors, activates the transcription of LEE1/ler directly; therefore, QseA indirectly activates the expression of other genes within the LEE pathogenicity island. The work in the first specific aim of this thesis examines the specific regulation of the LEE1/ler promoter by QseA through the use of genetic and biochemical methods. Quorum sensing E. coli regulator D, QseD, is a previously uncharacterized transcription factor that is repressed through quorum sensing in EHEC. QseD appears to play a significant role in the overall quorum sensing cascade, as it is involved in the modulation of both motility and type III secretion in EHEC. The second aim of this thesis is to study the role of QseD modulation in quorum sensing signaling in EHEC.Item The Developmental Transcription Factor Neurogenic Differentiation 1 in Migration and Survival of Neuroendocrine Carcinomas(2013-03-12) Osborne, Jihan K.; Minna, John D.; White, Michael A.; Johnson, Jane E.; Cobb, Melanie H.Differentiation and determination of cell fate during embryogenesis is decided by a collection of transcription factors, including the large family of basic-helix-loop-helix (bHLH) transcription factors. Neurogenic differentiation 1 (NeuroD1) is a bHLH transcription factor responsible for neuronal and neuroendocrine islet differentiation during development of the central and peripheral nervous systems and the pancreas respectively. NeuroD1 has also been shown to be anomalously expressed in a subset of aggressive neuroendocrine tumors. Initial examination of microarray data revealed that subsets of aggressive small cell lung cancers (SCLC) and certain neuroendocrine non-small cell lung cancers (NSCLC-NE) have high expression of NeuroD1 as compared to human bronchial epithelial cells (HBEC) and other non-small cell lung cancers (NSCLC). In several neuroendocrine carcinomas, including subsets of neuroendocrine lung cancers, melanoma and some undifferentiated prostate cell lines, NeuroD1 directly induces the expression of signaling pathways that support survival and migration. Loss-of-function/gain-of-function studies in cell lines from each of these cancer types reveled that NeuroD1 regulates both survival and the migration potential of neuroendocrine carcinomas that have lost or mutated p53. Subsequently, loss of p53 has been shown to up-regulate NeuroD1 expression in non-transformed HBECs and cancer cells with neuroendocrine features. The actions of NeuroD1 are carried out by downstream targets which include the signaling molecules, the tyrosine kinase, tropomyosin-related kinase B (TrkB), and the adhesion molecule, neural cell adhesion molecule (NCAM), and the ion channels, the nicotinic acetylcholine receptor subunit cluster of α3, α5, and β4 (nAChR), to name a few. Impaired expression of each of these downstream targets mirrors the various phenotypes associated with loss of NeuroD1. These findings ultimately have implications for the potential of NeuroD1 acting as a lineage-dependent oncogene in neuroendocrine carcinomas.Item Discovery and Characterization of the Polycomb Repressive Complex 1 of C. Elegans(2009-06-17) Karakuzu, Ozgur; Cameron, Scottunc-3 encodes the Caenorhabditis elegans homolog of the Olf-1/Early B cell factor family of transcription factors, which in vertebrates regulate development and differentiation of B lymphocytes, adipocytes, and cells of the nervous system. In the first chapter I analyze the role of unc-3 in determining the fates of neurons in ventral nerve cord (VNC). unc-3 mutants are uncoordinated in locomotion. I show that unc-3 represses a VC-like motor neuron program in the VA and VB motor neurons, which in wild-type animals control backwards and forwards locomotion, respectively. Our lab identified a physical interaction between UNC-3 and the C2H2 zinc finger transcription factor PAG-3, the mammalian homologs of which are coexpressed in olfactory epithelium and hematopoietic cells. Our data explain the locomotory defects of unc-3 mutants and suggest that interactions between unc-3 and pag-3 homologs in other species may be functionally important. The second chapter of the thesis is about the analysis of MIG-32 a RING protein similar to some Polycomb proteins that were identified in a yeast two hybrid screen with UNC-3 as bait. The Polycomb repression complex 2 (PRC2) methylates histone H3 lysine 27 at target genes to modify gene expression, and this mark is recognized by PRC1, which ubiquitylates histone H2A. In Caenorhabditis elegans, a complex of the MES-2, MES-3, and MES-6 proteins is functionally analogous to the PRC2 complex, but the functional analog of PRC1, and indeed whether C. elegans has such a complex, has been unclear. I describe here that MIG-32 is a homolog of BMI-1, a core component of PRC1. I also identify SPAT-3A as a homolog of Ring1B, a partner protein of BMI-1 in the PRC1 core complex. Mig-32 and spat-3 mutants have some defects that overlap with those of mes mutants. However, unlike the mes mutants, mig-32 and spat-3 mutants are fertile, despite lacking apparent H2A ubiquitylation. Migration and axon guidance of specific neurons were defective in mig-32 and spat-3 mutants. Our data suggest that mig-32 and spat-3 encode core components of a PRC1-like complex in C. elegans.Item The Functional Roles of Rho-Kinase and Matrix Metalloproteinases in Regulating Corneal Stromal Cell Mechanics in 3-D Collagen Matrices(2013-11-26) Zhou, Chengxin; Grinnell, Frederick; Petroll, W. Matthew; Luby-Phelps, Katherine; Tang, Liping; Alexandrakis, GeorgiosThe main focus of my research has been on understanding the biomechanical and biochemical mechanisms of cell-extracellular matrix (ECM) interactions during corneal wound healing, which may allow the development of new therapeutic strategies to promote corneal regeneration. Previous studies have established that the Rho GTPases play a central role in regulating the cytoskeletal changes associated with cell mechanical activity. A novel force monitoring system was successfully developed to investigate the role of Rho in corneal cell force generation in 3-D collagen matrices. Maximum tractional force generated by 9 million corneal fibroblasts in serum culture was around 265 Dynes. Inhibition of Rho kinase by Y-27632 induced a 69% force reduction. These results demonstrated that Rho/Rho kinase play a key role in mediating contractile force generation of corneal stromal fibroblasts in serum culture. I also investigated the functions of Rho GTPase signaling in corneal stromal fibroblast migration and cell-ECM interactions using a 3-D nested matrix construct. The experimental results showed that both the amount and the speed of corneal fibroblast migration and local collagen matrix reorganization were significantly inhibited by Y-27632. Following the inhibition, cells extended thinner dendritic processes into the outer matrix, and generated tractional forces at their leading edge. However, cells were unable to generate contractile forces needed to retract their tail and pull the cell body forward through the collagen matrix. I also studied the role of Matrix metalloproteinases (MMPs) in corneal cell mechanics, since these have been recognized as an influential component in extracellular matrix turnover and corneal repair. I first assessed the expression and collagenolytic activities of MMPs by primary corneal keratocyte in response to different signaling factors. I then studied the functions of MMPs in regulating keratocyte migration, cell-induced matrix contraction, and cell protrusive activity in 3-D collagen matrices. This study suggested that, in serum free PDGF culture, although collagenolysis was limited to a pericellular scale, primary corneal keratocytes utilized MMPs to facilitate cell migration, ECM contraction, cell spreading in 3-D collagen matrices. Thus MMPs may play a key role in facilitating cell-collagen matrix interactions by corneal keratocytes, without producing widespread disruption of corneal ECM structure.Item Identification of Developmental Signaling Pathways with a Novel Role in Regulating Zebrafish Primordial Germ Cell Migration(2013-07-01) Boldt, Clayton Ryan; Lum, Lawrence; Amatruda, James F.; Cobb, Melanie H.; Buszczak, MichaelNormal cell migration is critical for embryonic patterning, organ development and immune response. Primordial germ cell (PGC) migration in zebrafish has proven a valuable model for the study of cell motility. In zebrafish, PGC migration is guided principally by the chemokine Sdf-1a and its receptors Cxcr4b in germ cells, and Cxcr7b in somatic cells. While a role has also been suggested for PI3K signaling, the possible contribution of other signaling pathways in PGC migration is not fully understood. In this study, I used inhibitors of early developmental signaling pathways to identify those with a novel role in PGC migration. Among these compounds, the most significant effects were from the Tankyrase inhibitor IWR-1, which blocks β-catenin-dependent Wnt signaling. IWR-1 treatment during periods of active PGC migration results in fewer germ cells reaching the gonad. Treatment with the Porcupine inhibitor IWP-L6, which blocks Wnt production, did not result in substantial effects on PGC migration, suggesting that the effects of IWR-1 may not be dependent on β-catenin activity, but on the direct IWR-1 target, Tankyrase. Treatment with XAV939, an additional Tankyrase inhibitor, phenocopied the effects of IWR-1. Germ cell-targeted inhibition of Wnt signaling did not phenocopy the effects of IWR-1 and XAV939, arguing against a germ cell-autonomous role for Wnt signaling in PGC migration. We observed significant changes in the expression and patterning of sdf-1a, cxcr4b and cxcr7b following IWR-1 treatment, but not Wnt inhibition. Thus, I conclude that Tankyrase has an important role in patterning the PGC migratory environment during early development. I also sought to determine the eventual fate of ectopic PGCs in zebrafish. I created a transgenic line with GFP-labeled PGCs that has enabled us to follow ectopic germ cells during the first two weeks of development. Daily monitoring of ectopic PGCs revealed that they are not cleared, as in mice, but persist in their ectopic locations. In several instances, I observed ectopic germ cells undergoing morphological changes, followed by loss of GFP expression. The results of these observations lend credibility to the hypothesis that extragonadal germ cell tumors could arise from PGCs that do not migrate properly during development.Item PDGFR B Signaling in Mouse Epicardial and Mural Cells Influences Blood Vessel Remodeling(2009-06-15) Mellgren, Amy Marie; Tallquist, Michelle D.Platelet derived growth factor receptor beta (PDGFRbeta) is a receptor tyrosine kinase expressed in vascular smooth muscle cells (VSMC), which promotes proliferation and migration. We provide evidence of additional roles for the PDGFRbeta prior to the differentiation of VSMC. We show that PDGFRbeta, as well as PDGFRalpha, is expressed in epicardial and subepicardial mesenchymal cells, which are precursors for coronary VSMC. We demonstrate that PDGFRbeta-/- mice exhibit a lack of coronary VSMC and have disrupted endothelial vessels on the ventral surface of the heart; however, neither conditional ablation of the PDGFRbeta with an SM22 Cre Tg, which is expressed in differentiated VSMC, nor with a myocardinCre, which has an earlier expression profile and is believed to control VSMC differentiation, phenocopy the lack of coronary VSMC found in PDGFRbeta-/- mice. Further investigations into PDGFRbeta-/- mice revealed a defect in the function of the epicardium. The epicardium exhibited an altered cellular morphology and a decreased ability to migrate into the myocardium both in vivo and ex vivo. The decreased motility was associated with a nonpolarized distribution of actin and a lack of localization of Arp2/3 to the cell periphery. Moreover, these defects appeared to be dependent on the Src signaling pathway. This work thus establishes a novel in vivo role for the PDGFRbeta at a stage of coronary VSMC development during which the epicardium undergoes cytoskeletal rearrangement in order to efficiently migrate into the myocardium and form the mesenchymal precursors of coronary VSMC. In addition to this role in vasculogenesis, we demonstrate a role for the PDGFRbeta in angiogenesis. Using point mutations in PDGFRbeta we generated mice that possessed variations in the number of pericytes that were present in tissues, including the trachea and retina. We then utilized these mutant mouse lines to show that a decrease in pericytes affects the ability of the vasculature to respond to an angiogenic agent, Ang1. Moreover, this response is not secondary to hypoxia. This work emphasizes the value of targeting both VSMC and endothelial cells in therapies targeting vessel regeneration.Item Quantitative Analysis of Integrin Trafficking and Focal Adhesion Turnover to Study Integration of Early Endocytic Trafficking, Signaling and Migration(2019-01-22) Lakoduk, Ashley Marie; Danuser, Gaudenz; Ross, Theodora; Rosen, Michael K.; Schmid, SandraEarly endocytic trafficking is critical for regulating both receptor presence at the plasma membrane and cellular signaling. Clathrin-mediated endocytosis (CME) constitutes a major route of selective receptor internalization, yet little is knowing about how this process is regulated both temporally and spatially. We chose to investigate focal adhesion (FA) and beta-1 integrin turnover as a model system to better understand the spatial, temporal, and cargo-specific regulation of early endocytic trafficking. Importantly, the dynamic turnover of integrins and their associated adhesion complexes through endocytic and recycling pathways has emerged as key mechanism for controlling cancer cell migration and invasion. However, current tools available to study integrin trafficking do not provide adequate spatial information or can induce artifacts. Here, we report the generation and characterization of a neutral and monovalent antibody-based probe to study beta-1 integrin trafficking in cells. Using this tool, we report a novel regulatory mechanism of integrin turnover by a lipid kinase, PIPKI, and discover a mutant p53-driven endosomal signaling nexus that regulates beta-1 integrin recycling and cancer cell invasion. This work demonstrates the importance of the reciprocal crosstalk between early endocytic trafficking and receptor signaling in regulating both normal and tumor cell function.Item Quantitative Image Analysis in the Study of Neutrophil and Neutrophil-Like HL-60 Chemotaxis(2017-04-11) Zhang-Velten, Elizabeth Ren; Tu, Benjamin; Cobb, Melanie H.; Altschuler, Steven J.; Wu, Lani; Stull, James; Blount, PaulNeutrophils are fast-moving first responders of the innate immune system. External chemoattractant signals result in neutrophil polarization: the neutrophil forms a leading edge (front) which constantly protrudes and retracts actin-rich pseudopods, and a contractile myosin-enriched trailing edge (back) which is insensitive and directionally persistent. Previous work has suggested that polymerized actin and contractile actomyosin segregate to the neutrophil's morphological front and back, respectively, due to mutual inhibition. Beyond this initial establishment of spatially segregated domains, however, many questions remain unclear. In this work, I address two questions: (i) first, how do the front and back of the neutrophil demonstrate seemingly uncoupled behaviors despite these inhibitory links? (ii) and, second, at what fMLP concentration does neutrophil chemotaxis saturate, and how is this maximal concentration determined? With quantitative analysis of immunofluorescent fixed-cell images and live-cell migration videos, I demonstrate the role of microtubules in insulating the front and back modules of chemotactic neutrophils, and the role of ERK in driving neutrophil migration into maximal fMLP concentrations.Item Regulation of Cell Migration by WNK1(2012-12-06) Estrada, Armando, III 1980-; Taussig, Ronald; Albanesi, Joseph P.; Cobb, Melanie H.; Huang, Chou-LongCell motility is an immensely complex process that involves reorganization of the cytoskeleton, and consequent membrane deformation, triggered by a variety of motogenic stimuli, including growth and chemotactic factors, hormones, and elements of the extracellular matrix. My graduate research has focused on the regulation of cell motility by a serine/threonine protein kinase, WNK1 (With No lysine (K)1), so named because of the absence of a conserved lysine within the catalytic domain. Although WNK1 has been most thoroughly characterized for its role in controlling ion flux in the kidney, emerging evidence points to its essential participation in both intracellular membrane trafficking and overall cell movement. Having confirmed a previous observation that depletion of WNK1 in cultured cells interferes with wound closure in an established motility assay, I next sought to identify specific aspects of cytoskeleton remodeling that are impaired by reduction of WNK1 expression. The most dramatic effect of siRNA-mediated WNK1 depletion was an increase in the proportion of polymerized (F) actin. Surprisingly, this redistribution was accompanied by a corresponding increase in the proportion of active, unphosphorylated cofilin, an F-actin depolymerizing factor, most likely reflecting engagement of a feedback homeostatic mechanism. To understand the pathway(s) whereby WNK1 regulates stimulus-dependent actin dynamics, I examined three potential pathways known to impact cell motility: 1. Remodeling of subcellular cytoskeletal structures, including circular dorsal ruffles and stress fibers; 2. Activation of downstream effectors of phosphatidylinositol 3-kinase (PI3K), including Akt and LIMK1; 3. Regulation of proteins that control the cytoskeletal re-organization including the ERM proteins (Ezrin, Moesin, and Radixin), cofilin, and the Rho family of GTPases, including RhoA and Rac1.Item Roles of Myocardin-Related Transcription Factors in Muscle and Brain(2010-01-12) Mokalled, Mayssa H.; Olson, Eric N.Partnerships between DNA binding transcription factors and transcriptional cofactors govern gene transcription in various developmental and tissue contexts, particularly during cardiovascular and neuronal development. This dissertation aims at studying the in vivo relevance of the partnership between Serum response Factor (SRF) and its Myocardin Related Transcription Factor (MRTF) coactivators during development. I present here my studies on the functions of MRTFs during brain and muscle development. First, I show that MRTF-A and -B redundantly control neuronal migration and neurite outgrowth during brain development. Conditional deletion of these genes in the mouse brain disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. I also describe a previously unrecognized role for the MRTF/SRF pathway in the regulation of the Pctaire-1/Cdk5 kinase cascade to govern actin dynamics. I conclude that MRTFs function as essential coregulators of SRF to control actin dynamics during neuronal development via the Cdk5/Pctaire-1 kinase cascade. I also explore the role of MRTF-A and -B in cardiac development and function. Ablation of these MRTF genes in the embryonic heart causes` a range of cardiac defects, reflecting the sensitivity of cardiac function to MRTF gene dosage. Moreover, I show that the gene encoding Pctaire-1 kinase, whose functions in the heart are unknown, is also a target of MRTF/SRF signaling and a regulator of sarcomere assembly in the heart. Furthermore, by creating mice lacking the Myocardin related factor MASTR, I explore the in vivo developmental functions of MASTR. Germline deletion of MASTR alone does not cause any obvious defects in mice. However, deletion of MASTR in an MRTF-A null background causes perinatal lethality, which appears to be due to defective skeletal muscle growth and development. Thus, the results of my thesis research demonstrate that MRTFs are essential regulators of multiple developmental processes in brain, heart, and skeletal muscle. At the cellular level, MRTFs are essential regulators of the actin cytoskeleton. Disruption of MRTF functions, whether in neurons or in muscle cells, causes major cytoskeletal defects that impair brain and muscle development and function.Item Structural Analysis of Domain Swapping in the Protein Kinase: Crystal Structure of Human Ste20 OSR1 Kinase Domain(2007-12-18) Lee, Seung-Jae; Goldsmith, Elizabeth J.Ste20p (Sterile 20 protein) is a yeast MAP4K involved in the pheromoneresponsive MAPK cascade of the mating pathway. Recent studies reveal that its homologs in mammals, Drosophila melanogaster, Caenorhabditis elegans and other organisms constitute a large emerging group of protein kinases including 28 members in human. The Ste20p family has gained remarkable interest, due to the recent finding that it has various intracellular regulatory effects including the regulation of apoptosis and rearrangement of the cytoskeleton triggering cell-shape changes and cell motility. Moreover, from the viewpoint of structural biology, it is intriguing that the Ste20p family is characterized by the presence of a conserved kinase domain and a noncatalytic region of great diversity at various locations. Despite recent structural studies of Ste20p kinases such as PAK1 or TAO2, the mechanism of kinase regualtion in this family still remains to be investigated. Thus, in order to deepen our understanding of Ste20p from a structural viewpoint, the OSR1 kinase, which belongs to the GCK VI subfamily, was crystallized. In this dissertation, I present the crystal structure of the OSR1 kinase domain in an inactive conformation. This crystallographic result demonstrates the unexpected finding that the OSR1 kinase domain is dimerized in domain-swapped manner, which is a novel mode of protein-protein interaction in the protein kinase family. The detailed structural analysis shows that alpha EF helix and the P+1 loop region located in the activation segment were completely swapped between two monomers. The DFG magnesium binding loop and the N-terminal F-helix seem to function as anchors for the hinge loop, based upon the structural alignment with other domain-swapped Ste20p kinases, which were reported after I completed the OSR1 structure determination. Several Ste20p kinases also form domain-swapped dimers in a strikingly similar manner to OSR1.Item Unraveling the fundamentals of brain tumor cell migration(2015-08-14) Bachoo, Robert M.