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Treloar, Katrina K. "Mathematical models for collective cell spreading." Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/86960/1/Katrina_Treloar_Thesis.pdf.
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Collective cell spreading is frequently observed in development, tissue repair and disease progression. Mathematical modelling used in conjunction with experimental investigation can provide key insights into the mechanisms driving the spread of cell populations. In this study, we investigated how experimental and modelling frameworks can be used to identify several key features underlying collective cell spreading. In particular, we were able to independently quantify the roles of cell motility and cell proliferation in a spreading cell population, and investigate how these roles are influenced by factors such as the initial cell density, type of cell population and the assay geometry.
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Promwikorn, Waraporn. "Regulation of gene expression and cell cycle progression by cell shape." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250316.
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Jin, Hua. "The role of Abl tyrosine kinase in cell spreading." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3274697.
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Thesis (Ph. D.)--University of California, San Diego, 2007.<br>Title from first page of PDF file (viewed October 5, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Streicher, Pia. "Studying integrin-mediated cell spreading using a biomimetic system." Paris 6, 2008. http://www.theses.fr/2008PA066668.
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L'adhésion cellulaire dépendant des intégrines a été étudiée grâce à un système modèle réaliste. Nous avons développé une méthode basée sur celle initialement mise au point pour d'autres protéines dans l'équipe de P. Bassereau. Elle consiste à reconstituer l'intégrine αIIbβ3 dans des protéoliposomes (0. 1 -0. 2 µm de diamètre), puis à électroformer les vésicules géantes à partir des protéoliposomes partiellement séchés. Le succès de la reconstitution a été vérifié en analysant l'incorporation de la protéine et son activité biologique. La dynamique de l'adhésion ces vésicules sur des surfaces couvertes par le fibrinogène a été étudiée en détail. Nous avons identifié trois régimes et nous avons comparé les données expérimentales du régime 1) et 3) avec des prédictions théoriques. Dans les deux régimes, l'adhésion est limitée par la diffusion des ligands jusqu'à la zone adhésive.
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Vo, Brenda. "Novel likelihood-free Bayesian parameter estimation methods for stochastic models of collective cell spreading." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/99588/1/Brenda_Vo_Thesis.pdf.
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Biological processes underlying skin cancer growth and wound healing are governed by various collective cell spreading mechanisms. This thesis develops new statistical methods to provide key insights into the mechanisms driving the spread of cell populations such as motility, proliferation and cell-to-cell adhesion, using experimental data. The new methods allow us to precisely estimate the parameters of such mechanisms, quantify the associated uncertainty and investigate how these mechanisms are influenced by various factors. The thesis provides a useful tool to measure the efficacy of medical treatments that aim to influence the spread of cell populations.
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Redmann, Anna-Lena. "Kinetics of cell attachment and spreading on hard and soft substrates." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290385.
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A very important aspect for the functioning of an organism is that cells adapt their behaviour to external stimuli. They continuously interact with their environment, and biochemical and physical cues can activate cellular signalling, which leads to changes in cell behaviour such as proliferation and shape. Understanding cells' interactions with their environment is also important for understanding diseases. For example mechanosensing, which is the sensing of the cell's mechanical environment, has been associated with cancer development. In order for a cell to be able to sense its mechanical environment, it needs to form attachments to the environment. In my thesis, I have worked on three different tasks: the development of a new measurement technique and the study of initial cell adhesion and of cell spreading. When a cell from suspension first comes into contact with a substrate, it forms initial attachment bonds with proteins on the substrate surface. These bonds are mediated through integrins, which are transmembrane heterodimers, binding to the cell's environment on one side and to the cell's cytoskeleton on the other side. I study this initial cell attachment by measuring the force needed to detach cells, called cell adhesion strength. For these experiments I built a detachment device, which allows the detachment of cells from a substrate by vibrating the substrate in liquid. The device combines cell incubation, detachment and imaging. I measured the dependence of initial integrin bond formation on external factors such as incubation temperature and substrate stiffness. Once initial integrin bonds are formed, many different proteins are recruited to the adhesion site in order to form stronger adhesions. Amongst these proteins are signalling proteins, which direct the behaviour of the cell as a whole. One of the first cellular reactions to a substrate after initial integrin binding is cell spreading. This can be seen by the cell changing its shape from spherical to dome-like on the substrate. Because cell spreading is a very early response of a cell to a substrate, the onset time of spreading can be used as a quantitative measure for the time it takes the cell to sense a substrate and signal shape change. In my work, I look at the distribution of the time of initial cell spreading in a population of cells. I measure this distribution under different growth conditions such as pH, change of incubation medium from DMEM to PBS, substrate stiffness and incubation temperature. In my detachment experiments, I observe that vibration accelerates cell spreading in those cells which remain on the substrate. This is a connection between the detachment experiments and the cell spreading experiments and it shows how cells react to external forces. By changing the medium temperature in the cell detachment and cell spreading experiments, I am able to analyse the kinetics of these two processes. I use a signalling network model to analyse the internal cellular signalling path that leads from a spherical to a spread cell.
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Moore, Edward Andrew. "Cell attachment and spreading on physical barriers used in periodontal guided tissue regeneration /." Oklahoma City : [s.n.], 2002. http://library.ouhsc.edu/epub/theses/Moore-William-A.pdf.
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Gill, Amritpal Singh. "Development of a Novel Single-Cell Attachment and Spreading Platform Utilizing Fused-Fiber Nanonets." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/73504.
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Initial attachment to the extracellular matrix (ECM) and consequent spreading is a necessary process in the cell cycle of which little is known. Cell spreading has been well-recognized in 2D systems, however, the native fibrous ECM presents cells with 3D biophysical cues. Thus, using suspended fibers as model systems, we present the development of a novel platform (Cell-STEPs) capable of capturing cell attachment dynamics and forces from the moment a cell in suspension contacts the fiber. Cell-STEPs comprises of a custom glass-bottom petri dish with a lid to deliver a constant supply of CO2 to maintain pH. Fibrous scaffolds are attached in the dish to allow cellular investigations over extended periods of time. We find that cell-fiber attachment occurs in three progressive phases: initial attachment of cell to fiber (phase 0), rapid drop in circularity (phase 1), and increase in cell spread area (phase 2). Furthermore, using iterative inverse methods, forces involved in cell spreading through deflection of fibers were estimated. Our findings provide new insights in attachment biomechanics, including initial sensing and latching of cell to fiber with a negligible or protrusive force, followed by rapid loss in circularity through protrusion sensing at nearly constant spread area and minimal force generation, transitioning to a final phase of increased contractile forces until spread area and force saturation is observed. Also, anisotropic spreading of cells on single and two-fibers are closely related, while cells attached to several fibers take longer and spread isotropically. The Cell-STEPs platform allows, for the first time, detailed interrogations in the discrete and orchestrated adhesion steps involved in cell-fibrous matrix recognition and attachment along with simultaneous measurements of forces involved in cell attachment.<br>Master of Science
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Messmer-Blust, Angela F. "Murine Guanylate-Binding Protein-2: An interferon-induced GTPase that inhibits cell adhesion, cell spreading and MMP-9 expression." University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1263394455.
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Tse, Kathy Wan-Kei. "The role of Pyk2 and FAK in B cell migration, adhesion, and spreading." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/25041.
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The ability of the B cell receptor (BCR) to stimulate integrin-mediated adhesion, and induce cytoskeletal reorganization and cell spreading enhances the ability of B cells to bind and respond to antigens (Ag). The proper localization and trafficking of B cells in the secondary lymphoid organs are also critical for B cells to encounter Ags and to be activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that have been shown to regulate cell adhesion, morphology, and migration. However, their functions in B cells are not clear. The overall hypothesis of this thesis was that Pyk2 and FAK are downstream targets of BCR, integrin, and chemokine receptor signaling, and that they are involved in B cell morphological regulation, migration, and adhesion. I showed that the BCR and integrins collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, modifications that increase the kinase activity of Pyk2 and FAK. Activation of the Rap1 GTPase is critical for BCR-induced integrin activation and for BCR-induced reorganization of the actin cytoskeleton and I showed that inhibition of Pyk2 and FAK function by either gene knockdown or the use of chemical inhibitors impaired B cell spreading.
Marginal zone (MZ) B cells are innate-like B cells that are responsible for T cell-independent responses to microbial pathogens. The proper localization of MZ B cells is dependent on integrated migration and retention signals provided by the stromal cells in the spleen. Because MZ B cells are not found in Pyk2-/- mice, I hypothesized that Pyk2 and FAK are involved in MZ B cell retention in the spleen. I showed that Pyk2 and FAK are required for MZ B cell migration and that Pyk2 is required for integrin-dependent adhesion in response to chemoattractant stimulation. Moreover, I found that FAK is involved in chemokine-induced Akt phosphorylation in MZ B cells.
In summary, Pyk2 and FAK are downstream targets of the Rap GTPases and play a key role in regulating B cell morphology, migration, and adhesion.
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Mearns, Bryony Megan BABS UNSW. "Transglutaminase II: an integrator of fibroblast adhesion pathways in wound healing." Awarded by:University of New South Wales. BABS, 2006. http://handle.unsw.edu.au/1959.4/24166.
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Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.
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Ferro, Valerie Anne. "The role of endothelial cells in promoting adhesion, spreading and migration of B16F10 cells." Thesis, University of St Andrews, 1989. http://hdl.handle.net/10023/14067.
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For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must adhere and spread on the vascular endothelium before they can migrate through it to form secondary growths in the tissue beneath. In this study an in vitro assay was developed to study the behavourial interactions between B16F10 cells and Bovine aortic endothelial cells. It was hypothesized that molecules synthesized by the endothelial cells may be involved in the mediation of the adhesion, spreading and migration events and hence that such molecules may possibly be involved in the process of haematogenic metastasis. Endothelial derived extracts were obtained from the cell surface and from conditioned medium. The extracts were tested for their adhesion promoting abilities in a quick dot blot adhesion assay. To verify that these molecules promoted adhesion, antibodies were raised against the extracts. Partial characterisation of the molecules was achieved using SDS-PAGE and immunoprobing. The extracts were also tested for their spreading and migration promoting properties. An attempt was made to block the adhesion, spreading and migration events using antibodies directed against components of the extracts. Clearly, if endothelial-derived molecules are involved in metastasis, then preventing the mediation of adhesion, spreading and migration may ultimately have relevance in the clinical situation.
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Kovari, Daniel T. "Investigations of the spreading and closure mechanisms of phagocytosis in J774a.1 macrophages." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54882.
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Phagocytosis is the process by which cells engulf foreign bodies. It is the hallmark behavior of white blood cells, being the process through which those cells ingest and degrade pathogens and debris. To date a large amount of research has focused on documenting the existence and role of biochemical components involved with phagocytosis. Scores of signaling molecules have been implicated in the complex signal cascade which drives the process. These molecules are small (typically no larger than 5 nanometers) and operate in a crowded, chemically “noisy,” environment, yet they coordinate the cell's activity over comparatively expansive distances (as large as 20 micrometers). How these molecular processes scale-up to coordinate the activities of the cell over such massive distances is largely unknown. Using a planar analog of phagocytosis termed “frustrated phagocytosis,” we experimentally demonstrate that phagocytosis occurs in three distinct phases: initial cell-antigen binding, symmetric spreading, and late-stage contraction. Initial binding and symmetric spreading appears to be both mechanically and chemically similar to the quasi-universal cellular behaviors of adhesion and migration. Adhesion and migration have received extensive attention from the biophysics community in recent years. Leveraging these similarities, we adapt the biomechanical frameworks used in models of migration to phagocytosis. We show that macroscopic properties such as a cell's effective viscosity and membrane cortical tension can be used to model cell behavior during phagocytosis. Our experiments reveal that late-stage contraction distinguishes frustrated phagocytosis from other spreading behaviors. This contraction is myosin dependent. Additionally we demonstrate, for the first time, that late-stage contraction corresponds with formation of a contractile F-actin belt. Based on the dynamic contraction model (DC) developed to explain actin structure during cell migration we propose a DC model of phagocytosis which posits that contractile belt formation is the result of a late-stage myosin activity coupled with F-actin.
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Sasse, Ramona [Verfasser], and Benedikt [Akademischer Betreuer] Wirth. "Mutual information based parameter extraction for spreading cell colonies / Ramona Sasse ; Betreuer: Benedikt Wirth." Münster : Universitäts- und Landesbibliothek Münster, 2021. http://d-nb.info/1240763557/34.
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Lai, Jacqueline Cheuk-Yan. "Involvement of CD45 in early thymocyte development." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/3416.
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CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored.
The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development.
The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading.
The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified.
Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.
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Xu, Feng, Satoko Ito, Michinari Hamaguchi, and Takeshi Senga. "Disruption of Cell Spreading by the Activation of MEK/ERK Pathway is Dependent on AP-1 Activity." Nagoya University School of Medicine, 2010. http://hdl.handle.net/2237/14175.
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Hinoue, Atsushi. "Disruption of actin cytoskeleton and anchorage-dependent cell spreading induces apoptotic death of mouse neural crest cell cultured in vitro." Kyoto University, 2005. http://hdl.handle.net/2433/144698.
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Zeller, Kathrin Stephanie. "Integrin Signaling in Cell Adhesion and Mechanotransduction : Regulation of PI3K, AKT, and ROS." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-170267.
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Integrins are a family of conserved cell surface receptors found throughout the animal kingdom. They comprise 24 dimers in mammals, and regulate a number of processes including cell survival, differentiation, and migration. These complex cellular responses involve processes such as cell attachment, spreading, and various signaling pathways, which in turn depend on the composition of the extracellular environment, on its mechanical properties, and involved integrin types. This thesis focuses on identifying molecules that signal downstream of integrins and how integrin-induced signals may differ dependent on the type of mechanical stimulus that is given. In Paper I, we show that cell spreading and the activation of AKT is regulated by the catalytic PI3K isoform p110α. An intact β1 integrin cytoplasmic tail and actin polymerization was needed for spreading, whereas the presence of FAK or SRC, or the interaction between p110α and RAS was dispensable. Paper II reports that the RICTOR-mTOR complex (TORC2) acts as the kinase downstream of β1 integrins in order to phosphorylate AKT on Ser473, which was functionally linked to cell survival. β1 integrins activated both AKT1 and AKT2, but seemed to prefer AKT2. The investigation of several receptor types with regard to their requirement of TORC2, PAK, and ILK for AKT Ser473 phosphorylation revealed that different kinds of receptors engage specific enzyme combinations depending on cell type and context. In the third paper, we demonstrate that adhesion- and mechanical stretch-induced integrin signaling lead to divergent protein phosphorylation patterns, and that most signals from cell adhesion were not dependent on intracellular contractility. This indicates that integrin ligand binding and mechanical stretch induce signaling via distinct mechanisms. Reactive oxygen species (ROS) derived from different cellular sources modulated these responses. Stretching primarily induced phosphorylation of ERK1/2, and this signal was markedly increased by a derivative of the antioxidant ascorbate and extracellularly administered catalase. The robust AKT phosphorylation in response to adhesion was almost completely abolished with an inhibitor targeting mitochondrial ROS, whereas phosphorylation levels were only marginally affected in stretch assays. Similar results were obtained with siRNA knock-down of a critical subunit of ROS-producing NADPH oxidases.
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Huang, Yunjie. "ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION". UKnowledge, 2015. http://uknowledge.uky.edu/biochem_etds/20.
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Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets.
Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.
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Stefansson, Anne. "Mechanisms of Integrin Signal Transduction." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8221.
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<p>Integrins are a protein family of cell surface receptors, expressed in all cell types in the human body, except the red blood cells. Besides their importance in mediating physical connections with the surrounding environment, the integrin family members are also vital signalling mediators. They have no intrinsic kinase activity; instead the signals are transduced through conformational changes. </p><p>In this thesis, work is presented which is focused on molecular mechanisms of integrin signal transduction. The signal transduction was first studied from a structural point of view, determining the transmembrane domain borders of a few selected integrin family members and ruling out a signalling model involving a “piston-like” movement. </p><p>Then, downstream signalling events involved in the beta1 integrin-induced activation of Akt via the PI3kinase family were characterized. Our results identify a novel pathway for PI3K/Akt activation by beta1 integrins, which is independent of focal adhesion kinase (FAK), Src and EGF receptor. Furthermore, both beta1 integrins and EGF receptors induced phosphorylation of Akt at the regulatory sites Thr308 and Ser473, but only EGF receptor stimulation induced tyrosine phosphorylation of Akt.</p><p>Finally, signals from beta1 integrins underlying the morphologic changes during cell spreading were studied. A rapid integrin-induced cell spreading dependent on actin polymerisation was observed by using total internal reflection fluorescence (TIRF) microscopy. This integrin-induced actin polymerisation was shown to be dependent on PI3K p110alpha catalytic subunit and to involve the conserved Lys756 in the beta1-integrin membrane proximal part.</p>
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Schuler, Jeffrey Thomas. "Forward Chemical Genetics Drug Screen Yields Novel Proteases and Proteolytic Inhibitors of HGF–induced Epithelial–Mesenchymal Transition." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6257.
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Hepatocyte Growth Factor (HGF)–induced Epithelial–Mesenchymal Transition (EMT) is a complex cellular pathway that causes epithelial cell scattering by breaking cell–cell contacts, eliminating apical–basal polarity, and replacing epithelial markers and characteristics with mesenchymal markers. Early EMT events include a brief period of cell spreading, followed by cell compaction and cell–cell contact breaks. A forward chemical genetics drug screen of 50,000 unique compounds measuring HGF–induced cell scattering identified 26 novel EMT inhibitors, including 2 proteolytic inhibitors. Here, we show that B5500–4, one of the EMT inhibitors from the screen, blocks HGF–induced EMT by a predicted blocking of the protease furin, in addition to secondarily blocking Beta–Secretase (BACE).We also show that MMP–12 and MMP–9 are required for HGF–induced EMT to progress. MMP–12 is required for cell contraction, and its inhibition produces a continuous cell spreading phenotype.We also demonstrate that both furin and BACE activity are required for HGF–induced EMT to proceed, but that they are involved in separate pathways. We show that BACE inhibition leads to a failure of cell spreading in early EMT, and that EphA2 is a member of this pathway. We also demonstrate that it is likely BACE2, and not BACE1 that is responsible for early cell spreading. Furin is also required for HGF–induced cell scattering, but does not play a role in the cell spreading process. These findings highlight the importance of proteolytic activity at the earliest stages of HGF–induced EMT.
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Rudnicki, Mathilda Sophia. "Cell sensing on strain-stiffening substrates is not fully explained by the nonlinear mechanical property." Digital WPI, 2012. https://digitalcommons.wpi.edu/etd-theses/216.
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Cells respond to their mechanical environment by changing shape and size, migrating, or even differentiating to a more specialized cell type. A better understanding of the response of cells to surrounding cues will allow for more targeted and effected designs for biomedical applications, such as disease treatment or cellular therapy. The spreading behavior of both human mesenchymal stem cells (hMSCs) and 3T3 fibroblasts is a function of substrate stiffness, and can be quantified to describe the most visible response to how a cell senses stiffness. The stiffness of the substrate material can be modulated by altering the substrate thickness, and this has been done with the commonly-used linearly elastic gel, polyacrylamide (PA). Though easy to produce and tune, PA gel does not exhibit strain-stiffening behavior, and thus is not as representative of biological tissue as fibrin or collagen gel. Fibroblasts on soft fibrin gel show spreading similar to much stiffer linear gels, indicating a difference in cell stiffness sensing on these two materials. We hypothesize cells can sense further into fibrin and collagen gels than linear materials due to the strain-stiffening material property. The goal of this work is to compare the material response of linear (PA) and strain-stiffening (fibrin, collagen gel) substrates through modulation of effective stiffness of the materials. The two-step approach is to first develop a finite element model to numerically simulate a cell contracting on substrates of different thicknesses, and then to validate the numerical model by quantifying fibroblast spreading on sloped fibrin and collagen gels. The finite element model shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 10µm. Due to the strain-stiffening behavior, the nonlinear material experiences a more drastic increase in effective stiffness at these low thicknesses. Experimentally, the gradual response of cell area of HLF and 3T3 fibroblasts on fibrin and collagen gels is significantly different (p<0.05) from these cell types on PA gel. This gradual increase in area as substrate thickness decreases was not predicted by the finite element model. Therefore, cell spreading response to stiffness is not explained by just the nonlinearity of the material.
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Song, Jaekyung Cecilia. "Protein Kinase C-δ and Protein Kinase C-ε Cooperatively Enhance Epithelial Cell Spreading via Transactivation of Epidermal Growth Factor Receptor and Actin-Dependent Phosphorylation of Focal Adhesion-Associated Proteins". Cincinnati, Ohio : University of Cincinnati, 2005. http://www.ohiolink.edu/etd/view.cgi?acc%5Fnum=ucin1132198567.
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Thesis (Ph. D.)--University of Cincinnati, 2005.<br>Title from electronic thesis title page (viewed Sept. 13, 2007). Includes abstract. Keywords: Protein Kinase C; Cell spreading; Cell migration; Epithelial Cells; Epidermal Growth Factor Receptor; Transactivation; Focal Adhesion; Actin; Focal Adhesion Kinase; Src; Paxillin Includes bibliographical references.
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Hatzikirou, H., K. Böttger, and A. Deutsch. "Model-based Comparison of Cell Density-dependent Cell Migration Strategies." Cambridge University Press, 2015. https://tud.qucosa.de/id/qucosa%3A39048.
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Here, we investigate different cell density-dependent migration strategies. In particular, we consider strategies which differ in the precise regulation of transitions between resting and motile phenotypes. We develop a lattice-gas cellular automaton (LGCA) model for each migration strategy. Using a mean-field approximation we quantify the corresponding spreading dynamics at the cell population level. Our results allow for the prediction of cell population spreading based on experimentally accessible single cell migration parameters.
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Wilson, Cameron. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins." Queensland University of Technology, 2005. http://eprints.qut.edu.au/16096/.
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Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were conducted on a limited range of surface textures. Titanium samples were ground to produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.
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Wilson, Cameron John. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins." Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16096/1/Cameron_Wilson_Thesis.pdf.
Full textAbstract:
Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were
conducted on a limited range of surface textures. Titanium samples were ground to
produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.
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Willett, Mark. "Investigating the localisation and trafficking of the mammalian eIF4F complex in NIH3T3 fibroblasts during cell spreading, adhesion and normal growth conditions." Thesis, University of Sussex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437454.
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Sandmann, Rabea [Verfasser], Sarah [Akademischer Betreuer] Köster, and Florian [Akademischer Betreuer] Rehfeldt. "Blood Platelet Behavior on Structured Substrates : From Spreading Dynamics to Cell Morphology / Rabea Sandmann. Betreuer: Sarah Köster. Gutachter: Sarah Köster ; Florian Rehfeldt." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1078420084/34.
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Nguyen, Beth P. "Integrin alpha 6 beta 4 ligation to laminin 5 and phosphoinositide 3-OH kinase define differences in alpha 3 beta 1-laminin 5 and alpha 2 beta 1-collagen spreading : implications for epidermal wound repair /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/9286.
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Cavalher, Felicia Peterson. "Caracterização funcional das isoformas de splicing do gene ADAM23." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-10052013-130119/.
Full textAbstract:
A ADAM23 é uma glicoproteína transmembrana pertencente à família ADAM (A Disintegrin and Metalloprotease) que apresenta a estrutura protéica típica dos membros desta família, mas não possui atividade de metaloprotease. O gene ADAM23 apresenta três isoformas de splicing, α, β e γ, que codificam proteínas com porções C-terminais distintas. As isoformas α e β codificam proteínas com domínios transmembranas diferentes, enquanto γ provavelmente consiste em uma isoforma secretada ou citoplasmática de ADAM23. Foi demonstrado que o gene ADAM23 está epigeneticamente silenciado em tumores de mama de estágios mais avançados e que seu silenciamento está associado a um maior risco de desenvolvimento de metástases e a um pior prognóstico. Recentemente, foi descrito que a proteína ADAM23 interage diretamente com a integrina αVβ3 na linhagem tumoral de mama MDA-MB-435, sendo capaz de modular seu estado conformacional, controlando sua ativação. Utilizando RNAi, observou-se que o silenciamento completo do gene ADAM23 (i.e., as três isoformas) aumenta os níveis de αVβ3 em conformação ativa na superfície das células MDA-MB-435, promovendo um incremento de sua capacidade migratória e adesiva. No presente trabalho, avaliamos por reações de amplificação em tempo real o perfil de expressão das três isoformas de splicing do gene ADAM23 em cinco tecidos normais (mama, cólon, cérebro, próstata e pâncreas) e em doze linhagens tumorais derivadas destes tecidos. Observamos diferenças nos níveis de expressão das isoformas em todas as amostras avaliadas, tanto dentro de uma determinada amostra, como quando comparamos tecidos normais entre si ou com linhagens tumorais. A isoforma γ é a mais expressa em todos os tecidos normais (exceto em cérebro) e em todas as linhagens tumorais. Em tecido normal de mama e de próstata e nas doze linhagens tumorais, ADAM23α é a segunda isoforma mais expressa, sendo β a menos expressa. Constatamos também que a fração representada por cada isoforma, em relação à expressão total do gene ADAM23, está alterada nas linhagens tumorais, em comparação aos tecidos normais correspondentes. Com o intuito de elucidar a função das isoformas de ADAM23 separadamente, utilizamos shRNAs (short hairpin RNAs) para reduzir a expressão de cada isoforma de modo individual e específico na linhagem tumoral MDA-MB-435, e avaliamos seu efeito na proliferação, na morfologia, na adesão e no espraiamento celular. Verificamos que a redução da expressão da isoforma γ aumentou significativamente a taxa de proliferação das células MDA-MB-435 cultivadas em modelo tridimensional. Demonstramos também que ADAM23γ participa da regulação da morfologia e da capacidade de espraiamento das células MDA-MB-435 em condições padrão de cultivo (i.e., meio de cultura completo e placas não-sensibilizadas com substratos) e em componentes específicos da matriz extracelular, como fibronectina, colágeno I e matrigel. A isoforma α também está envolvida no controle da morfologia e do espraiamento da linhagem MDA-MB-435, porém, de modo distinto da isoforma γ. Já ADAM23β não interfere na morfologia das células MDA-MB-435 e tem efeito marginal no espraiamento celular apenas em condições padrão de cultivo. Em conjunto, nossos resultados demonstram que as isoformas de ADAM23 são diferencialmente expressas em tecidos normais e tumorais, e exercem funções biológicas distintas.<br>ADAM23 is a transmembrane glycoprotein that belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins and exhibits the typical protein structure of the family members, but it doesn\'t have metalloprotease activity. The ADAM23 gene has three splicing isoforms, α, β and γ, that code for proteins with different C-terminal regions. Isoforms α and β code for proteins with different transmembrane domains, while γ probably constitute a secreted or cytoplasmatic isoform of ADAM23. It has been demonstrated that the ADAM23 gene is epigenetically silenced in advanced stage breast tumors and that its silencing is associated with a higher risk of developing metastases and with a worse prognosis. Recently, it was described that ADAM23 protein interacts directly with αVβ3 integrin in the breast tumor cell line MDA-MB-435, modulating its conformational state and controlling its activation. Using RNAi, it was observed that the complete silencing of ADAM23 gene (the three isoforms) raises the levels of αVβ3 in its active conformation in the surface of MDA-MB-435 cells, promoting an increase in its migratory and adhesive capacity. In the present work, we evaluated by real time PCR the expression pattern of the three splicing isoforms of ADAM23 gene in five normal tissues (breast, colon, brain, prostate and pancreas) and in twelve tumor cell lines derived from these tissues. We observed differences in the expression levels of the three isoforms in all samples, either within a specific sample or comparing normal tissues among them or with tumor cell lines. Isoform γ has the highest expression in all normal tissues (except for brain) and in all tumor cell lines evaluated. In breast and prostate normal tissues and in all tumor cell lines, ADAM23α is the second most expressed isoform, while β is the less expressed. We also noticed that the ratio represented by each isoform, relative to the total expression of ADAM23 gene, is altered in the tumor cell lines, compared to the corresponding normal tissues. With the aim to elucidate the function of ADAM23 isoforms separately, we used shRNAs (short hairpin RNAs) to reduce the expression of each isoform specifically in the MDA-MB-435 tumor cell line, and studied its effects in proliferation, morphology, adhesion and cell spreading. We observed that the reduced expression of isoform γ significantly increased the proliferation rate of MDA-MB-435 cells cultivated in tridimensional system. Also, we demonstrated that ADAM23γ participates in the regulation of cell morphology and spreading of MDA-MB-435 cells, both in standard culture conditions (cell culture media with fetal serum and in plates not sensitized with substrates) and in specific components of extracellular matrix, such as fibronectin, collagen type I and matrigel. Isoform α is also involved in the control of morphology and spreading of MDA-MB-435 cell line, although in a distinct manner from isoform γ. ADAM23β doesn\'t interfere in the morphology of MDA-MB-435 cells and plays a discrete role in cell spreading only under standard culture conditions. Together, our results demonstrate that ADAM23 isoforms are differently expressed in normal and tumoral tissue, and play distinct biological roles.
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Copley, LaRae. "Investigation of the function of myotubularin through the examination of protein-protein interactions and exclusion of MTMR1 as a frequent cause of X-linked myotubular myopathy." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1080146560.
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Venkova, Larisa. "Régulation du volume cellulaire en réponse aux déformations." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS396/document.
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Dans les tissus, les cellules génèrent et sont soumises en permanence à des forces mécaniques. Les perturbations biochimiques à l'intérieur des cellules, ainsi que les altérations de leur environnement mécanique peuvent modifier l'équilibre physiologique et mener à des pathologies, comme le cancer. Bien que les propriétés mécaniques puissent être modifiées à l'échelle du tissus, la compréhension de la mécanique au niveau de la cellule unique demeure importante. En particulier, la différenciation, la migration des cellules immunitaires et le caractère invasif d'un cancer dépendent fortement des propriétés mécaniques des cellules uniques. Les déformations mécaniques peuvent induire un changement de la surface et du volume cellulaires. Nous nous intéressons particulièrement à la régulation du volume cellulaire chez les cellules mammifères dans le contexte de déformations à différentes échelles de temps. Jusqu'à présent, la régulation du volume dans ce contexte n'a été que très peu étudiée, en raison de la difficulté d'obtention de mesures précises, et du fait que le volume de la cellule est généralement considéré comme constant. Nous avons développé une méthode de mesure du volume cellulaire reposant sur l'exclusion de fluorescence, qui nous permet d'effectuer des mesures de volume précise au niveau de la cellule unique. Dans cette étude, nous nous sommes concentrés sur la régulation du volume cellulaire au cours de l'étalement dynamique sur un substrat (échelle de temps : minutes). Nous avons démontré qu'il existe différents régimes de régulation du volume lors de l'étalement : les cellules réduisent, augmentent ou ne modifient pas leur volume, en fonction de l'état du cortex d'actomyosine et de la vitesse d'étalement. Nous avons constaté que les cellules s'étalant plus vite ont tendance à perdre davantage de volume. Notre hypothèse est que lors d'une extension rapide de lamellipode dépendante d'Arp2/3, l'actine tire sur la membrane et génère une tension et l'activation de transport ionique, s'accompagnant d'une perte de volume compensatoire. L'inhibition de la polymérisation de l'actine ou de sa ramification dépendante d'Arp2/3 réduit la vitesse d'étalement et ainsi la perte de volume. Nous avons ensuite montré que l'inhibition de la contractilité augmente la vitesse d'étalement et la perte de volume. Cependant, l'inhibition d'Arp2/3 dans des cellules à faible contractilité conduit à un étalement rapide sans perte de volume. En effet, l'inhibition d'Arp2/3 induit des bulles de membranes, une déformation rapide n'induirait donc pas de perte de volume car la cellule peut relâcher la tension en dépliant la membrane. Nous avons également montré que la régulation du volume en réponse à une compression mécanique rapide (échelle de temps : millisecondes) indépendante de l'adhérence dépend également de l'état du cortex d'actomyosine. Les cellules perdent jusqu'à 30% de leur volume lorsqu'elles sont confinées, car la membrane plasmique est attachée au cortex et ne peux pas être dépliée en réponse à l'augmentation de la tension. La perturbation du cortex d'actine induit le détachement de la membrane et limite la perte de volume. Enfin, nous avons montré que la réponse du volume à un choc osmotique (échelle de temps : secondes) est plus que complexe que décrite dans la littérature. Nos données indiquent qu'au niveau de la cellule unique, la réponse initiale du volume au changement de l'osmolarité extérieure n'est pas un processus passif uniforme. En utilisant la technique du choc osmotique, nous avons également confirmé que les cellules ont un large excès de membrane repliée dans des réservoirs. Nos résultats montrent que le volume et l'aire cellulaires sont couplés par l'homéostasie de la tension de surface, et, étant donné que les déformations induisent une augmentation de la tension de surface, elles conduisent à des modifications du volume et de l'aire de la cellule<br>The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area
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Gustavsson, Anna. "Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling." Doctoral thesis, Umeå : Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-183.
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Deveraux, Solenne. "Modélisation de la mécanique de la cellule et son noyau dans le cadre de la migration confinée." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLC063/document.
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Les cellules possèdent une capacitéfondamentale à leur survie : la migration. Del’embryogénèse aux métastases tumorales, lorsde la migration, les cellules doivent se faufiler àtravers des mailles sub-nucléaires pour atteindreleur localisation cible. Pour ce faire, ellespeuvent adapter leur mode locomotion ou leurspropriétés mécaniques à l’environnement quiles entoure. La cellule ainsi que son noyausubissent d’importantes déformations lors de lamigration en milieu confiné. Le noyau étantl’organelle le plus gros et le plus rigide, il peutlimiter la capacité migratoire de la cellule. Sespropriétés mécaniques sont donc décisives afinde migrer à travers un environnement complexe.Dans la littérature, les signaux moléculairespendant le processus migratoire ont étéabondamment décrits, mais la modélisationmécanique d’une cellule en migration peut-ellenous révéler de nouveaux éléments sur lesmécanismes sous-jacents ?La migration cellulaire est un procédé d’unecomplexité mécano-biologique telle, que tous sesaspects ne peuvent être modélisés à ce jour. Nousen choisissons donc trois que nousdévelopperons ici. Nous nous intéressonsd’abord à l’interaction mécanique entre le noyauet le cytoplasme lors d’une constriction de lacellule, puisque la plasticité du noyau sembleavoir un rôle primordial. Nous étudions ensuitele chimneying, un mode migratoire sansadhésion dont le mécanisme repose sur desforces de friction couplées à la poroélasticité ducytoplasme. Enfin, les substrats avec des micropilierssont depuis peu utilisés pour étudier lespropriétés mécaniques de la cellule et de sonnoyau, mais la mécanique de ce phénomène estpeu comprise. Nous modélisons le processus parlequel le noyau se déforme afin de déterminer s’ilest poussé ou tiré dans l’espace inter-piliers<br>One of the fundamental properties incells is their ability to migrate. Fromembryogenesis to tumor metastasis, migratingcells must overcome mechanical obstacles toreach their intended location, squeezing throughsub-cellular and sub-nuclear gaps. It can be doneby adapting the locomotion mode to thesurrounding environment or by tuning the cell’sown mechanical properties. Migrating in aconfined space leads to intensive deformation ofthe cell and thus its nucleus. Being the largestand stiffest organelle, the nucleus can hamperthe migratory process. Its mechanical propertieshence are key to a successful migration in acomplex environment. Molecular signals behindcell migration have been extensively studied inthe literature, but what can computationalmechanics modeling unveil about themechanisms behind cell migration?Cell migration is such a complex mechanobiologicalprocess, that all aspects cannot bemodeled at once for now. We choose threedistinct situations for in-depth study. We firstseek to understand the mechanical interplaybetween the nucleus and the cytoplasm, sincenuclear plasticity seems decisive for migrationthrough sub-nuclear gaps. Second, weinvestigate the mechanics of chimneying, aspecific confined migratory mode, in which noadhesion in needed for the cell to move forward.Poroelasticity, coupled with friction, appears asthe key to successful locomotion. Eventually,cell spreading on micro-pillared substrates hasrecently been developed to study nuclearmechanical properties. The mechanism behindthis process being however unclear, we designeda large deformation model to determine whetherthe nucleus is being pushed or pulled in theinter-pillars gaps
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Monteiro, Eric. "Contributions aux méthodes numériques pour traiter les non linéarités et les discontinuités dans les matériaux hétérogènes." Phd thesis, Université Paris-Est, 2010. http://tel.archives-ouvertes.fr/tel-00601050.
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Motivé par l'étude de tissus biologiques, ce travail contribue aux développements d'outils numériques permettant de prédire la réponse mécanique de matériaux hétérogènes non linéaires dans lesquels les énergies d'interfaces deviennent prépondérantes. Ainsi, une méthode d'homogénéisation multi échelle combinée à une technique de réduction de modèle basée sur la décomposition orthogonale aux valeurs propres est proposée dans un cadre thermique et hyperélastique. Les énergies d'interfaces entre les différentes phases des composites sont décrites par un modèle d'interface cohérent et prises en compte numériquement par une approche liant la méthode des éléments finis étendus et la méthode level-set. Une étude de l'étalement d'une cellule vivante entre deux lamelles fixes est ensuite réalisée. Les deux modèles utilisés pour les simulations montrent que l'assemblage cortex d'actine-membrane plasmique ne joue qu'un rôle minime dans la réponse mécanique cellulaire
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Salazar, Montano Ylia [Verfasser]. "Microenvironmental Th9 and Th17 lymphocytes induce epithelial-mesenchymal transition in lung cancer cells thereby promoting metastatic spreading / Ylia Maria Salazar Montano." Gießen : Universitätsbibliothek, 2020. http://d-nb.info/1223461866/34.
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Salazar, Montano Ylia Maria [Verfasser]. "Microenvironmental Th9 and Th17 lymphocytes induce epithelial-mesenchymal transition in lung cancer cells thereby promoting metastatic spreading / Ylia Maria Salazar Montano." Gießen : Universitätsbibliothek, 2020. http://d-nb.info/1223461866/34.
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Henninger, Nils. "Inhibiting Axon Degeneration in a Mouse Model of Acute Brain Injury Through Deletion of Sarm1." eScholarship@UMMS, 2017. http://escholarship.umassmed.edu/gsbs_diss/900.
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Traumatic brain injury (TBI) is a leading cause of disability worldwide. Annually, 150 to 200/1,000,000 people become disabled as a result of brain trauma. Axonal degeneration is a critical, early event following TBI of all severities but whether axon degeneration is a driver of TBI remains unclear. Molecular pathways underlying the pathology of TBI have not been defined and there is no efficacious treatment for TBI.
Despite this significant societal impact, surprisingly little is known about the molecular mechanisms that actively drive axon degeneration in any context and particularly following TBI. Although severe brain injury may cause immediate disruption of axons (primary axotomy), it is now recognized that the most frequent form of traumatic axonal injury (TAI) is mediated by a cascade of events that ultimately result in secondary axonal disconnection (secondary axotomy) within hours to days.
Proposed mechanisms include immediate post-traumatic cytoskeletal destabilization as a direct result of mechanical breakage of microtubules, as well as catastrophic local calcium dysregulation resulting in microtubule depolymerization, impaired axonal transport, unmitigated accumulation of cargoes, local axonal swelling, and finally disconnection. The portion of the axon that is distal to the axotomy site remains initially morphologically intact. However, it undergoes sudden rapid fragmentation along its full distal length ~72 h after the original axotomy, a process termed Wallerian degeneration.
Remarkably, mice mutant for the Wallerian degeneration slow (Wlds) protein exhibit ~tenfold (for 2–3 weeks) suppressed Wallerian degeneration. Yet, pharmacological replication of the Wlds mechanism has proven difficult. Further, no one has studied whether Wlds protects from TAI. Lastly, owing to Wlds presumed gain-of-function and its absence in wild-type animals, direct evidence in support of a putative endogenous axon death signaling pathway is lacking, which is critical to identify original treatment targets and the development of viable therapeutic approaches.
Novel insight into the pathophysiology of Wallerian degeneration was gained by the discovery that mutant Drosophila flies lacking dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously recapitulated the Wlds phenotype. The pro-degenerative function of the dSarm gene (and its mouse homolog Sarm1) is widespread in mammals as shown by in vitro protection of superior cervical ganglion, dorsal root ganglion, and cortical neuron axons, as well as remarkable in-vivo long-term survival (>2 weeks) of transected sciatic mouse Sarm1 null axons. Although the molecular mechanism of function remains to be clarified, its discovery provides direct evidence that Sarm1 is the first endogenous gene required for Wallerian degeneration, driving a highly conserved genetic axon death program.
The central goals of this thesis were to determine (1) whether post-traumatic axonal integrity is preserved in mice lacking Sarm1, and (2) whether loss of Sarm1 is associated with improved functional outcome after TBI. I show that mice lacking the mouse Toll receptor adaptor Sarm1 gene demonstrate multiple improved TBI-associated phenotypes after injury in a closed-head mild TBI model. Sarm1-/- mice developed fewer beta amyloid precursor protein (βAPP) aggregates in axons of the corpus callosum after TBI as compared to Sarm1+/+ mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phosphorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after TBI. Strikingly, whereas wild type mice exhibited a number of behavioral deficits after TBI, I observed a strong, early preservation of neurological function in Sarm1-/- animals. Finally, using in vivo proton magnetic resonance spectroscopy, I found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1-/- mice compared to controls immediately following TBI. My results indicate that the Sarm1-mediated prodegenerative pathway promotes pathogenesis in TBI and suggest that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after TBI.
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Holmqvist, Kristina. "The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells." Doctoral thesis, Uppsala University, Department of Medical Cell Biology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3943.
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<p>Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels. Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors. My aim has been to identify the role of Shb in FGF- and VEGF-signalling in endothelial cells. Shb was found to be phosphorylated in a Src-dependent manner upon both FGF- and VEGF-stimulation. This was confirmed using fibroblasts overexpressing temperature sensitive v-Src. Furthermore, Shb-induced cell spreading on collagen of immortalised brain endothelial (IBE) cells was also Src-dependent. FGF stimulation led to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding domain of Shb. IBE cells overexpressing wild-type or R522K Shb (inactive SH2 domain) displayed increased FAK activation on collagen.</p><p>The SH2-domain of Shb was found to bind to tyrosine 1175 in the VEGFR-2 in a phosphotyrosine dependent manner using PAE cells expressing VEGFR-2. Furthermore, by use of siRNA, Shb knock-down experiments revealed that Shb regulates FAK activity, cellular migration and stress fiber formation in response to VEGF stimulation of VEGFR-2. In summary, Shb binds to both FGFR-1 and VEGFR-2 and regulates the activity of FAK and thereby stress fiber formation and cellular migration, which are necessary for formation of new blood vessels. IBE cells with an inactive SH2 domain of Shb displayed disorganised formation of tubular structures in the tube formation assay, while overexpression of wild-type Shb led to accelerated tubular morphogenesis.</p><p>Taken together, my data show that the adaptor protein Shb plays an important role in the process angiogenesis, in response to angiogenic tyrosine kinase receptors, by interacting with FAK and regulating spreading, stress fiber formation and cellular migration.</p>
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Abounit, Saïda. "Molecular and cellular mechanism of α-synuclein assemblies transfer between neuronal cells : role of Tunneling nanotubes". Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112063.
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Les synucléionopathies représentent un groupe de maladies neuro-dégénératives incurables du système nerveux central. Elles regroupent entre autres la maladie de Parkinson, l’atrophie multi-systématisée et la maladie à corps de Lewy. Toutes ces maladies se caractérisent par un déclin progressif des fonctions motrices, cognitives, comportementales et autonomiques. La mal-conformation et l’agrégation de la protéine α-synuclein qui forme des inclusions intraneuronales sont des éléments communs à toutes les synucleinopathies. Ces inclusions portent le nom de corps de Lewy et se forment dans des neurones ou cellules gliales appartenant à des régions cérébrales spécifiques. Elles sont vraisemblablement à l’origine de la perte progressive de neurones dans certaines parties du cerveau. Dans le cas de la maladie de Parkinson et dans d’autres maladies neuro-dégénératives, il a été démontré que la pathologie se propage anatomiquement d’une manière spécifique et prévisible au niveau cérébrale. Ceci suggère donc que la progression de la maladie est étroitement liée au transfert des agrégats d’α-synucléine. Ce procédé est très similaire à celui impliqué dans la maladie du prion qui elle en revanche est infectieuse. Par ailleurs, des inclusions neuronales d’α-synucléine ont été identifiées dans des neurones dopaminergiques d’origine fœtaux qui avaient été transplanté dans des cerveaux de patients parkinsoniens. Cette étude a permis d’envisager pour la première fois la possibilité de la transmission d’inclusions d’α-synucléine entre les neurones. Bien que de nombreuses études aient démontré la propagation d’α-synucléine in vitro et in vivo, le mécanisme permettant ce transfert n’est pas clairement établi. Par conséquent, ma thèse s’attache à étudier le mécanisme de transfert d’assemblages d’α-synucléine (i.e., oligomères et fibrilles). Dans un premier temps, j’ai apporté la preuve que les assemblages d’α-synucléine transfèrent de manière efficace entre les cellules neuronales via les Tunneling nanotubes (TNT). Les TNT sont définis comme étant des ponts membranaires riches en F-actine et permettant de connecter physiquement le cytoplasme de cellules éloignées. Au niveau subcellulaire, j’ai démontré que les assemblages d’α-synucléine qui transfèrent se trouvent dans des lysosomes. En revanche, après le transfert, ces assemblages se retrouvent libres dans le cytoplasme. J’ai également mis en évidence qu’à la suite du transfert, permis par les TNT, les fibrilles d’α-synucléine sont capables de recruter et d’induire l’agrégation de l’α-synucléine soluble afin de perpétuer le processus d’agrégation à l’infinie. Ces résultats indiquent que les TNT peuvent représenter un moyen efficace permettant le transfert d’assemblages d’α-synucléine. Cette découverte offre de nouvelles opportunités pour le développement de nouveaux agents neuro-protectifs contre la propagation des synucléinopathies<br>Synucleinopathies are a group of fatal neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, characterized by a chronic and progressive decline in motor, cognitive, behavioral, and autonomic functions. The hallmark of these diseases is the misfolding and aggregation of α-synuclein protein accumulating into intracellular inclusions Lewy bodies in neurons and glial cells which leads to the loss of neurons in specific brain regions. In the case of Parkinson’s disease and other neurodegenerative diseases, the pathology was shown to progress throughout the brain in a specific and predictable manner suggesting that the progression of the diseases is linked to the transfer of aggregated α-synuclein that is reminiscent of prion diseases that are infectious. Importantly, upon transplantation of fetal dopaminergic neurons in the brain of Parkinson’s patients, neuronal inclusions were found in the grafted neurons strongly suggesting that α-synuclein inclusions could transmit between neurons. While several studies showed α-synuclein propagation in vitro and in vivo the mechanism of intercellular transfer remains elusive. The aim of my thesis was to study the mechanism of transfer of α-synuclein assemblies (i.e., oligomers and fibrils) involved in Parkinson’s pathogenesis. I evidenced that α-synuclein assemblies transferred efficiently via tunneling nanotubes (TNT), F-actin based membranous bridges connecting the cytoplasm of remote cells. I demonstrated that, at the sub-cellular level, the transferred α-synuclein assemblies were specifically confined in lysosomes and that upon transfer a large amount of α-synuclein was found free in the cytosol of acceptor cells. Finally, I showed that after TNT-mediated transfer α-synuclein fibrils recruited and seeded the aggregation of the soluble α-synuclein protein in order to perpetuate aggregation. The identification of TNT as an efficient means of α-synuclein transfer opens new avenues to the development of novel therapies targeting the spreading into the brain of amyloidogenic proteins involved in neurodegenerative diseases
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廖乾廷. "numerical simulation of cell spreading and protrusion." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/43941693301365920356.
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Choo, Lai Mun, and 朱麗雯. "The function of human MOB2 in cell spreading in fibrosarcoma cells." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/40238958167824388773.
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碩士<br>東海大學<br>生命科學系<br>99<br>Cell spreading is an initial mechanism for cell migration which plays a vital role in cancer development. Cell spreading has been shown to act as one of the key regulating steps between static and metastatic transition of a cancer cell. Hence, by identifying regulatory networks controlling cell spreading, it may provide valuable information and therapeutic strategies for preventing tumor metastasis. Both cell spreading and cell migration involve actin polymerization at the leading edge of plasma membrane follow by cell retraction at the rear end of cells. The molecular mechanisms in regulating cell spreading and cell migration have been extensively studied but remain unclear. Studies from yeast, Drosophila to mammalian cells have shown that MOB2 protein plays an important role in controlling the cell morphology changes by affecting cell polarity and rearrangement of actin cytoskeleton. Currently there is no research done to study the function of Mob2 in cell spreading and cell migration. In this study, we identified hMOB2 protein which plays a significant role in promoting cell spreading in HT1080 human fibrosarcoma cells. Our results showed that hMOB2 was detected at the leading edge of migrating HT1080 human fibrosarcoma cell. To study whether hMOB2 was involved in cell motility, we downregulated hMOB2 expression using RNA interference and found that cell spreading was delayed in HT1080 cells. In addition, we observed that overexpression of hMOB2 enhanced cell spreading in HT1080 cells and enhanced its accumulation at the leading edge. Furthermore, to determine the possible functional domain in cell motility, we successfully generated A107G, Y110A point mutated hMOB2 stable cell lines. Over-expressed point mutated hMOB2 expression delayed cell spreading and suppressed its accumulation at the leading edge. These observations suggested that hMOB2 affects cell spreading by regulating its expression at leading edge. No significant difference was observed in the migration rate between the different HT1080 cell populations when the percentage of gap closure was determined. However, over-expressed wild type hMOB2 induced broad lamellipodial structures and moved as a coherent group when compared with parent cells. These studies provided additional information on the molecular mechanisms which control cell spreading.
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Tu, Hsin-Mou, and 杜信謀. "Finite Element Analysis of Bio-cell Spreading and Phagocytosis." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/74476416189886343995.
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Cheng-EnLu and 呂承恩. "Routability-Driven Post-Optimization of Placement Using Cell Spreading Technique." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/59839231735619670683.
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Wong, Nelson Kwan Yin. "CD44 signaling in T cells leading to cell spreading and its regulation by CD45." Thesis, 2006. http://hdl.handle.net/2429/18417.
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CD44 is a widely expressed adhesion molecule that has been implicated in
mediating cellular signaling. In this dissertation, the signaling pathway initiated by CD44
that leads to actin rearrangement and cell spreading in T cells was studied. The results
indicate that engagement of CD44 leads to actin-dependent clustering of this adhesion
molecule. CD44 clustering then initiates the recruitment of signaling proteins, including
the Src-family kinases (SFK) Lck and Fyn, phosphatidylinositol-3 kinase (PI3K), and
non-receptor related focal adhesion kinase Pyk2. The outcome of actin rearrangement
and cell spreading resultant of CD44 signaling was determined by CD45, a
transmembrane tyrosine phosphatase. In the absence of CD45, elongated cell spreading
and F-actin polymerization along the longitudinal axis of the cells were observed. This
was accompanied by the accumulation of tyrosine phosphorylation at CD44
microclusters. Moreover, Pyk2 phosphorylation was also associated with the CD44-
induced elongated cell spreading. The CD44-induced signaling pathway that leads to
Pyk2 phosphorylation and elongated cell spreading involves the activities of SFK,
phospholipase C (PLC), and phosphatidylinositode-3-kinase (PI3K), as well as actin
polymerization and calcium mobilization. These signaling components identified are
also involved in T-cell receptor (TCR)/CD3 signaling, which is initiated during T cell
activation; however, the CD44 pathway was distinct. This was supported by the
observations that LAT (linker for activation of T cells) phosphorylation and ERK
activation were not involved in CD44 signaling, while these events are observed in
TCR/CD3 signaling. In the presence of CD45, BW5147 T cells formed F-actin rings and
spread round on immobilized CD44 antibody. The formation of F-actin structures in
CD45+ BW5147 T cells also required Src family kinase activity. Results from confocal
microscopy studies suggest that CD45 was recruited to CD44 microclusters and this was
associated with the prevention of sustained Lck activation.
Overall, this work shows that CD44 mediates signals that result in actin
reorganization and cell spreading in T cells; however, the outcome of these events is
regulated by CD45. This is likely due to the negative regulatory effect of CD45 on SFK
during CD44 signaling.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate
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Hsiu-MeiChen та 陳秀梅. "The Role of Integrin-β1 in Leiomyomal Cell Spreading and Proliferation". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/7a65eq.
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博士<br>國立成功大學<br>基礎醫學研究所<br>101<br>Uterine leiomyoma is a benign tumor derived from uterine smooth muscle layer. It is one of the most common gynecological diseases in women of reproductive age. Symptoms of leiomyoma include pelvic pressure, abnormal uterine bleeding, and infertility. Due to the presence of excessive amount of extracellular matrix (ECM), leiomyoma is also called fibroid. Besides contributing to the huge volume in leiomyoma, ECM may play an important role in the development of leiomyoma. Integrins are the major ECM-receptor on cell surface. Binding of ECM to integrins leads to cell adhesion and initiates a cascade of signaling for cell spreading, migration, proliferation, and differentiation. However, the role of integrin-ECM in leiomyomal pathogenesis remains largely unclear. The aim of this study is to elucidate the pathological processes of leiomyoma regulated by integrin, especially integrin β1. Results from my study showed that the expression of integrin β1 in leiomyomal cells was greater than that in myometrium. Although knockdown of integrin β1 did not affect cell adhesion on fibronectin, laminin, and collagen, it significantly reduced cell spreading and stress fiber formation in leiomyomal cells, which leads to a decrease in focal adhesion and contraction force. Furthermore, knockdown of integrin β1 inhibited the phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, resulting in reduction of cyclin D1 and cyclin A expression and leiomyomal cell proliferation. Treatment with rhodostomin, a small protein that disrupts interaction of ECM and integrins, reduced the expression of cyclin D1 and cyclin A and cell proliferation. Finally, I demonstrated that overexpression of integrin β1 is induced by transforming growth factor βs (TGF βs). Taken together, these data demonstrate that overexpression of integrin β1 in leiomyomal cells is induced by TGF β1 and β3. Interaction of ECM and overexpressed integrin β1 enhances initiates a cascade of signaling transduction pathways to increase cell spreading and proliferation. Results of this study also provide novel information in designing a treatment regimen by targeting the disruption of ECM-integrin interaction using rhodostomin or similar RGD-containing small molecules.
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Chen, Ying-Ti, and 陳映荻. "Evidence for the Involvement of Thrombomodulin in Cell Spreading and Migration." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/87516128485901231231.
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碩士<br>中國醫藥大學<br>醫學檢驗生物技術學系碩士班<br>98<br>Thrombomodulin（TM）, a cell surface-expressed glycoprotein, is critical for thrombin-mediated activation of protein C. Recent evidence has revealed that TM also has protein C- and thrombin-independent physiological function. In this study, we found that depletion of TM could regulate the morphology of the human keratinocyte cell line, HaCaT. These cells showed an enhanced migratory activity. Phalloidin staining revealed that TM-knockdown induced lamellipodia protrusion at the free edges of colonies. To investigate the mechanism of TM regulated the cytoskeleton reorganization, we detected intersectin-1 complexed with TM, intersectin-1 by using a combination of immunoprecipitation（IP）system and liquid chromatography tandem mass spectrometry （LC-MS/MS）. Co-immunoprecipitation and co-localization studies showed that TM interacted with actin around the adherens junction. In addition, a decrease in TM causes a translocation of intersectin-1 from cytoplasma to the lamellipodia. Our data suggest that loss of endogenous TM expression results in predisposes to acquisition of mesenchymal characteristics. The morphology-regulated and motility-suppressive functions of TM in HaCaT are mediated in part via interaction with intersectin-1.
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Chang, Chu-Lung, and 張主龍. "L-Caldesmon Dependent Mechanical Changes of Cell Spreading and Adhesion Force in Osteoclastogenesis." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/84570975147666939765.
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碩士<br>國立中興大學<br>生命科學系所<br>105<br>Caldesmon (CaD) is an actin regulator, expressing two isoforms in smooth muscle cells (h-CaD) and non-muscle cells (l-CaD), respectively. Both isoforms of CaD are capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization. However, little is known about the role of l-CaD in the control of cell-cell fusion in osteoclastogenesis. To determine the functional role of the increased l-CaD expression in osteoclastogenesis, Raw264.7 cells transfected with fusion DNA constructs containing EGFP and l-CaD and followed by RANKL induction were investigated. In comparison with no transfection, overexpressing l-CaD significantly increased osteoclastogenic gene expressions for CTSK, c-fos, NFATc1, and beta 3 integrin in cells. Analyses with TRAP staining also indicated that l-CaD overexpression could accelerate osteoclast (OC) cell differentiation. On the other hand, si l-CaD decreased the potential for RANKL-induced OC differentiation. To determine whether l-CaD is involved in modulating cell-cell profusion in osteoclastogenesis, Atomic Force Microscopy (AFM) was used to resolve the mechanical changes of cell spreading and adhesion in RANKL-induced OCs with or without l-CaD overexpression. Cell spreading is determined by height scanning area, where the percentage of outmost and peripheral areas to total scanning area represents the tendency of cell spreading out of the center. In addition, cell-cell fusion was measured by the cell fusion index. Our data obtained showed that overexpression of l-CaD increased the cell-spreading and adhesion force that might facilitate cell-cell fusion into multinucleate OCs. Clearly, the regulation of l-CaD expression is important event for osteoclastogenesis.
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Ferguson, Caroline. "Mechanical Forces Regulate Cartilage Tissue Formation by Chondrocytes via Integrin-mediated cell Spreading." Thesis, 2009. http://hdl.handle.net/1807/19322.
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In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.
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Bastien, Jayson I. L. "Endosomal membrane dynamics underlying cell spreading: A role for the small GTPase Arf6." Thesis, 2012. https://doi.org/10.7916/D8R78N8R.
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Cell migration is an orchestrated and highly coordinated multi-step process that is central to the development and maintenance of multicellular organisms. Dysregulated migration however, is associated with pathological states such as tumor formation and metastasis; thus a clear understanding of the molecular mechanisms that drive this process is critical to the development of counteracting therapeutics. Cell migration and adhesion-dependent cell spreading share a number of features. For example, both processes rely on the activation of mechanisms for the coordinated spatial and temporal assembly/disassembly of focal adhesions, as well as mechanisms controlling actin rearrangements and directed vesicular trafficking. Actin remodeling and vesicular trafficking events are in turn, implicated functions of a variety of small GTPases of the Ras superfamily, which include the Rho and Arf subfamilies. Thus towards efforts of further characterizing the molecular pathways that drive cell spreading, I pursued aims to examine the role of a specific member of the Arf subfamily Arf6, in this process. In contrast to other studies which have primarily used constitutively active or dominant negative mutants of Arf6 to study its cellular function, we employed mouse genetics. In this system, mouse embryonic fibroblasts (MEFs) were derived and immortalized from mice genetically manipulated for the acute deletion of Arf6 using a tamoxifen inducible Cre/loxP recombination system. Acute deletion of Arf6 in these MEFs resulted in a kinetic delay in transferrin recycling as well as in cell spreading. The spreading delay correlated with reduced trafficking of cholera toxin B-labeled intracellular membranes to the plasma membrane. Cholera toxin-B labels the ganglioside GM1, which is enriched in lipid rafts. These specialized membrane domains are thought to serve as signaling hubs bearing many proteins that in turn, mediate trafficking steps required for cell spreading/migration. I further report that the trafficking of these specialized membranes to the plasma membrane involves the retromer complex, a coat-like multi-protein complex primarily known for mediating retrograde transport from endosomes to the trans-Golgi network. Altogether, my studies have confirmed genetically, an involvement of Arf6 in cell spreading and raft trafficking, and established a link between these membrane microdomains and the retromer complex. In separate studies, I have also investigated the role of phospholipase D2 (PLD2) in endocytic trafficking and found that similarly derived cultures exhibit alterations in the expression levels of various trafficking related proteins as well as defects in transferrin and epidermal growth factor receptor trafficking. These results suggest a role for PLD2 and possibly its enzymatic product phosphatidic acid, in these events.