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1
Mortara, R. A. "Microfilament-membrane interactions in isolated P815 filopodia." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372923.
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RAIMO, SERENA. "UNRAVELING A NEW ROLE OF TFEB IN FILOPODIA FORMATION." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/562675.
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The microphthalmia family (MITF, TFEB, TFE3, and TFEC) of transcription factors is emerging as global regulators of cancer cell survival and energy metabolism, both through the promotion of lysosomal genes as well as newly uncharacterized targets. During my Ph.D. thesis project, I revealed a new set of TFEB target genes that when activated could contribute to cell migration and invasiveness in cancer. During my work, I found that TFEB regulates the filopodial initiator's factors IRSp53 and EPS8 causing a change in cell shape and an increase in filopodia number that correlates with an augmented motility and invasiveness of the cell. On the contrary, depletion of TFEB and TFE3 leads to down-regulation of EPS8 and IRSp53, and a decrease of filopodia numbers. I confirmed the entire study in the Melanoma cell line (501Mel), a model of cancer, that are cells with a high degree of motility, showing that also in this system, there is an increase in the number of filopodia as well as of EPS8 and IRSp53 levels. This phenotype was completely reversed by depletion of MITF or TFEB and TFE3, demonstrating that the upregulation of these transcription factors could contribute to the invasive phenotype of melanoma cells.
Altogether these data revealed a new role of MITF transcription factors as regulators of a transcriptional program that could control metastatic cancer initialization.
3
Gauthier-Campbell, Catherine. "Regulation of filopodia dynamics is critical for proper synapse formation." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/722.
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Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts.
We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation.
We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process.
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Arstikaitis, Pamela. "The role of filopodia in the formation of spine synapses." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/32688.
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In the mammalian brain, excitatory (glutamatergic) synapses are mainly located on dendritic spines; bulbous protrusions enriched with F-actin. Dendritic filopodia are thin protrusions thought to be involved in the development of spines. However, limited evidence illustrating the emergence of spines from filopodia has been found. In addition, the molecular machinery required for filopodia induction and transformation to spines is not well understood. Paralemmin-1 has been shown to induce cell expansion and process formation and is concentrated at the plasma membrane, in part through a lipid modification known as palmitoylation. Palmitoylation of paralemmin-1 may also serve as a signal for its delivery to subcellular lipid microdomains to induce changes in cell morphology and membrane dynamics making it a candidate synapse-inducing molecule. Using live imaging as well as loss and gain-of-function approaches, our analysis identifies paralemmin-1 as a regulator of filopodia induction, synapse formation, and spine maturation. We show neuronal activity-driven translocation of paralemmin-1 to membranes induces rapid protrusion expansion, emphasizing the importance of paralemmin-1 in paradigms that control structural changes associated with synaptic plasticity and learning. Finally, we show that knockdown of paralemmin-1 results in loss of filopodia and compromises spine maturation induced by Shank1b, a protein that facilitates rapid transformation of newly formed filopodia to spines.
To investigate the role of filopodia in synapse formation, we contrasted the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. Expression of the palmitoylated protein motifs found in growth associated protein 43kDa, enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, siRNA knockdown of NLG-1, reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, reduced filopodia number, but increased the stabilization of the initial contact with axons. These results suggest that increased filopodia stability and not density may be the rate-limiting step for synapse formation.
5
Lee, Kwonmoo. "Self-assembly of filopodia-like structures on supported lipid bilayers." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/62648.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Physics, 2010.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 98-121).
Filopodia are finger-like protrusive structures of cells, comprised of actin bundles, which can serve as sensory organelles. To probe their pathway of assembly we have reconstituted filopodia-like structures (FLSs) by applying frog egg extracts to supported lipid bilayers containing phosphatidylinositol(4,5)bisphosphate, PI(4,5)P 2. The FLSs recapitulate important characteristics of filopodia - they assemble parallel actin bundles from the lipid membrane and they form in the presence of capping activity. Known filopodial tip components such as Diaphanous-related formin and VASP localize to the membrane base of the structures, and bundling protein fascin to the shaft. Actin subunits assemble at the tip and translocate into the shaft. FLS assembly requires negativelycharged lipid membranes, with specific requirements for PI(4,5)P 2 and, for maximal efficiency, phosphatidyl-serine. The focal nature of FLSs is not a result of templating by PI(4,5)P2 microdomains but instead by the self-organization of tip complex assembly on uniform PI(4,5)P 2-enriched regions. BAR domain protein toca-1 recruits N-WASP then the Arp2/3 complex and actin assembly follow. Elongation proteins Diaphanous-related formin, VASP and fascin are recruited later. The Arp2/3 complex is absolutely required for FLS initiation but is not required for elongation, which may involve multiple factors including formins. We propose a model for filopodia formation involving an initial clustering of Arp 2/3 complex regulators, self-assembly of filopodial tip complexes on the membrane, resulting in the outgrowth of parallel actin bundles.
by Kwonmoo Lee.
Ph.D.
6
De, Arpan. "Role of RHO- Family Guanosine Triphosphatase Effectors in Filopodia Dynamics." Bowling Green State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1440176135.
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Lourenco, da Conceicao Luz Marta. "Cellular mechanisms involved in Wnt8 distribution and function in zebrafish neurectoderm patterning." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1228815553128-55176.
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Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.
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Ezeanochie, Tochukwu Chinedu. "Modelling and Simulation of Filopodial Protrusion." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32781.
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The effect of substrate surface topology on the interaction of living cells with inanimate substrates is a well-established phenomenon. When cells are placed on biomaterials, they outgrow protrusions called filopodia that sense surface features in their immediate surroundings and initiate the formation of stable cell adhesion complexes closer to the cell body. Adhesion proteins permit filopodia to constantly explore the surrounding microenvironment. A better understanding of the relationship of filopodia with surface features is highly relevant for exploiting custom-made surfaces to guide cell activity.
In this work, mathematical modeling and simulation were used to describe different phenomena related to the interaction of a filopodium with its microenvironment, with the aim of reproducing experimentally observed phenomena associated to filopodia growth and interactions with substrates. The Kelvin Voigt model was used for the viscoelastic response of filopodia. Result predict filopodia protrusion under test conditions and helps improving our understanding on the effect of substrate topology on the biomechanical response of filopodial extensions.
9
Evers, Jan Felix. "The role of dendritic filopodia in postembryonic remodelling of dendritic architecture." [S.l. : s.n.], 2005. http://www.diss.fu-berlin.de/2005/153/index.html.
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Michiels, Rebecca [Verfasser], and Alexander [Akademischer Betreuer] Rohrbach. "Investigation of filopodia dynamics in macrophage cells by photonic force microscopy." Freiburg : Universität, 2019. http://d-nb.info/1185977295/34.
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Ademuyiwa, Omolade Mary. "The Role of Calcium Flux in the Regulation of Filopodia Dynamics." Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1562940435730486.
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Pandey, Pratima. "Role of Protein Kinase C (PKC) Isoforms in Regulation of Filopodia Dynamics." Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1451317928.
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Kondo, Hanae. "Spatio-temporal properties of membrane-localized actin nucleating complexes." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/289704.
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The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane.
14
Bohil, Aparna Bhaskar Cheney Richard E. "Myosin-X is a molecular motor central to filopodia formation, adhesion, and signaling." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,713.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Molecular Physiology - School of Medicine." Discipline: Cell and Molecular Physiology; Department/School: Medicine.
15
Sigal, Yury J. Bankaitis Vytas A. "Lipid phosphatases and related proteins from regulation of phospholipid metabolism to filopodia formation /." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1010.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Developmental Biology." Discipline: Cell and Developmental Biology; Department/School: Medicine. Includes 7 supplemental QuickTime movies.
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Oak, Youbean. "Filopodia-independent roles of the actin bundling protein fascin in promoting cell motility." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13104236.
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Fascin is an actin bundling protein whose overexpression has in recent years been systematically linked to increased metastasis and poor outcome in cancer patients. It is well established that fascin expression correlates with enhanced cell migration; however, the underlying mechanisms are poorly understood. We combined various methods of high-resolution live cell imaging and computational analysis to investigate the role of fascin in increasing cell motility. We found that fascin promotes collective migration in normal epithelial cells and that this behavior is in agreement with protrusive activities at the single cell level. Traction force measurements indicated that fascin expression level is negatively correlated with traction stress levels and that a cell expressing high levels of fascin protrudes over longer distances than cells with lower levels. Together this led to the hypothesis that fascin distributes cell traction more efficiently, which lowers the load on individual adhesions and actin filaments growing against increasing membrane tension during one protrusion cycle. Measurements of adhesion formation and maturation indicate that fascin expression indeed promotes nascent adhesion formation over a wide area behind the leading edge. In metastatic cells with high fascin expression, we observed decreased invasion upon fascin knock down. These observations demonstrate a role for fascin in promoting cell motility in normal and neoplastic cells, in part by templating nascent adhesions at the leading edge.
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Price, Regan R. "Clic Modulates Filopodia Formation Downstream of Cdc42 and its Effectors in Drosophila Hemocytes." Ohio University Honors Tutorial College / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1338571019.
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Marchenko, Olena O., Sulagna Das, Ji Yu, Igor L. Novak, Vladimir I. Rodionov, Nadia Efimova, Tatyana Svitkina, Charles W. Wolgemuth, and Leslie M. Loew. "A minimal actomyosin-based model predicts the dynamics of filopodia on neuronal dendrites." AMER SOC CELL BIOLOGY, 2017. http://hdl.handle.net/10150/624039.
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Dendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis, but the underlying mechanisms are poorly understood. To study filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity, and substrate adhesion gives rise to various filopodial behaviors. We formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility, and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.
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Lourenco, da Conceicao Luz Marta. "Cellular mechanisms involved in Wnt8 distribution and function in zebrafish neurectoderm patterning." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A23716.
Full textAbstract:
Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.
20
DeMuth, John Gary. "Role of p21-activated Kinase (PAK)-Nck in the Formation of Filopodia and Large Protrusions." Bowling Green State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1265898682.
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Malwade, Santosh. "Shape and quantitative analysis of factor #4 (filopodia) and factor #7 (massive protrusions) in tumorigenic cells." Bowling Green, Ohio : Bowling Green State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=bgsu1207859857.
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Mebane, Leslie Marie. "The role of Ena/VASP and associated proteins in regulation of neuronal morphology and filopodia architecture." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/65293.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references.
During central nervous system development cortical neurons extend a primary axon and multiple collateral branches to connect to numerous synaptic targets. While many guidance cues and their receptors have well-characterized roles in cortical axon guidance, the pathways that link these signals to cytoskeletal remodeling remain poorly understood. The Ena/VASP family of proteins function as key signaling molecules that influences actin reorganization in response to environmental cues, and has been implicated in many aspects of development. My work has focused on defining the mechanisms by which the brain-specific ubiquitin ligase, Trim9, regulates cytoskeletal dynamics in response to the axon guidance cue Netrin-1 and its receptor DCC. I have shown Trim9 binds the cytoplasmic tail of DCC and also binds Ena/VASP proteins and Myosin-X, which are cytoskeletal effectors downstream of Netrin-1. I discovered that inhibition of Trim9 ubiquitin ligase activity specifically blocks Netrin-1 induced cortical branching. I uncovered an interaction between Trim9 and the microtubule-associated protein, Map Ib, a regulator of microtubule stability and axon branching. My data demonstrates that Trim9 coordinates Netrin- 1 induced axon branching via its interaction with the cytoplasmic tail of DCC and cytoskeletal-associated proteins. I have also investigated the role of several actin-associated proteins in regulation of the actin ultra-structure. I used platinum replica electron microscopy to study the architecture of actin in neurons null for the Ena/VASP family, which failed to form axons. We determined the defect in axon formation is due to an inability to form bundled actin filaments and filopodia. In addition, splice isoforms Mena, a member of the Ena/VASP family, are tightly regulated during cancer metastasis and we determined these splicing changes influence the assembly of actin protrusions. My findings have helped to elucidate how environmental signals affect actin cytoskeletal dynamics and how changes in the cytoskeleton influence development.
by Leslie Marie Mebane.
Ph.D.
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Malwade, Santosh Ramkrishna. "Shape and Quantitative Analysis of Factor #4 (Filopodia) and Factor #7 (Massive Protrusions) in Tumorigenic Cells." Bowling Green State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1207859857.
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Zhong, Lei. "ROLES OF NEUROTRANSMITTERS IN THE REGULATION OF NEURONAL ELECTRICAL PROPERTIES AND GROWTH CONE MOTILITY." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_diss/134.
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In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA).
The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.
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Santiago, Joanne Collazo. "COFILIN NAVIGATES CELLULAR CYTOSKELETON AND INVASION RESPONSES TO TGF-β TOWARDS PROSTATE CANCER METASTASIS." UKnowledge, 2013. http://uknowledge.uky.edu/toxicology_etds/7.
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Cofilin’s activity to nucleate actin filament assembly, is regulated by phosphorylation at a single site on the amino terminus, Serine 3. Phosphorylation at this site abolishes the ability of ADF/cofilin to bind to F-actin and inhibits its severing function. This work characterizes the ability of dephosphorylated cofilin (mutation at Serine 3 site) to navigate prostate cancer actin cytoskeleton and metastatic properties in response to TGF-β. TGF-β increased Lim Domain Kinase 2 (LIMK-2) activity leading to cofilin phosphorylation and decrease actin filament severing in wild type cofilin (WTCFL) PC-3 cells. Constitutively active cofilin in Serine 3 cofilin mutants (S3ACFL) promoted prostate cancer cell filopodia formation, actin severing and directed TGF-β mediated migration and invasion. Co-culture of prostate cancer cells with prostate cancer associated fibroblasts induced cell invasion in WTCFL and S3ACFL cells. Active cofilin further enhanced the invasive response, even in the presence of a TGF-β-neutralizing antibody, implicating the contribution of the microenvironment. Active cofilin led to a significant increase in prostate cancer cell metastatic potential in vivo and cofilin correlated with metastasis in a mouse model of prostate tumor progression. In human prostate cancer, cofilin expression was significantly higher in metastasis compared to the primary tumors. Cofilin thus emerges as a regulator of the actin cytoskeleton remodeling capable of coordinating the cellular responses to TGF- β towards prostate cancer metastasis. Understanding how cancer cells interprete TGF-β signals from the microenvironment, is critical for defining the mechanism via which TGF- β function is switched from a growth suppressor to a metastasis promoter. Here we show that in prostate cancer, TGF-β action is directed by active cofilin enabling actin cytoskeleton changes and metastatic behavior. The significant association of cofilin with prostate cancer metastatic progression supports its predictive and targeting value in metastasis.
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Kiso, Marina. "Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network." Kyoto University, 2018. http://hdl.handle.net/2433/235980.
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Bisi, S. "NOVEL IRSP53 (INSULIN RECEPTOR SUBSTRATE PROTEIN OF 53 KDA) FUNCTIONS IN ACTIN CAPPING, FILOPODIA FORMATION AND POLARITY ESTABLISHMENT." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/471381.
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IRSp53 (Insulin Receptor Substrate protein of 53 kDa) is a multidomain scaffolding protein that possesses an I-BAR domain (responsible for protein dimerization and membrane deformation), a partial CRIB motif (for the interaction with active CDC42), a SH3 domain (through it binds actin-related proteins) and additional protein interaction modules at the C-terminus.
We have studied a previously unknown capping role of IRSp53 and the functional consequences of its interaction with VASP. In particular, we showed that following CDC42 binding and activation, IRSp53 is able to recruit and cluster VASP both in vitro and on the plasma membrane, thus fostering VASP processive elongation activity. The elongation of actin filaments by VASP, together with the membrane-deforming ability of IRSp53, combine to promote filopodia initiation downstream of CDC42. Thus, IRSp53 is essential for coordinating these activities in vitro and in cells in culture.
In the second part of this thesis, an IRSp53 role in the polarity program was investigated. We showed that IRSp53 is apically localized in 3-D cyst models of epithelial cells, and IRSp53 depletion leads to a multilumen phenotype in MDCK and Caco2 spheroids. Moreover, using MDCK cysts as a model system, we noted that IRSp53 is enriched early at the prospective lumen initiation site (called AMIS), even before the apical marker podocalyxin. Finally, IRSp53 removal affects podocalyxin localization in MDCK cysts at the early stages.
We speculate that IRSp53 might be involved in the trafficking of the apical protein podocalyxin, we are currently investigating the molecular mechanism responsible for this putative role.
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Barrett-Freeman, Conrad. "Effects of advection on non-equilibrium systems." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/5846.
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We study a number of non-equilibrium models of interest to both active matter and biological physicists. Using microscopic agent-based simulation as well as numerical integration of stochastic PDEs, we uncover the non-trivial behaviour exhibited when active transport, or an advection field, is added to out of equilibrium systems. When gravity is included in the celebrated Fisher-Kolmogoro Petrovsky Piscouno (F-KPP) equation, to model sedimentation of active bacteria in a container, we observe a discontinuous phase transition between a `sedimentation' and a `growth' phase, which should in principle be observable in real systems. With the addition of multiplicative noise, the resulting model contains, as its limits, both the bacterial sedimentation previously described and the fluctuating hydrodynamic description of Directed Percolation (DP), an important and well-studied non-equilibrium system whose physics incorporate many universal features which are typical of systems with absorbing states. We map out the phase diagram describing all the systems in between these two limiting cases, finding that adding an advection term, however small, immediately lifts the resulting system out of the DP universality class. Furthermore, we find two distinct low-density phases separated by a dynamical phase transition reminiscent of a spinodal transition. Finally, we attempt to improve the current diffusion-limited model for the growth of filopodia, which are intriguing networks of actin fibres used by moving cells to sense their environment. By the addition of directed transport of actin monomers to the fibre tip complex by myosin molecular motors, we show that, under appropriate conditions, the resulting dynamics may be more efficient that transport by diffusion alone, which would result in filopodial lengths better corresponding to experimental observation.
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Morrow, Anne. "A Role for the Phosphoinositide Lipid Kinase PI4KIIIbeta in Breast Oncogenesis and Akt Activation." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30418.
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The lipid kinase phosphatidylinositol 4-kinase III β (PI4KIIIβ) phosphorylates phosphatidylinositol (PtdIns) to generate PI(4)P in the Golgi. PI4KIIIβ is likely involved in the development of breast cancer as it has been reported genetically amplified in a subset of human breast tumours and is a downstream effector of the eukaryotic elongation factor 1 alpha 2 (eEF1A2), a transforming gene that is amplified and highly expressed in approximately 60% of human breast tumours. The goal of my thesis is to investigate a role for PI4KIIIβ in breast oncogenesis.
We show that PI4KIIIβ is highly expressed in approximately 20% of primary human breast tumours. Overexpression of PI4KIIIβ in an invasive breast ductal carcinomas cell line, BT549, increased the production of filopodial actin filament protrusions and enhanced in vitro proliferative capacity. Enhanced PI4KIIIβ expression did not impact the migratory rate of these breast cancer cells.
We found that PI4KIIIβ expression activates Akt kinase in the BT549 breast cancer cell line. PI4KIIIβ overexpression led to an increase in the plasma membrane abundance of the PI3K derived PI(3,4,5)P3/PI(3,4)P2 lipids, upstream activators of Akt signalling. PI(4)P and PI(4,5)P2 are precursors to PI(3,4,5)P3 and PI(3,4)P2 generation, however, no changes in the overall cellular abundance or localization of PI(4)P or PI(4,5)P2 were detected in PI4KIIIβ-overexpressing cells. Inhibition of PI4KIIIβ kinase activity, using the drug Pik93, had no effect on PI4KIIIβ-mediated Akt activation. Additionally, ectopic expression of a catalytically inactive PI4KIIIβ also led to increased Akt activity and PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance. Together, this implies that PI4KIIIβ regulates Akt independently of PI(4)P generation. The PI4KIIIβ interacting protein, Rab11, is likely involved in PI4KIIIβ mediated Akt activation, as RNAi-mediated depletion of Rab11 suppressed the effect of PI4KIIIβ overexpression on Akt activation. Furthermore, PI4KIIIβ overexpression altered cellular Rab11 distribution and led to enhanced recruitment of PI4KIIIβ and Rab11 to recycling endosomes.
Therefore, PI4KIIIβ is highly expressed in a subset of breast tumours and upregulated PI4KIIIβ expression enhances filopodia production and cell growth in vitro. Enhanced PI4KIIIβ expression increases PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance and Akt activation independently of its kinase function, through a mechanism that likely involves Rab11. This work suggests that PI4KIIIβ impacts breast oncogenesis by regulating PI3K/Akt signalling through Rab11 and endosomal trafficking.
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Gupta, Rohini [Verfasser], and Robert [Gutachter] Blum. "Intracellular self-activation of the TrkB kinase domain causes FAK phosphorylation and disrupts actin filopodia dynamics / Rohini Gupta ; Gutachter: Robert Blum." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1231715049/34.
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Amarachintha, Surya Prakash. "Cellular Architecture and Cytoskeletal Structures Involved in Cell Haptotaxis." Bowling Green State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1332102412.
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Franko, Jennifer Lynne. "Regulation of Effector/Memory T Cell Activation by Inducible Co-Stimulator (ICOS)." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1228358364.
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Watson, Joanna. "Structural and biochemical insight into the interactions of Cdc42 with TOCA1 and N-WASP." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/268520.
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Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.
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Fox, Helen Mary. "Toca-1 driven actin polymerisation at membranes." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275610.
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Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.
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Pulipakkam, Radhakrishnan Uvaraj. "Studies on Zebrafish Thrombocyte Function." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc984278/.
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Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on thrombocyte function in zebrafish could enhance fundamental knowledge on hemostasis and may provide future target candidates for therapies.
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Hardy, Holly. "Cofilin and drebrin mediated regulation of the neuronal cytoskeleton in development and disease." Thesis, University of Exeter, 2017. http://hdl.handle.net/10871/31746.
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The brain is a highly complex structure; neurons extend axons which follow precise paths to make connections with their targets. This extension is guided by a specialised and highly motile structure at the axon tip -the growth cone- which integrates guidance cues to steer the axon through the environment. Aberrant pathfinding is likely to result in developmental impairments causing disruption to brain functions underlying emotion learning and memory. Furthermore, pre-existing connections are constantly remodelled, the ability to do so declines with age, and can have huge impacts on quality of life and well-being. Examining how changes in growth cone behaviour triggered by external cues occurs is crucial for understanding processes in both development and disease. Controlled reorganisation of growth cone cytoskeletal components, such as actin filaments, generate membrane protrusions forming lamellipodia and filopodia. Filopodium formation is commonly associated with sensing the mechanical and chemical environment of the cell. Despite our understanding of the guidance choices that can be made, how filopodia transmit information at a molecular level leading to profound changes in morphology, motility and directionality remains largely unknown. Various actin-binding proteins regulate the number, stability and branching of filopodia. They may therefore have a key role in priming or abrogating the ability of the growth cone to respond to a given guidance cue. I have shown that the actin binding proteins drebrin and cofilin, whilst displaying opposing molecular activities on actin filaments, work synergistically in a temporally regulated manner. A fluorescent membrane marker combined with tagged cofilin and drebrin enabled accurate correlation of cofilin and drebrin dynamics with growth cone morphology and filopodial turnover in live neurons. In contrast to previous in vitro experiments, cofilin was found to enhance the effect of drebrin to promote filopodia formation in intact neurons, and that growth cone spread was significantly constrained when cofilin was knocked down. Importantly, this adds to our understanding of how the two actin binding proteins contribute to directed motility in neuronal growth cone filopodia during guidance. Furthermore, following acute treatment with low concentrations of the repulsive guidance cue semaphorin-3A, neuronal growth cones expressing cofilin displayed increased morphological complexity and filopodial stability. This suggests that traditional collapse signals may serve as pause signals allowing neurons to increase the surface area to sense the environment adequately and enable precise wiring decisions. Remodeling of the cytoskeleton is perturbed in a number of degenerative diseases including Alzheimer's, Huntington's, and Amyotrophic Lateral Sclerosis. These conditions are associated with widespread synaptic loss, resulting in memory loss, cognitive impairment, and movement disorders which leads to severe deterioration in quality of life for those afflicted in addition to wider negative socioeconomic impacts. How widespread synaptic loss occurs is poorly understood. One common characteristic is neuronal stress which can be initiated through different conditions such as neuroinflammation, energetic stress, glutamate excitotoxicity, and accumulation of misfolded proteins, all of which have been associated with perturbation of the actin cytoskeleton and the initiation of the cofilin-actin rod stress response. Dysfunction of the cytoskeleton can lead to the disruption of synaptic activity by blocking the delivery of elements such as organelles and proteins required for maintenance of the synapse. Modulating this stress response offers an approach to protecting the integrity of normal synaptic function. Actin interacting protein-1 is a conserved actin binding protein that enhances the filament disassembly activity of cofilin. I have discovered that AIP-1 has a potent ability to prevent the formation of cofilin rods which are thought to contribute to the neuronal dysfunction in several neurodegenerative disorders, even when they are treated with amyloid-β or subjected to metabolic stress. This is the first study to demonstrate a molecular mechanism for preventing rod formation in the presence of a neuronal stressor and has the potential to protect against rod formation by other stressors associated with disease such as inflammation and excitotoxicity. AIP-1 offers the exciting possibility of a means to reverse cofilin rod formation and the subsequent cytoskeletal pathology associated with dementia and has potential for therapeutic exploitation in human disease. Furthermore, it is the first study to demonstrate that AIP-1 localises to areas of rapid actin remodeling in neuronal growth cones. Exploiting the action of AIP-1 therefore represents an exciting and novel therapeutic avenue to tackle neurodegeneration.
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Garcia, Mikael. "Rôle du couplage N-cadhérine/actine dans les mécanismes de motilité et de différentiation synaptique dans les neurones." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22055/document.
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Les protéines d’adhésions homophiles N-cadhérine jouent un rôle majeur dans le développement du cerveau, notamment en agissant sur la croissance et la plasticité synaptique. Au cours de ma thèse, j’ai étudié le rôle de la N-cadhérine dans ces deux processus en utilisant des neurones issus de cultures primaires déposés sur des substrats micropatternés. Ces substrats sont recouverts de N-cadhérine purifiée afin d’induire des adhésions N-cadhérines sélectives au niveau de micro-motifs régulièrement espacés. Mes deux premières études sont basées sur le modèle d’embrayage moléculaire, décrivant le processus par lequel la motilité du cytosquelette d’actine se couple aux adhésions au niveau de la membrane cellulaire afin de générer des forces de traction aux zones de contact avec le substrat, permettant ainsi l’avancée cellulaire (Giannone et al., 2009). Plusieurs études ont mis en avant l’existence d’un tel modèle (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), cependant le mécanisme exact permettant d’expliquer ce couplage mécanique de l’actine aux protéines d’adhésions reste mal connu. Via des techniques de pinces optiques, des travaux précédemment menés dans l’équipe ont prouvé l’existence d’un couplage entre le flux d’actine et les adhésions N-cadhérine permettant la migration du cône de croissance (Bard et al., 2008). Cette technique n’a cependant pas permis la visualisation directe de l’engagement d’un tel mécanisme. Nous avons donc couplé l’utilisation des substrats micro-patternés à la microscopie haute résolution sptPALM/TIRF afin de visualiser directement la dynamique des protéines impliquées dans l’embrayage moléculaire. Dans le premier article, j’ai montré pour la première fois l’existence d’interactions transitoires entre le flux d’actine et les adhésions N-cadhérines au niveau du cône de croissance, reflétant un embrayage glissant à l’échelle de la molécule unique (Garcia et al., en préparation). Dans le second article, en travaillant sur des neurones plus matures, nous avons pu montrer l’engagement d’un embrayage moléculaire trans-synaptique entre adhésions N-cadhérines et flux d’actine permettant la stabilisation du filopode dendritique et ainsi sa transition en épine mature (Chazeau/Garcia et al., en préparation). J’ai également participé à une troisième étude dans laquelle j’ai observé l’effet des substrats micropatternés recouverts de N-cadhérine, sur la synaptogenèse. J’ai ainsi pu prouver que la N-cadhérine déposée sur les micro-motifs, stimule la croissance dendritique et axonale et joue un rôle prépondérant dans la maturation morphologique des neurones. Cependant, la N-cadhérine est incapable d’induire la formation de synapses contrairement aux protéines d’adhésion neurexine/neuroligine ou SynCam (Czöndör et al., 2013)The homophilic adhesion molecule N-cadherin plays major roles in brain development, notably affecting axon outgrowth and synaptic plasticity. During my PhD work, I addressed the role of N-cadherin in these two processes, using primary neurons cultured on micro-patterned substrates. These substrates are coated with purified N-cadherin to trigger selective N-cadherin adhesions in a spatially controled manner. My two first studies are based on the “molecular clutch” paradigm, by which the actin motile machinery is coupled to adhesion at the cell membrane to generate forces on the substrate and allow cells to move forward (Giannone et al., 2009). Many publications have provided evidence for such a mechanism (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), but the exact mechanisms underlying the molecular coupling between the actin retrograde flow and adhesion proteins remain elusive. The team previously inferred, using optical tweezers, that a molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration (Bard et al., 2008), but could not achieve a direct visualization of the engagement process with this technique. Here, we combined the use of micropattern substrates with high resolution microscopy sptPALM/TIRF to visualize directly the dynamics of the main proteins involved in the molecular clutch. In my first paper, I reveal for the first time transient interactions between the actin flow and N-cadherin adhesions in growth cones, reflecting a slipping clutch process at the individual molecular level (Garcia et al., in preparation). In a second study, working with more mature neurons, we revealed that engagement of a molecular clutch between trans-synaptic N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines (Chazeau/Garcia et al., in preparation). I also participated to a third study, where I observed the effect of N-cadherin coated substrates on synaptogenesis. I showed that, although N-cadherin on micro-patterned substrates stimulated axonal and dendritic elongation and played a major role in morphological maturation, it was not able to induce synapse formation like neurexin/neuroligin or SynCAM adhesions (Czöndör et al., 2013)
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Ferrari, I. "THE ROLE OF THE IRSP53-LIN7 COMPLEX IN CELL MEMBRANE DYNAMICS." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/250654.
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The insulin receptor substrate protein of 53 kDa is a critical factor in determining neuronal polarisation, as it initiates membrane protrusions to form filopodia and neurites by coupling membrane deformation with F-actin polymerization. With its C-terminal tail, IRSp53 can bind to PDZ domain containing proteins including LIN7, a small scaffold protein possessing a single L27 domain, necessary for membrane recruitment. Here, we investigated the role of the IRSp53-LIN7 complex in cellular mechanisms that heavily rely on membrane deformation, such as the formation of filopodia and neurites or mitochondrial fission.
Concerning the role of the IRSp53:LIN7 complex in filopodia and neurite induction, our findings indicate that the formation of actin-filled filopodia and neurites depends on motifs mediating IRSp53-LIN7 association and filopodia tip localisation. We further showed that co-expression of LIN7 with IRSp53 enhanced the formation of filopodia protrusions in neuronal NSC34 cells, while also preventing the appearance of actin-deficient protrusions induced by the overexpression of IRSp53 alone. The positive regulatory role of LIN7 in IRSp53-mediated extension of filopodia was further demonstrated by live-cell imaging experiments in neuronal N2A cells. Moreover, LIN7 silencing in N2A cells prevented the extension of filopodia and neurites, induced by either the ectopic expression of IRSp53 or serum starvation. Defective neuritogenesis could be rescued by the expression of RNAi-resistant full length LIN7 or chimeric L27-IRSp53, whereas the expression of full length IRSp53 or the LIN7ΔPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7 silenced cells. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100 insoluble complexes, otherwise occurring in differentiated cells. Collectively, this first set of data identify in LIN7 a novel regulator of the filopodia- and neurites-promoting activity of IRSp53, whose role is to spatially restrict its activity to the plasma membrane for filopodia and neurite initiation, and to further promote the stabilisation of these actin–rich structures.
More recently, we tested the hypothesis of a role for the IRSp53-LIN7 complex in the modification of intracellular membranes. To this regard, we found that endogenous LIN7 and IRSp53 localized in punctuate structures along mitochondria, a fact that prompted us to further investigate the possible effects of modifications in the expression levels of IRSp53/LIN7 on mitochondrial morphology. Eukaryotic cells, in fact, strictly regulate the overall morphology of their mitochondrial network thanks to the existence of protein complexes able to control fission and fusion events. We found that, upon overexpression of LIN7 and/or IRSp53, mitochondria morphology was altered, with a significant increase in the percentage of cells showing a less interconnected mitochondrial network compared to GFP-transfected control cells, a phenotype that was blocked by co-expression of the K38A dominant negative mutant of the fission protein Drp1. Downregulation of endogenous LIN7 and/or IRSp53 by shRNA, on the other hand, increased the amount of cells with highly fused mitochondria. Mitochondria hyperfusion in the downregulated cells was associated with an increased resistance to NaN3-induced mitochondrial fragmentation, and by the appearance of cells with aberrantly shaped and often multi-lobed nuclei, a phenotype that we also found in Drp1 K38A-expressing cells, and that others reported to be caused by defective mitochondrial fragmentation during mitosis. Collectively our data strongly suggest a Drp1-dependent function of LIN7 and IRSp53 on mitochondrial division apparatus.
Taken together, our data unravel a role of the IRSp53-LIN7 complex on membrane dynamics that is not restricted to plasma membranes, as previously thought, but may also apply to other cellular mechanisms that heavily rely on membrane deformation, as our data on mitochondrial morphology have shown.
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Welshhans, Kristy. "Neuronal growth cone dynamics are regulated by a nitric oxide-initiated second messenger pathway." unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-09282007-114034/.
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Thesis (Ph. D.)--Georgia State University, 2007.Vincent Rehder, committee chair; Sarah Pallas, Walter William Walthall, committee members. Electronic text (248 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 28, 2008; title from file title page. Includes bibliographical references (p. 218-248).
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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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Bugyei, Francis Kyei. "Polarity and Endocytic Traffic in the Mammalian Cell." Bowling Green State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1404222497.
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Hasan, Amr [Verfasser]. "Role of neuronal cell adhesion molecules in regulating filopodial dynamics and synapse formation in the drosophila visual system / Amr Hasan." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1216104972/34.
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Kohler, Felix [Verfasser], and Alexander [Akademischer Betreuer] Rohrbach. "Photonic force based investigations of filopodial dynamics and coupled molecular motors = Photonische Kraft basierte Untersuchungen zur Filopodiendynamik und gekoppelte molekulare Motoren." Freiburg : Universität, 2013. http://d-nb.info/1123477345/34.
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Eliautou, Sandra. "Signalisation déclenchée par le complexe GPIb-V-IX et émission de filopodes dans la plaquette sanguine." Strasbourg, 2009. https://publication-theses.unistra.fr/public/theses_doctorat/2009/ELIAUTOU_Sandra_2009.pdf.
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L’hémostase primaire est la première étape du processus de réparation tissulaire lors d’une lésion de la paroi vasculaire. Elle permet la constriction du vaisseau lésé et la formation d’un clou plaquettaire qui obstrue la brèche. Les plaquettes sanguines jouent un rôle majeur dans ce processus grâce à leurs fonctions d’adhésion, de sécrétion et d’agrégation. Ces étapes d’activation sont accompagnées de changements morphologiques avec au stade initial le passage d’une forme discoïde à une forme sphérique avec extension de filopodes. Les filopodes pourraient favoriser l’interaction entre ligands et récepteurs et ralentir les plaquettes soumises au flux sanguin lors de l’interaction avec la matrice sous-endothéliale. L’émission de filopodes est notamment observée suite à l’interaction du complexe GPIb-V-IX plaquettaire avec le Facteur Willebrand (FW) dans l’étape initiale du processus hémostatique. La voie de signalisation en aval du complexe GPIb-V-IX implique une src kinase, l’activation de PLCγ2, la production d’IP3 et la mobilisation des stocks internes de Ca2+. Les mécanismes et les acteurs moléculaires impliqués dans l’émission de filopodes sont mal connus dans la plaquette. L’objectif de ce travail a été d’étudier ces mécanismes en aval de GPIb-V-IX en caractérisant plus précisément cette voie de signalisation et en évaluant des acteurs impliqués dans l’émission de filopodes dans d’autres types cellulaires. Ces acteurs potentiels ont été évalués en utilisant des souris génétiquement modifiées et des inhibiteurs pharmacologiques. L’émission de filopodes dans la souris a été étudiée dans un modèle d’adhésion sur FW recombinant de souris et également en réponse au fibrinogène et à des agonistes solubles comme l’ADP et la thrombine. La src kinase Lyn et la tyrosine Btk, connues pour participer à l’activation de PLCγ2 en aval de GPVI, ont été évaluées dans des souris déficientes pour Lyn et Btk et exprimant une forme activée constitutive de Lyn (Lyn up/up). Les résultats d’adhésion sur FW indiquent que ces deux kinases ne jouent pas de rôle en aval de GPIb. La GTPase Cdc42, PKCδ et VASP, qui ont été impliquées dans l’émission de filopodes dans différentes cellules, dont la plaquette, ont été évaluées en aval du complexe GPIb-V-IX. Un rôle a été révélé uniquement pour Cdc42 qui semble spécifique de cette signalisation. Deux autres GTPases de la famille Rho, Rac1 et RhoA, ne participent pas à l’émission de filopodes en aval de GPIb. La Myosine IIA, connue pour son rôle dans la contraction du cytosquelette intra plaquettaire, a également été évaluée dans des souris déficientes pour le gène MYH9. L’absence de Myosine IIA empêche l’émission de filopodes en aval de GPIb mais également en réponse aux agonistes solubles comme l’ADP. Afin d’évaluer d’autres acteurs pour lesquels il n’existe pas de lignées murines ou d’inhibiteurs, nous avons établi une lignée cellulaire humaine HEK transfectée par le complexe GPIb-IX capable d’adhérer sur FW et d’émettre des filopodes. Le rôle de RalA, une Ras GTPase susceptible de lier le complexe GPIb via la Filamine, a été évalué dans cette lignée par extinction shRNA montrant une émission normale de filopodes et que cette GTPase n’est probablement pas impliquée en aval de GPIb. Ce travail a permis d’identifier Cdc42 et la Myosine IIA comme étant impliquées dans la signalisation GPIb-V-IX menant à l’émission de filopodes et d’éliminer plusieurs acteurs potentiels (Lyn, Btk, PKCδ, VASP, RalA, Rac1 et RhoA). Le rôle potentiellement important de l’émission de filopodes en aval de ce récepteur à un stade précoce du processus hémostatique justifie de compléter sa caractérisation dans le but d’identifier de nouvelles cibles antiplaquettaires.
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Schäfer, Claudia [Verfasser]. "Analyse der Zellwanderung am Beispiel von Keratinozyten: Zusammenspiel von Filopodien, Krafterzeugung und Matrix-Sekretion / Claudia Schäfer. Mathematisch-Naturwissenschaftliche Fakultät." Bonn : Universitäts- und Landesbibliothek Bonn, 2010. http://d-nb.info/1022190199/34.
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Durkaya, Göksel. "Nanoscopic Investigation of Surface Morphology of Neural Growth Cones and Indium Containing Group-III Nitrides." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/phy_astr_diss/43.
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This research focuses on the nanoscopic investigation of the three-dimensional surface morphology of the neural growth cones from the snail Helisoma trivolvis, and InN and InGaN semiconductor material systems using Atomic Force Microscopy (AFM). In the analysis of the growth cones, the results obtained from AFM experiments have been used to construct a 3D architecture model for filopodia. The filopodia from B5 and B19 neurons have exhibited different tapering mechanisms. The volumetric analysis has been used to estimate free Ca2+ concentration in the filopodium. The Phase Contrast Microscopy (PCM) images of the growth cones have been corrected to thickness provided by AFM in order to analyze the spatial refractive index variations in the growth cone. AFM experiments have been carried out on InN and InGaN epilayers. Ternary InGaN alloys are promising for device applications tunable from ultraviolet (Eg[GaN]=3.4 eV) to near-infrared (Eg [InN]=0.7 eV). The real-time optical characteristics and ex-situ material properties of InGaN epilayers have been analyzed and compared to the surface morphological properties in order to investigate the relation between the growth conditions and overall physical properties. The effects of composition, group V/III molar ratio and temperature on the InGaN material characteristics have been studied and the growth of high quality indium-rich InGaN epilayers are demonstrated.
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Gorojans, Gregor. "Der Einfluss von Konstituenten der extrazellulären Matrix und motogener Mediatoren auf Parameter der epithelialen Migration Dynamik von Lamellipodien und Ausbildung von Filopodien und des "Migration Track" in humanen epidermalen Keratinocyten /." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982006810.
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Salim, Cláudio. "Expression de la protéine géante AHNAK après lésion de la moelle épinière et dans le système nerveux périphérique : études fonctionnelles sur les cellules de Schwann in vitro." Paris 6, 2007. http://www.theses.fr/2007PA066507.
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Le gène ahnak de rat a été identifié lors d’un criblage différentiel visant à déterminer les protéines impliquées dans les lésions de la moelle épinière. Après la lésion chez le rat, AHNAK est produite en quantité accrue de 48h à 6 mois. AHNAK est présente dans la composante fibrotique de la cicatrice, exprimée par des types cellulaires variés. Sa localisation en bordure des cavités suggère une participation dans la formation d’une barrière protégeant le parenchyme sain après lésion. Dans le système nerveux périphérique, AHNAK est constitutivement exprimée par les neurones sensoriels de petits et de moyens diamètres, ainsi que par les cellules satellites, et les cellules de Schwann du nerf. Pendant la myélinisation la protéine AHNAK est redistribuée d’un compartiment péri-myélinique du cytoplasme externe, à une distribution diffuse et associée à des vésicules au niveau de la membrane abaxonale. In vitro AHNAK et dystroglycan sont associés aux filopodes de cellules de Schwann non confluentes. L’interférence de l’expression de ahnak, induit la rétraction des processus et le détachement du substrat, s’accompagnant d’une chûte du taux de bêta- dystroglycan et la délocalisation de son partenaire Dp116. AHNAK pourrait participer à la stabilisation du complexe d’attache contenant le dystroglycan et contribuer ainsi au maintien de la myélineAhnak gene in rat has been first identified by a differential screening that aimed in identifying proteins overexpressed in a spinal cord injury. After a spinal injury in rat, AHNAK is expressed by different types of cells invading the lesion epicenter as soon as 48h after injury. Those cells constitute the fibrotic component of the glial scar, and produce ahank at least until 6 months after injury. AHNAK expressing cells delineate the inner border of cystic cavities in the lesion epicenter, suggesting that AHNAK may participate in the formation of a tissue-protective barrier. In the peripheral nervous system, AHNAK is constitutively expressed by sensory neurons of the dorsal root ganglia, satellite cells, and Schwann cells from the nerve. During myelination in rat, AHNAK is redistributed from a strictly perimyelinic compartment of the external cytoplasm, to a more diffuse distribution associated with the outer surface of vesicles, and with the abaxonal plasma membrane. In non confluent Schwann cells in vitro, AHNAK and the laminin-receptor dystroglycan are associated with filopodia-like cell extensions. Ahnak interference in Schwann cells induces retraction of cell processes and detachment from laminin coated surfaces, associated with a reduction of the Schwann cell content in beta-dystroglycan and a nuclear translocation of Schwann cell specific dystrophin Dp116 which normally binds beta dystroglycan with the actin cytoskeleton. . We suggest AHNAK to be implicated in targeting and/or scaffolding of the dystroglycan-associated complex to the abaxonal membrane. Thus, similarly to periaxin with which it shares certain features, AHNAK may contribute to SC-basal lamina interaction, and myelin formation and/or maintenance
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Laurichesse, Quentin. "Caractérisation génétique des précurseurs de tendons appendiculaires au cours des étapes précoces de la métamorphose chez Drosophila melanogaster : rôle du Krüppel-like factor Dar1 dans le développement des précurseurs de tendons appendiculaires." Thesis, Université Clermont Auvergne (2017-2020), 2019. http://www.theses.fr/2019CLFAC072.
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La mise en place de l’architecture musculo-squelettique émerge d’un développement coordonné inter-tissulaire entre muscles et tissus conjonctifs, dont l’agencement requiert une communication permanente lors des stades embryonnaires. En dépit de l’absence de squelette interne, la patte de la drosophile possède à l’instar des vertébrés des longs tendons internes, auxquels sont rattachés les muscles. Ces sites d’attachements partagent avec mammifères, à la fois l’aspect fonctionnel des tendons à savoir rattacher les muscles et permettre la locomotion, mais également des aspects moléculaires, avec l’identification d’orthologues requis pour leur développement et homéostasie. Chez la drosophile, les tendons appendiculaires sont les seuls sites d’attachement à proposer une telle architecture. Sur la base de ces observations et connaissances, nous avons pris le parti d’étudier plus précisément quels pouvaient être les gènes responsables du développement de ces tendons. Mes travaux de thèse ont consisté à la mise en place d’une méthode cellule-spécifique adaptée à des populations rares que sont les précurseurs de tendons appendiculaires, dans le but d’étudier leur transcriptome. Les données de RNA-seq obtenues ont permis de mettre en exergue environ 900 gènes, dont 68 facteurs de transcription enrichis au sein des précurseurs de tendons. Suite à crible ARN interférent, j’ai identifié le gène dar1 qui est spécifiquement exprimé au sein des tendons de la patte au moment de la métamorphose. La perte de ce gène entraîne une perte des longs tendons appendiculaires et induit une désorganisation drastique de l’architecture musculaire. De manière intéressante, Dar1 est un représentant de la famille des Krüppel-like factor, dont les orthologues KLF4 et 5 sont retrouvés au sein de données transcriptomiques de certains tendons chez les vertébrés, sans que leurs rôles n’aient encore pu être élucidés. A travers cette étude, Dar1 est aujourd’hui proposé comme marqueur spécifique des tendons appendiculaires, et tend à mettre en lumière des relations potentiellement intéressantes entre les tendons de drosophile et les multiples tissus conjonctifs des vertébrésMusculoskeletal development is a coordinated process that requires the integration of multiple cues and the interaction between muscles and connective tissues (CT). Despite the lack of internal skeleton, the drosophila leg, like the vertebrate limb, shows long internal tendons, which are connected with muscle fibres. These muscle attachment sites share similar function with their mammalian counterpart; they transmit the strength generated by the muscles to allow locomotion. They also share well-known molecular orthologs that are required for their development and homeostasis. Thus, the study of this long internal tendons within the drosophila leg is of great interest to understand the development of this sort of structure. Based on these observations and knowledge, we decided to investigate the genes that are responsible for the development of such particular tendons. We focused on leg tendon precursors, which in fly, develop into tube-like CT structures. We developed a cell-specific approach to isolate tendon precursors and perform RNAseq analysis. This experiment led us to identify approximately 900 transcripts enriched in tendon precursors, in which 68 of them encode for transcription factors (TF). Amongst them, the Krüppel-like factor Dar1 is specifically expressed in tendon leg precursors during the early stages of metamorphosis. Tissue sections of fly legs with attenuated dar1 expression revealed aberrant leg muscle organization with a loss of internal appendicular tendons. These results suggest that Dar1 plays a key role in tendon development. Interestingly, Dar1 orthologs KLF- 4 and 5 are also expressed in mouse tendon precursors and studies conducted on chicken explants suggest that it could impact CT development. This work allowed Dar1 to be identified as a specific marker of long tendon of the leg that could also be required for the development of connective tissues in the vertebrate limb
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Hsu, Tsi-Hsuan, and 許慈軒. "Dynamics of cancer cell filopodia characterized by super-resolution bright-field optical microscopy." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/43131461009902805930.
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碩士國立陽明大學
生醫光電工程研究所
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In this thesis, we explore the dynamics of cellular filopodia with diameters around 200 nm by using super-resolution bright-field optical microscopy. Filopodia are positively related to cell motility because of the low optical contrast of filopodia, we use an optical technique with nanometer depth sensitivity, non-interferometric widefield optical profilometry (NIWOP), to obtain the high contrast signal for super-resolution image restoration. The image contrast of NIWOP comes from the topographic variations of sample, rather than fluorescence labeling. In super-resolution NIWOP, the lateral resolution is improved to 130 nm. Compared with the original optical resolution of 250 nm, the lateral resolution is reduced by 50%. At present, our system has an image-acquisition rate of 20 frames/min. In order to confirm that the lateral resolution and the image-acquisition rate is suitable for observing the dynamics of cancer-cell filopodia, we compare the numbers and dynamics of filopodia before and after the treatment of epidermal growth factor (EGF), which is known to affect the dynamics of filopodia. In our work, The cell line is human lung adenocarcinoma cell lines CL1-0, which is less invasive, and therefore with fewer filopodia. We found that the treatment of EGF raises the number of filopodia by nearly a factor of 2. In addition, we observe the growth and activities of single filopodia of a CL1-0 cell. In the culturing condition, we measure that the filopodia exhibit an average elongation rate of 90 nm/sec, and an average shrinkage rate of 75 nm/sec. After the treatment of EGF, the elongation and shrinkage rates increase to 110 nm/sec and 100 nm/sec respectively. With the treatment of EGF, the increase on the number and the dynamics of filopodia implies the enhancement of cell motility. We also use the super-resolution NIWOP to observe the effects of a new factor on the number of CL1-0 filopodia. After the new factor treatment, the average number of filopodia also increases, and the maximum occurs at 8-10 minutes afterwards. This result verifies that the new factor could affect the filopodia of CL1-0, and it may promote the metastasis of lung cancer cells. Moreover, with super-resolution NIWOP, we observed that the number of filopodia in gene A-transfected CL1-0 is more than that in mock-transfected CL1-0 cells. We speculate that gene A could also enhance cancer cell migration and invasion. It is proposed that filopodia could sense the chemical materials around a cell. However nobody demonstrates that filopodia can be used to characterize the chemical gradient around a cell. Therefore, we apply super-resolution NIWOP to a transparent microfluidic cell culturing chip to demonstrate the chemo-gradient sensitivity of filopodia on a single cell. Owing to the specially designed fluidic field in the chip, we may control the concentration of chemical materials around a single cell. Placing a CL1-0 cell at high gradient of EGF, the number of filopodia on the high-concentration side is evidently more than that on the low-concentration side. This experiment proves that the quantity of filopodia depends the chemo-gradient of EGF, even on the same cell. The non-fluorescence observation technique developed in this thesis is very attractive for living cell analyses. Without the high-intensity illumination required by fluorescence imaging, cells stay in their natural states before and after the observation. The results are thus more directly related to their behavior in native environments. On a CL1-0 lung cancer cell, the high contrast and resolution of the super-resolution NIWOP images make the characterization of filopodia much easier and more accurate. Furthermore, the imaging-acquisition rate is as fast as 20 frame/min, which is suitable for dynamic observations. With super-resolution NIWOP, we can easily observe the effect on filopodia by the treatment of various factors, genes or chemical gradient of drug and then conjecture that if they can enhance or suppress cancer-cell migration and invasion.