Dissertations / Theses on the topic 'Actin'
To see the other types of publications on this topic, follow the link: Actin.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Actin.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Wang, Hui. "Structural studies of actin and actin-binding proteins." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/10916.
Full textAbstract:
Actin is involved in cell movement, maintaining cell shape and anchoring cytoskeletal proteins. These functions are regulated by many actin-binding proteins, including those of the gelsolin superfamily. Gelsolin superfamily members regulate actin organization by severing, capping F-actin, nucleating the formation of F-actin and/or bundling F-actin. Although abundant structures are available for gelsolin and gelsolin fragments in complexes with actin, the detailed mechanisms for gelsolin activation, and for gelsolin severing and capping of F-actin are still unknown. Structures for gelsolin family members and their complexes with actin help elucidate these mechanisms.
In this thesis, I describe the purification, crystallization and solution of the structures of the following four proteins and protein complexes, including human G1-G3/actin complex, a novel equine G1-G3/actin complex, human villin domain V6 and actin monomer. The structure of human G1-G3/actin indicates cooperative binding of calcium ions in G2 and G6 is responsible for opening the G2/G6 latch to expose the F-actin binding site on G2. A new equine G1-G3/actin structure suggests G2-G3 can adopt a CapG-like conformation and reveals novel interactions between gelsolin and actin. The villin V6 structure implies a common spring-loaded activation mechanism in the gelsolin superfamily. Finally, a new actin monomer structure is the first reported for G-actin in an ATP state, without ABPs or modification. All these structures contribute to our understanding of actin's physiological roles and their regulation by the gelsolin superfamily.
2
Hull, Richard Alan. "Actin and actin-binding proteins in higher plants." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279874.
Full text
3
Heisler, David Bruce. "Role of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1501519777175964.
Full text
4
Gholami, Azam. "Actin-based motility." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-72151.
Full text
5
Yeoh, Sharon I.-Wen. "Molecular interactions of human actin depolymerizing factor and cofilin with actin." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621255.
Full text
6
Gallinger, Julia. "WH2 domains and actin variants as multifunctional organizers of the actin cytoskeleton." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-161698.
Full textAbstract:
Actin is one of the most abundant proteins in eukaryotic cells and regulation of the microfilament system is crucial for a wide range of cellular functions including cell shape, cell motility, cell division and membrane dynamics. The aim of this thesis was (1) to gain a better understanding of the function of distinct actin binding domains in the regulation of the actin cytoskeleton and (2) to elucidate the role of actin variants.
WH2 domains (WH2, Wiskott-Aldrich syndrome protein homology 2) are ubiquitous multifunctional regulators of actin dynamics. The protein Spire contains four central WH2 domains A-B-C-D with about 20 amino acids each and the cyclase-associated protein CAP2 contains only one WH2 domain. Under certain conditions, they can (1) nucleate actin polymerization, (2) disintegrate actin filaments and (3) sequester actin monomers. Here, the influence of selected Drosophila melanogaster Spire-WH2 and Mus musculus CAP2-WH2 domain constructs on actin dynamics was tested in vitro. To act as a filament nucleator, at least two WH2 domains are required, and nucleation of actin polymerization was only observed at substoichiometric concentrations of WH2 domains over actin. At higher concentrations, the sequestering activity of WH2 domains takes over. Preformed and purified SpireWH2-actin complexes act as extremely efficient nuclei for actin polymerization, even at superstoichiometric WH2 concentrations, under which free WH2 domains would sequester actin. All analyzed constructs, including these with only a single WH2 domain, sequester actin as well as they can disrupt filaments. This latter and most peculiar behavior of WH2 domains was observed in fluorometric, viscometric and TIRF assays. The WH2 domains seem to have such a high affinity for actin that they can forcefully sequester monomers even from filaments and filament bundles, thus breaking the whole structures. Taken together, the data clearly show that SpireWH2-actin complexes are the intermediates that account for the observed nucleating activity, whereas free WH2 domains can disrupt filaments and filament bundles within seconds, again underlining the intrinsic versatility of this regulator of actin dynamics. These data have been confirmed by crystallography in collaboration with the groups of Prof. Dr. Tad Holak and Prof. Dr. Robert Huber (Martinsried, Germany).
Besides the well-studied conventional actins many organisms harbor actin variants with unknown function. The model organism Dictyostelium discoideum comprises an actinome of a total of 41 actins, actin isoforms and actin-related proteins. Among them is filactin, a highly conserved actin with an elongated N-terminus. The 105 kDa protein has a distinct domain organization and homologs of this protein are present in other Dictyosteliidae and in some pathogenic Entamoebae. Here, the functions of filactin were studied in vivo and in vitro. Immunofluorescence studies in D. discoideum localize endogenous and GFP-filactin in the cytoplasm at vesicle-like structures and in cortical regions of the cell. A most peculiar behavior is the stress-induced appearance of full length filactin in nuclear actin rods. To perform in vitro analyses recombinant filactin was expressed in Sf9 cells. Fluorescence studies with the filactin actin domain suggest that it interferes with actin polymerization by sequestering G-actin or even capping filaments. Gel filtration assays propose a tetrameric structure of full length filactin. Protein interaction studies suggest that filactin is involved in the ESCRT (endosomal sorting complexes required for transport) pathway which is responsible for multivesicular body formation. The data on filactin suggest that only the conventional actins are the backbone for the microfilamentous system whereas less related actin isoforms have highly specific and perhaps cytoskeleton-independent subcellular functions.Aktin ist als Bestandteil des Zytoskeletts eines der häufigsten Proteine in allen eukaryontischen Zellen. Eine genaue Regulation des Mikrofilamentsystems ist essentiell für Zellform, Zellmigration, Zellteilung und Membrandynamik. Ziel dieser Arbeit war (1) die Funktion von ausgewählten Aktin Bindedomänen in der Regulation des Aktin Zytoskeletts zu untersuchen und (2) die Funktion von Aktinvarianten zu verstehen. WH2 Domänen (WH2, Wiskott-Aldrich Syndrom Protein Homologie 2) sind kurze, konservierte Sequenzmotive (ca. 20 Aminosäuren), welche bevorzugt monomere Aktinmoleküle binden. Von besonderem Interesse waren Drosophila melanogaster Spire-WH2 und Mus musculus CAP2-WH2 Konstrukte. Das Protein Spire enthält vier WH2 Domänen (A-B-C-D) wohingegen CAP2 (Cyclase-assoziiertes Protein 2) nur eine WH2 Domäne besitzt. Diese WH2 Domänen können unter bestimmten Bedingungen (1) die Aktinpolymerisation stimulieren, (2) Aktinfilamente zerstückeln und (3) Aktinmonomere sequestrieren. Für die Nukleation der Aktinpolymerisation müssen mindestens zwei hintereinander angeordnete WH2 Domänen vorhanden sein und unterstöchiometrische Mengen an WH2 Domänen im Vergleich zur Aktinkonzentration vorliegen. Bei höheren WH2 Konzentrationen überwiegt die Sequestrierungsaktivität. Polymerisationsexperimente mit vorgefertigten SpireWH2-Aktin Komplexen bestätigen, dass diese Komplexe für die beobachtete Nukleation der Aktinpolymerisation verantwortlich sind. Im Gegensatz zu ungebundenen WH2 Domänen sind diese WH2-Aktin Komplexe selbst bei überstöchiometrischen WH2 Konzentrationen äußerst effiziente Nukleatoren. Alle untersuchten WH2 Konstrukte zeigen die bereits bekannte Bindung an G-Aktin, können aber auch vorgeformte Aktinfilamente sogar auseinanderreißen. Diese letztere und besonders auffällige Eigenschaft von WH2 Domänen wurde in fluorometrischen, viskometrischen und TIRF Experimenten nachgewiesen. Anscheinend ist die Affinität der WH2 Domänen zu Aktinmonomeren so stark, dass sie diese aus den Filamenten entfernen können und damit ganze Filamente und Filamentbündel zerstückeln. Für die Multifunktionalität der analysierten konservierten WH2 Domänen spricht zusammenfassend, dass sie neben der Aktinfilament Nukleation auch Filamente und Filamentbündel innerhalb von Sekunden fragmentieren können. Diese Daten wurden in Kollaboration mit den Gruppen Prof. Dr. Tad Holak und Prof. Dr. Robert Huber (Martinsried) durch kristallographische Versuchsansätze bestätigt. Neben den gut untersuchten konventionellen Aktinisoformen liegen oft auch Aktinvarianten vor, deren Funktion bisher unbekannt ist. Der Modellorganismus Dictyostelium discoideum besitzt mit seinen 41 Aktinen und Aktin-verwandten Proteinen ein umfangreiches „Aktinom”. Dazu gehört auch das Protein Filaktin (105 KDa), eine besonders außergewöhnliche Aktinvariante, die neben der konservierten Aktin-ähnlichen Domäne zusätzlich einen verlängerten N-Terminus mit einer definierten Domänenstruktur besitzt. Homologe von Filaktin wurden bisher in Dictyosteliden und einigen pathogenen Entamoeben identifiziert. Im zweiten Teil dieser Arbeit wurden die Funktionen von Filaktin in vivo und in vitro analysiert. Immunfluoreszenz Experimente zeigen, dass Filaktin mit konventionellem Aktin kolokalisiert und zusätzlich im Zytoplasma an Vesikel-artigen Strukturen zu sehen ist. Ein besonderes Merkmal von Filaktin ist zudem, dass es Teil von Stress-induzierten, intranukleären, stäbchenförmigen Proteinaggregaten, sogenannten „nuclear rods” ist. Für umfassende in vitro Experimente wurden rekombinante Filaktin Konstrukte mithilfe von Sf9 Insektenzellen exprimiert. Die Ergebnisse von fluorometrischen und viskometrischen Experimenten deuten darauf hin, dass die Aktin Domäne von Filaktin Aktinmonomere sequestrieren oder sogar Aktinfilamente verkappen kann. Gelfiltrationsexperimente ergaben zusätzlich, dass Filaktin wohl als Tetramer vorliegt. Außerdem verbinden Protein-Interaktionsstudien Filaktin mit dem ESCRT Signalweg (Endosomal Sorting Complexes Required for Transport), der unter anderem bei der Entstehung von multivesikulären Körpern wichtig ist. Zusammengefasst besteht das Mikrofilamentsystem vermutlich hauptsächlich aus konventionellen Aktinen, wohingegen spezielle Aktinvarianten andere zusätzliche und sogar Zytoskelett-unabhängige Funktionen übernehmen können.
7
Broderick, Michael James Francis. "The utrophin-actin interface." Thesis, University of Glasgow, 2005. http://theses.gla.ac.uk/30889/.
Full textAbstract:
The spectrin superfamily is a diverse group of proteins variously involved in cross- linking, bundling and binding to the F-actin cytoskeleton. These proteins are modular in nature and interaction with actin occurs, at least in part, via CH domain containing ABDs. The actin binding domains of the spectrin superfamily proteins are all very similar in overall structure however the functions of the individual proteins differ greatly. Utrophin is a member of the spectrin superfamily and has been used extensively to investigate and model the association of actin-binding domains with F- actin; however, much controversy exists as to whether binding occurs when the domain is in an open or a closed conformation. The data herein specifically investigates the importance of the utrophin ABD inter- CH domain linker to the conformation of the domain and how this domain associates with F-actin. We provide evidence that this particular region of the ABD is particularly sensitive to mutation and that the conformation of the domain when in solution cannot be altered by affecting the electrostatic environment surrounding the protein. It has been assumed previously that the utrophin ABD adopts a closed and compact configuration in solution similar to the fimbrin crystal structure conformation; however we present evidence that suggests this is not the case. It has been proposed that the utrophin ABD may open from this closed conformation to bind F-actin in a more open manner, we present data that demonstrates that opening of the domain is not essential to F-actin binding and that there is very little conformation change associated with the domain upon interaction with F-actin. It appears that the utrophin ABD can bind F actin in two conformations. This supports current models of utrophin ABD binding where interaction with F-actin occurs in either an open or closed conformation. The data presented here provides an interesting insight into the utrophin ABD/F-actin interaction and raises many questions regarding the evaluation of current binding models. Future research stemming from this work will serve to further the understanding of how utrophin and related actin-binding proteins interact with F-actin.
8
McGrath, James L. (James Lionel). "Actin dynamics in the cell cytoplasm and the role of actin associated proteins." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/50446.
Full text
9
Singh, Anish D. "Regulation and function of the non-muscle [beta]-actin and [gamma]-actin genes." Phd thesis, Department of Paediatrics and Child Health, Faculty of Medicine, 2004. http://hdl.handle.net/2123/11556.
Full text
10
Kruth, Karina Annette. "Effects of three deafness-causing gamma-actin mutations on actin structure and function." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1475.
Full textAbstract:
Hearing requires proper function of the auditory hair cell, which is critically dependent upon its actin-based cytoskeletal structure. Eleven point mutations in gamma (γ) nonmuscle actin have been identified as causing progressive autosomal dominant nonsyndromic hearing loss (DFNA20/26); however, exactly why these mutations lead to deafness is unclear. Organization, stability, and repair of the hair cell cytoskeleton are highly regulated by actin binding proteins (ABPs), and two of the mutations, K118M and K118N, are located near an area of the actin monomer believed to be important in actin-ABP interactions. A third mutation, D51N, is located in a region of the actin monomer believed to be important for polymerization dynamics and stability in filamentous actin. I therefore hypothesized that the K118M/N mutations cause hearing loss due to impaired regulation of the actin cytoskeleton within the hair cell, whereas the D51N mutation likely interferes with polymerization dynamics and actin filament stability or flexibility.
The goal of my thesis was to investigate the effects of these three mutations, K118M, K118N, and D51N, on actin dynamics and regulation. I show in Chapter 2 that the K118M/N mutations differentially affect regulation of actin by the Arp2/3 complex, but also, surprisingly, that the K118N mutation accelerates polymerization dynamics. Chapter 3 details a continued investigation of the K118M/N mutations to ascertain their effects on actin structure and dynamics, particularly with regard to how they may affect polymerization. Chapter 4 provides both an in vivo and in vitro characterization of the D51N mutation, which revealed that not only does the mutation significantly accelerate actin polymerization, it also causes significant effects on yeast mitochondrial morphology and cytoskeletal regulation.
The work detailed within this thesis provides new insight into how the K118M/N and D51N mutations affect actin structure and dynamics and how these effects could lead to deafness. More importantly, this work provides a strong foundation for many future studies, ranging from structural investigation of the K118N and D51N actins as F-actin mimics, to the potential role of mitochondria in actin-based disease.
11
Hayot, Caroline. "Mise au point d'une stratégie pharmacologique originale pour l'obtention de composés anti-cancéreux anti-migratoires." Doctoral thesis, Universite Libre de Bruxelles, 2006. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210860.
Full textAbstract:
La migration cellulaire est une étape clé intervenant à un stade précoce de la dissémination des cellules cancéreuses dans l’organisme, et est donc responsable de la formation des métastases qui tuent environ nonante pourcent des patients atteints de cancer. De plus, ces cellules migrantes résistent à l’apoptose grâce à l’activation constitutive de voies de signalisation anti-apoptotiques, et développent donc une résistance vis-à-vis des traitements anti-cancéreux actuels qui sont généralement pro-apoptotiques. Nous avons pris pour cible ce processus de migration cellulaire dans l’espoir d’identifier des agents anti-migratoires qui permettraient de lutter contre la formation des métastases et de restaurer chez les cellules migrantes une certaine sensibilité aux traitements pro-apoptotiques.Dans la première partie de notre travail, nous avons analysé les effets anti-angiogéniques et anti-migratoires des agents anti-tubuline. Nous avons confirmé que le Taxol® présentait une action anti-angiogénique à des concentrations non-cytotoxiques. Nous avons ensuite démontré que d’autres agents anti-tubuline exerçaient la même action que le Taxol®, et que cette action leur était spécifique. Nous avons montré que certains de ces agents étaient également capables de réduire la migration de lignées cellulaires tumorales, toujours à des concentrations non-cytotoxiques, et que cette action pouvait s’exercer via une affectation du cytosquelette d’actine.
Dans la deuxième partie du présent travail, nous avons démontré l’importance de la mise au point d’une approche pharmacologique originale permettant l’identification de composés à action anti-migratoire puisque l’outil utilisé par le U.S. National Cancer Institute pour le criblage de nouvelles molécules anti-cancéreuses ne permet pas de discerner l’activité anti-migratoire des molécules testées.
Enfin dans la troisième partie de ce travail, après avoir souligné la raison du choix de l’actine comme cible pour inhiber la migration cellulaire, nous avons développé une stratégie pharmacologique in vitro originale de découverte de composés anti-actine à activité anti-migratoire. Grâce à une approche divisée en plusieurs étapes, à savoir un essai de cytotoxicité, une étude de la dynamique de la polymérisation d’actine en tubes ou sur cellules entières, et des essais de migration bidimensionnelle sur cellules individuelles ou sur population cellulaire, nous avons montré d’une part que des molécules connues pour affecter le cytosquelette actinique étaient capables d’affecter la migration cellulaire, et d’autre part que la méthodologie que nous avons développée permettait bien l’identification de composés affectant l’actine et capables de réduire la migration de cellules tumorales. En conclusion, cette stratégie in vitro pourrait être utilisée dans l’identification de nouvelles molécules à activité anti-migratoire pour lutter contre le cancer.
Doctorat en sciences pharmaceutiques
info:eu-repo/semantics/nonPublished
12
Storz, Tobias-Alexander. "Statische und dynamische Lichtstreuung an Lösungen von Aktinfilamenten." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963434861.
Full text
13
Glenz, Martin H. "Heterodimere F-actin capping proteine." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=981289509.
Full text
14
Osborn, Eric A. (Eric Alan) 1975. "Actin remodeling in motile cells." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28600.
Full textAbstract:
Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004.Includes bibliographical references.
Non-muscle cell shape change and motility depend primarily on the dynamics and distributions of cytoplasmic actin. In cells, actin cycles between monomeric and polymeric phases tightly regulated by actin binding proteins that control cellular architecture and movement. Here, we characterize actin remodeling in shear stress stimulated endothelial cells and in actin networks reconstituted with purified proteins. Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Alignment requires 24 h of exposure to flow, but the cells respond within minutes to flow by diminishing their movements by 50%. Although movement slows, actin filament turnover times and the amount of polymerized actin in cells decreases, increasing actin filament remodeling in individual cells composing a confluent endothelial monolayer to levels used by disperse, non-confluent cells for rapid movement. Hours later, motility returns to pre-shear stress levels, but actin remodeling remains highly dynamic in many cells. We conclude that shear stress initiates a cytoplasmic actin remodeling response that is used to modify endothelial cell shape instead of bulk cell translocation. We determine the steady state dynamics of purified actin filament networks in the entangled state and after orthogonal cross-linking with filamins using a novel, non-perturbing fluorescence system. Human filamin A or Dictyosteliun discoidium filamin slow actin filament turnover by [approximately] 50% and recruit much of a significant population of actin oligomers that we measure are present in polymerized purified actin solutions into the immobile filament fraction. Surprisingly, these observations occur at very low stoichiometry to actin, approximately requiring only one
(cont.) filamin molecule bound per actin filament, similar to the amount required for actin filament gelation in vitro. Networks formed with filamin truncates localize this activity to the actin binding domain and reveal that dimerization and orthogonal cross-linking are not required for dynamic stabilization. Re-expression of filamin A with or without the actin binding domain in human melanoma cells that naturally lack this protein support the findings in purified actin networks. These results indicate that filamin cross-linking stabilizes filament dynamics by, slowing filament subunit cycling rates and by either decreasing spontaneous filament fragmentation or promoting filament annealing.
by Eric A. Osborn.
Ph.D.
15
McGrath, James L. (James Lionel). "Measuring actin dynamics in endothelium." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/38015.
Full text
16
Ahrens, S. "Extracellular actin in innate immunity." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1433762/.
Full textAbstract:
The innate immune system is capable of responding to tissue injury by detecting the abnormal exposure of intracellular, often ubiquitously expressed, molecules referred to as damage-associated molecular patterns (DAMPs). DAMPs are normally sequestered inside healthy cells but become exposed to the extracellular environment upon loss of membrane integrity during cell death. Exposed DAMPs are then recognised by receptors of the innate immune system. One such DAMP receptor is DNGR-1 (CLEC9A), which is expressed on CD8+ DCs, a rare but specialised subset of DCs involved in regulating T cell responses. Loss of DNGR-1 on CD8+ DCs impairs cross-presentation of dead-cell associated antigens to CD8+ T-cells indicating that DNGR-1 couples DAMP recognition to the generation of cytotoxic T cell immune responses. Prior to the work presented in this thesis, the DAMP ligand for DNGR-1 had not been identified. Using a variety of experimental approaches, I demonstrated that this ligand corresponds to filamentous actin (F-actin), a component of the cytoskeleton of all cells. Given its extreme evolutionary conservation, abundance and ubiquitous expression, as well as its association with tissue damage in a range of inflammatory conditions, actin possesses ideal DAMP characteristics. Thus, I further hypothesised that actin may engage receptors other than DNGR-1 and act as a universal and evolutionarily ancient sign of cell damage that is more generally detected by metazoans as a means of inducing sterile inflammation and/or tissue repair. In order to test this hypothesis, I made use of the Drosophila melanogaster model system. I found that actin injection into flies stimulates strong activation of the stress-induced JAK/STAT pathway without triggering immune defence pathways. Given the conservation of innate defence mechanisms in invertebrates and vertebrates, it is tempting to speculate that understanding the recognition of actin in Drosophila melanogaster will provide useful insights into the induction of inflammation in mammals.
17
Marzook, Noorul Bishara. "Lights, Camera, Actin: Divergent roles of beta- and gamma-cytoplasmic actin in vaccinia virus infection." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/16859.
Full textAbstract:
Intracellular pathogens require access to host cells for their replication and spread. The host actin cytoskeleton represents a physical barrier to them, although many have evolved ways to circumvent, or hijack, this system to their advantage. Vaccinia virus (VACV) can manipulate the host actin cytoskeleton to facilitate dissemination. It expedites its cellular egress by nucleating actin beneath its particles, creating filamentous actin (F-actin) comets that propel virions across the cell surface. Tagging VACV proteins with fluorescent markers is used to study virus-host interactions, and define host molecular mechanisms, particularly within dynamic actin pathways. To this end, we developed a novel, optimised protocol for generating recombinant VACV. We then used this to create a recombinant VACV expressing Lifeact-GFP, a fluorescent marker that can highlight F-actin on infection, enabling live tracking of VACV comets via real-time fluorescence microscopy. F-actin comprises two cytoplasmic isoforms: β- and γ-actin. Despite differing only by four N-terminal amino acids, recent studies outlined their distinct localisations and functions in cell lines and whole organisms. We performed a detailed study of their roles in VACV actin-based motility. Initiation of comet formation appears to have an essential requirement for β-actin. Conversely, speed of virus movement was enhanced when γ-actin was silenced, indicating a moderating effect on the rate of actin polymerisation by this isoform. We aimed to define the site of β-actin dependency for VACV actin-based motility by biochemical pull-down assays. This represents the first investigation of the role of actin isoforms in pathogen motility, implicating the importance of their relative distribution in initiating VACV-induced actin comets. Further studies may underpin the importance of β- over γ-actin in other organisms using actin-based motility, providing a route to curb actin-assisted spread of intracellular pathogens.
18
Ferrer, Jorge M. 1976. "Mapping the actin and actin binding proteins interactions : from micromechanics to single molecule force spectroscopy." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40950.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.
Mechanical forces play an important role in cell morphology, orientation, migration, adhesion and can even induce apoptosis. The eukaryotic cell is equipped with a dynamic frame, known as the cytoskeleton, that provides the cell's structural integrity in order to sustain and react to such forces. Therefore, understanding the mechanical properties of the cytoskeleton is an important step towards building models describing cell behavior. Filamentous actin (F-actin), as one of the major constituents of the cytoskeleton, has been the target of extensive in vitro studies to determine its mechanical properties in bulk. However, there is still a lack in the understanding of how the molecular interactions between F-actin and the proteins that arrange these filaments into networks regulate the dynamic properties of the cytoskeleton Here we present a novel, single molecule assay to test the rupture force of a complex formed by an actin binding protein (ABP) linking two actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin, a crosslinker, and a-actinin, a bundler. We measured rupture forces of 28-73 pN and 30-56 pN for filamin/actin and a-actinin/actin respectively, suggesting that the former is a slightly stronger interaction. Moreover, since no ABP unfolding events were observed at our force levels, our results suggest that ABP unbinding is a more relevant mechanism than unfolding for the temporal regulation of the mechanical properties of the actin cytoskeleton. In addition, we explore the micro-scale properties of F-actin networks reconstituted in vitro.
(cont.) Using imaging and microrheology techniques we characterized the effects of filament length and degree of crosslinking on the structural arrangement and mechanical properties of F-actin networks. We found that the mechanical properties of these networks are length-scale dependent. Also, when probed with active methods, the F-actin networks exhibited strain hardening followed by a gradual softening at forces -30 pN, in good agreement with the single molecule rupture force of 28-73 pN. Thus, with the combination of single molecule and network studies, we can expand the knowledge-base on the regulation and control of the cellular machinery starting from the molecular building blocks.
by Jorge M. Ferrer.
Ph.D.
19
Sofia, Denise Michela. "Characterization of profilin and actin depolymerizing factors expression and function in the testis." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:16-opus-76643.
Full text
20
Jeffries, T. E. "The use of pyrene-labelled actin to investigate the interaction between actin and other muscle proteins." Thesis, University of Bristol, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384441.
Full text
21
Wear, Martin Alexander. "Biochemical studies on gelsolin : actin complexes and experiments to form a minimal, defined-length actin filament." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/23253.
Full textAbstract:
In this thesis we report the formation of a putative "capped-actin-minifilament" complex. This was created by combining the gelsolin:actin2 ternary (G:A2) and the actin:DNaseI binary (A:D) complexes together (1:1 molar ratio) under polymerising conditions (100mM KC1; 2mM MgC12 in the presence of 0.2mM CaC12). Size-exclusion data indicates the formation of a significantly larger species (in relation to G:A2), with an apparent stiochiometry of G:A3:D (gelsolin:actin:DNaseI, respectively). Kinetic and modelling evidence (Weber et al, 1994) suggests that the binding of two DNaseI molecules at the pointed-end of filaments is not possible due to a steric clash. Using DNaseI's ability to bind at the pointed-ends of actin monomers, we have probed the disposition of the monomers in the G:A2 complex. Size-exclusion, native-PAGE and fluorescence enhancement data (performed with NBD-Actin) indicate the formation of a stable, co-operative complex with a stoichiometry of G:A2:D2 (gelsolin:actin:DNaseI, respectively). The apparent Kd of A:D binding to the gelsolin:actin binary complex (G:A) is ~ 50nM, and is equivalent to the binding of G-Actin alone (Kd ~ 39nM). Our data are consistent with DNaseI having no effect on the interaction of actin monomers with gelsolin, and with the spatial orientation of monomers in G:A2 being different to those at the barbed-end of filaments. In contrast to this, data from fluorescence enhancement experiments with rhodamin-phalloidin (an actin filament specific binding molecule) provide evidence for the actin monomers, within the putative "minifilament", being in a filamentous-like conformation. We observe a specific binding, with significant levels of fluorescence enhancement (~ 3 - 4 fold), of rhodamine-phalloidin to the "minifilament", with an apparent Kd of ~4.6mM. We have also examined the possibility of replacing gelsolin (as the barbed-end capping protein) with a cloned polypeptide fragment derived from tensin, a component of focal adhesions.
22
Matthews, Jermey N. A. "Thermodynamics and relaxation during actin polymerization." College Park, Md. : University of Maryland, 2005. http://hdl.handle.net/1903/2346.
Full textAbstract:
Thesis (Ph. D.) -- University of Maryland, College Park, 2005.Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
23
Moniz, de Sa Mario. "The evolution of angiosperm actin genes." Thesis, University of Ottawa (Canada), 1995. http://hdl.handle.net/10393/10062.
Full textAbstract:
Forty-four actin genes from five angiosperm species, whose evolutionary relationships are well characterized, were PCR-cloned and sequenced. Phylogenetic analysis of 34 of these actin genes, along with those previously published, indicate that plant actin genes are monophyletic and underwent a rapid radiation early in land plant evolution. Six sets of putative orthologues have been identified and their sequences were used to calculate rates of evolution. The synonomous rate of substitution $(5.44\times10\sp{-9}$/site/year) is similar to that of other nuclear protein-encoding genes but the non-synonomous rate $(0.13\times10\sp{-9}$/site/year) is 4-10 times higher than that of vertebrate actin genes. Relative-rate tests do not support a faster rate in plants than in vertebrates. Evidence is also provided that some members of the actin multigene family in maize are undergoing gene conversion. Finally, we show that some plant actin genes have undergone intron loss probably as a consequence of a gene conversion event between the genomic copy and the reverse transcript.
24
Niedermayer, Thomas. "On the depolymerization of actin filaments." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2013/6360/.
Full textAbstract:
Actin is one of the most abundant and highly conserved proteins in eukaryotic cells. The globular protein assembles into long filaments, which form a variety of different networks within the cytoskeleton. The dynamic reorganization of these networks - which is pivotal for cell motility, cell adhesion, and cell division - is based on cycles of polymerization (assembly) and depolymerization (disassembly) of actin filaments. Actin binds ATP and within the filament, actin-bound ATP is hydrolyzed into ADP on a time scale of a few minutes. As ADP-actin dissociates faster from the filament ends than ATP-actin, the filament becomes less stable as it grows older. Recent single filament experiments, where abrupt dynamical changes during filament depolymerization have been observed, suggest the opposite behavior, however, namely that the actin filaments become increasingly stable with time. Several mechanisms for this stabilization have been proposed, ranging from structural transitions of the whole filament to surface attachment of the filament ends.
The key issue of this thesis is to elucidate the unexpected interruptions of depolymerization by a combination of experimental and theoretical studies. In new depolymerization experiments on single filaments, we confirm that filaments cease to shrink in an abrupt manner and determine the time from the initiation of depolymerization until the occurrence of the first interruption. This duration differs from filament to filament and represents a stochastic variable. We consider various hypothetical mechanisms that may cause the observed interruptions. These mechanisms cannot be distinguished directly, but they give rise to distinct distributions of the time until the first interruption, which we compute by modeling the underlying stochastic processes. A comparison with the measured distribution reveals that the sudden truncation of the shrinkage process neither arises from blocking of the ends nor from a collective transition of the whole filament. Instead, we predict a local transition process occurring at random sites within the filament.
The combination of additional experimental findings and our theoretical approach confirms the notion of a local transition mechanism and identifies the transition as the photo-induced formation of an actin dimer within the filaments. Unlabeled actin filaments do not exhibit pauses, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis.
This destabilization can be identified with an acceleration of the depolymerization prior to the interruption. In the final part of this thesis, we theoretically analyze this acceleration to infer the mechanism of ATP hydrolysis. We show that the rate of ATP hydrolysis is constant within the filament, corresponding to a random as opposed to a vectorial hydrolysis mechanism.Aktin ist eines der am häufigsten vorkommenden und am stärksten konservierten Proteine in eukaryotischen Zellen. Dieses globuläre Protein bildet lange Filamente, die zu einer großen Vielfalt von Netzwerken innerhalb des Zellskeletts führen. Die dynamische Reorganisation dieser Netzwerke, die entscheidend für Zellbewegung, Zelladhäsion, und Zellteilung ist, basiert auf der Polymerisation (dem Aufbau) und der Depolymerisation (dem Abbau) von Aktinfilamenten. Aktin bindet ATP, welches innerhalb des Filaments auf einer Zeitskala von einigen Minuten in ADP hydrolysiert wird. Da ADP-Aktin schneller vom Filamentende dissoziiert als ATP-Aktin, sollte ein Filament mit der Zeit instabiler werden. Neuere Experimente, in denen abrupte dynamische Änderungen während der Filamentdepolymerisation beobachtet wurden, deuten jedoch auf ein gegenteiliges Verhalten hin: Die Aktinfilamente werden mit der Zeit zunehmend stabiler. Mehrere Mechanismen für diese Stabilisierung wurden bereits vorgeschlagen, von strukturellen Übergängen des gesamten Filaments bis zu Wechselwirkungen der Filamentenden mit dem experimentellen Aufbau. Das zentrale Thema der vorliegenden Dissertation ist die Aufklärung der unerwarteten Unterbrechungen der Depolymerisation. Dies geschieht durch eine Kombination von experimentellen und theoretischen Untersuchungen. Mit Hilfe neuer Depolymerisationexperimente mit einzelnen Filamenten bestätigen wir zunächst, dass die Filamente plötzlich aufhören zu schrumpfen und bestimmen die Zeit, die von der Einleitung der Depolymerisation bis zum Auftreten der ersten Unterbrechung vergeht. Diese Zeit unterscheidet sich von Filament zu Filament und stellt eine stochastische Größe dar. Wir untersuchen daraufhin verschiedene hypothetische Mechanismen, welche die beobachteten Unterbrechungen verursachen könnten. Die Mechanismen können experimentell nicht direkt unterschieden werden, haben jedoch verschiedene Verteilungen für die Zeit bis zur ersten Unterbrechung zur Folge. Wir berechnen die jeweiligen Verteilungen, indem wir die zugrundeliegenden stochastischen Prozesse modellieren. Ein Vergleich mit der gemessenen Verteilung zeigt, dass der plötzliche Abbruch des Depolymerisationsprozesses weder auf eine Blockade der Enden, noch auf einen kollektiven strukturellen Übergang des gesamten Filaments zurückzuführen ist. An Stelle dessen postulieren wir einen lokalen Übergangsprozess, der an zufälligen Stellen innerhalb des Filaments auftritt. Die Kombination von weiteren experimentellen Ergebnissen und unserem theoretischen Ansatz bestätigt die Vorstellung eines lokalen Übergangsmechanismus und identifiziert den Übergang als die photo-induzierte Bildung eines Aktindimers innerhalb des Filaments. Nicht fluoreszenzmarkierte Aktinfilamente zeigen keine Unterbrechungen, woraus folgt, dass ältere Filamente in vivo durch die ATP-Hydrolyse destabilisiert werden. Die Destabilisierung zeigt sich durch die Beschleunigung der Depolymerisation vor der Unterbrechung. Im letzten Teil der vorliegenden Arbeit untersuchen wir diese Beschleunigung mit theoretischen Methoden, um auf den Mechanismus der ATP-Hydrolyse zu schließen. Wir zeigen, dass die Hydrolyserate von ATP innerhalb des Filaments konstant ist, was dem sogenannten zufälligen Hydrolysemechanismus entspricht und im Gegensatz zum sogenannten vektoriellen Mechanismus steht.
25
Thangavelu, Madan. "The actin gene family of tobacco." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335212.
Full text
26
Begg, Carolyn E. "Studies of mouse actin genomic clones." Thesis, University of Glasgow, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280030.
Full text
27
Carrel, Hyman A. (Hyman Andrew) 1979. "Giant vesicles compressed by actin polymerization." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/16646.
Full textAbstract:
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Physics, 2004.Includes bibliographical references (p. 45-46).
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Actin polymerization plays a critical role in generating propulsive force to drive many types of cell motility. The discovery of actin based motility of the bacterial pathogen Listeria monocytogenes has lead to clearer understandings of the essential ingredients required for cell motility. The biophysical mechanisms by which these proteins generate forces is the subject of intense investigation. A novel system to study force generation by this polymerization engine is introduced by combining the well characterized mechanical properties of synthetic Giant Vesicles with the well understood biochemistry of actin polymerization. Giant Vesicles mimic the structural features of eukaryotic cell membranes. We find that Giant Vesicles coated with a protein that catalyzes actin polymerization form thick actin shells which produce a compressive force. The polymerization force directed at the membrane interface causes the membrane to rupture. In the resulting collapse we find that the shell thickens inward with a constant radial velocity and is characterized by radial lines of lipid and actin. We show that actin polymerization is the primary force driving the collapse.
by Hyman A. Carrel.
S.M.
28
Shin, Jennifer Hyunjong 1974. "Dynamics and statics of actin assemblies." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/27043.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.Includes bibliographical references (p. 95-101).
The conversion of chemical energy into mechanical forces that powers cell movements is a ubiquitous theme across biology. The acrosome reaction of Limulus sperm is a simple example of such a dynamical transformation where a 60 [mu]m-long crystalline bundle of actin filaments, tightly cross-linked by actin bundling protein scruin, straightens from a coiled conformation and extends from the cell in five seconds. This spring-like mechanism represents a third type of actin-based motility that is distinctly different from the better known polymerization or myosin-driven processes. To identify the basis and mechanism for this movement, we examine the possible sources of chemical and mechanical energy and show that the stored elastic energy alone is sufficient to drive the reaction. We also provide an estimate of the maximum force generated during the uncoiling by stalling the bundle using an agarose gel. Finally, we provide a simple mathematical model that rationalizes the dynamics of uncoiling. Motivated by the very stiff cross-linking in the bundle induced by scruin, we next turn to a model system of scruin mediated cross-linked actin networks where the elastic response is dominated by the properties of actin. While the biological significance of the actin cross-linking proteins is well documented, little is known about how bundling and cross-linking quantitatively affects the microstructure and mechanical properties of actin networks. We quantify the effect of scruin on actin networks using imaging techniques, co-sedimentation assays. multi-particle tracking (MPT), and bulk rheology and demonstrate how a simple entropic elasticity model for a semi-flexible polymer network explains the linear elastic regime of the actin-scruin network.
by Jennifer Hyunjong Shin.
Ph.D.
29
Lee, Hyungsuk. "Mechanical properties of F-actin network." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/50588.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Includes bibliographical references.
Cells sense, generate and respond to forces in their surroundings through cytoskeletal dynamics. Actin, the most abundant protein found in eukaryotic cells, is organized into various cytoskeletal structures that provide physical support for the cell and play important roles in numerous cellular processes. Assembly of F-actin into higher-order structures is regulated by over 100 actin binding proteins (ABPs). Although extensive measurements to estimate the mechanical properties of ABP/F-actin networks showed that they are nonlinear and viscoelastic, a full understanding of the origin of such fascinating behaviors is lacking. This thesis presents a multi-scale approach to identify the factors that determine the mechanical properties of F-actin networks from the macroscopic level to the single-molecule level. The mechanical properties of F-actin networks were probed by passive and active methods using optical tweezers. For the passive approach the thermal fluctuations of colloidal spheres are monitored to estimate the frequency-dependent complex shear modulus of an F-actin network. In the active approach, the response of an embedded microsphere to a driving force is tracked to obtain the strain-dependent viscoelasticity. The developed methods were applied to F-actin networks cross-linked with various ABPs such as filamin and a -actinin, with and without gelsolin to control filament length. Microstructures of those networks were also characterized in terms of filament length, mesh size, and degree of bundling.
(cont.) Comparison between cross-linked F-actin with two different length scales of actin filament suggested that network connectivity is another critical parameter in determining mechanical properties. To better understand how the cross-linking protein responds to an external force, a single molecule assay was used to measure the rupture force of a complex formed by an ABP filamin linking two actin filaments. Both force-induced unbinding and unfolding of filamin were observed at the critical force of 70 ± 23pN and 57 ± 19pN, respectively, although unbinding occurred more frequently. Similar pulling experiments were also performed on cross-linked F-actin networks and an abrupt transition was observed in the force trace indicating network rupture. The critical forces at transitions exhibited a similar loading-rate dependence to that observed for rupture forces in the single molecule measurements. Nonlinear behavior observed in strain-dependent microrheology was found to be irreversible. Combined results of molecular unbinding, network rupture, and irreversible network properties suggest that unbinding rather than unfolding is a dominant mechanism governing the mechanical properties of cross-linked F-actin networks. In addition, the mechanical behavior of F-actin networks subjected to an external prestress was investigated using a shear device. Visualization of sheared F-actin networks showed the structural evolution including mesh deformation, filament alignment, and network rupture.
(cont.) Measurement of mechanical properties as a function of external strain demonstrated that some regions exhibited strain-hardening while the others showed strain-softening. Aligned stretching of actin filaments observed at high strain seemed to play a role in strain-stiffening. By comparing the behaviors of an F-actin network cross-linked with wildtype and mutant FLNa, it was demonstrated how molecular structure of the ABP alters the mechanical behavior of F-actin network.
by Hyungsuk Lee.
Ph.D.
30
Robertson, Alec 1974. "Material properties of actin filament bundles." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/46628.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Includes bibliographical references (p. 119-127).
Actin is an ubiquitous structural protein fundamental to such biological processes as cell motility and muscle contraction. Our model system is the acrosomal process of the Limulus sperm which extends a 60 ýtm long actin bundle during reproduction. It is an example of a biological spring where the force of elongation derives from twist energy stored within the bundle during spermatogenesis. In addition to actin the acrosome comprises only one other protein: scruin, an actin-binding protein specific to Limulus that decorates and crosslinks actin filaments into a crystalline bundle. Our goal is to reconstitute the structure of the acrosome using these two proteins in order to further elucidate the role of scruin in actin bundle crosslinking.A multi-scale approach is presented wherein the bending rigidity of scruin bundles and their constituent filaments are probed individually, then inter-related by simple mechanical models. Material properties of filaments and bundles are measured using a combination of optical tweezers, electron and fluorescence microscopy. We find that scruin bundles reconstituted from acrosome fragments display an ordered structure, with a bending rigidity orders of magnitude higher than their individual filaments. Actin bundles formed by depletion exhibit similar behavior, revealing an intrinsic regime of coupled actin bundle formation. Bundle elastic moduli are eight orders of magnitude stiffer than reconstituted networks and an order of magnitude softer than the native acrosome, highlighting scruin's ability to dictate a wide range of material properties depending on the formation conditions.
by Alec P. Robertson.
Ph.D.
31
Brown, Jennifer. "Investigating the actin cytoskeleton in cancer." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7266/.
Full textAbstract:
Dynamic alterations in the actin cytoskeleton, under the regulation of the Rho/ROCK pathway, permit cell motility, cell-to-cell and cell-to-matrix adhesion, and have also been shown to participate in apoptosis and cell proliferation. These facets of cellular behaviour all have the capacity to become dysregulated in cancer; components of the Rho/ROCK pathway are known to play varying roles in these processes, both within primary tumours and within the tumour microenvironment. The LIM kinases are phosphorylated and activated by ROCK, leading to inactivation of cofilin and subsequent stabilisation of actin filaments. In addition, LIM kinase 2 serves as a p53 target and is upregulated in response to DNA damage. In some solid tumours (e.g. breast and prostate), LIM kinase levels are elevated. However, we found that LIM kinase 2 expression is downregulated in colon cancer, with a progressive reduction noted with advancing tumour stage. I found that LIMK2 expression in colon cancer is under epigenetic regulation, with hypermethylation of the promoters leading to transcriptional silencing; this implicates LIMK2 as a tumour suppressor gene in this context. This has potential translational implications as loss of LIMK2 could be utilised as a biomarker to stratify patients in the future. Elevated mechanical tension within the tumour microenvironment is known to be an adverse prognostic indicator due to its association with desmoplasia. ROCK activation has previously been shown to increase epidermal tissue stiffness and thickness, but little was known about the mechanisms by which this occurs. I found that ROCK activation leads to the deposition of extracellular matrix components, with a presumed consequent further increase in stromal stiffness. This indicates that a positive feedback cycle is established in the tumour microenvironment, maintaining a fibrotic stromal reaction that permits tumour progression. These results highlight the disparate roles that the actin cytoskeleton and constituents of the Rho/ROCK pathway play in tumour initiation and propagation, indicating the need for further research.
32
Lieleg, Oliver. "Model systems of the actin cortex." kostenfrei, 2008. http://mediatum2.ub.tum.de/doc/672641/672641.pdf.
Full text
33
Scoville, Damon Charles. "Filament dynamics and actin binding factors." Diss., Restricted to subscribing institutions, 2008. http://proquest.umi.com/pqdweb?did=1693066541&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
Full text
34
Hu, Xiaohua. "Actin polymerization dynamics at the leading edge." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/39940.
Full textAbstract:
Actin-based cell motility plays crucial role throughout the lifetime of an organism. While the dendritic nucleation model explains the initiation and organization of the actin network in lamellipodia, two questions need to be answered.
In this study, I reconstructed cellular motility in vitro to investigate how actin filaments are organized to coordinate elongation and attachment to leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel beads or thin glass nanofibers coated with N-WASP WCA domains. During sustained motility, physiological concentrations of Mg2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, filament capping, or actin shell formation. Addition of other types of crosslinkers restored both comet tail attachment and particle motility. We propose a model in which polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.
I lowered actin, profilin, Arp2/3, and CP concentrations to address the generation of actin filament orientation during the initiation of motility. In the absence of CP, Arp2/3 nucleates barbed ends that grow away from the nanofiber surface and branches remain stably attached to nanofiber. CP addition causes shedding of short branches and barbed end capture by the nanofiber. Barbed end retention by nanofibers is coupled with capping, indicating that WWCA
iii
and CP bind simultaneously to barbed ends. In pull-down assays, saturating CP addition only blocks WWCA binding to barbed end by half. Labeled WWCA bound to barbed ends with an affinity of 14 pM and unlabeled WWCA with an affinity of 75 pM. CP addition increased WWCA binding slightly at low CP concentrations and decreased WWCA binding to 50% at high CP concentrations. Molecular models of CP and WH2 domains bound respectively to the terminal and penultimate actin subunit showed no overlap and that CP orientation might blocks WWCA dissociation from the penultimate subunit. Simultaneous binding of CP and WWCA to barbed ends is essential to the establishment of filament orientation at the leading edge.Ph. D.
35
Gallinger, Julia [Verfasser], and Michael [Akademischer Betreuer] Schleicher. "WH2 domains and actin variants as multifunctional organizers of the actin cytoskeleton / Julia Gallinger. Betreuer: Michael Schleicher." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1043906355/34.
Full text
36
Buencamino, Raphael Hector Domingo. "Novel roles of actin binding proteins in Listeria monocytogenes actin-based motility revealed within a cellular context." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3297798.
Full text
37
Uhde, Jörg. "Mikrorheometrie passiver und aktiver Aktinnetzwerke." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972234802.
Full text
38
Baroni, Luciana. "Caracterização molecular da actina do Apicomplexa Neospora caninum." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-25022013-103829/.
Full textAbstract:
Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies, a NcAct tem maior identidade/similaridade com a actina de Eimeria tenella (97%/99%), seguida da actina de Plasmodium falciparum PfACT1 (93%/97%), da actina de Babesia bovis (86%/94%) e PfACT2 (80%/92%). Quando localizada com anticorpo anti-?-actina C4, NcAct apresenta-se em duas bandas de 43 e 45 kDa em gel de acrilamida 1D e em nove isoformas em gel de acrilamida 2D. Todas as identidades das bandas e spots foram confirmados por espectrometria de massas (MS/MS). Além disso, NcAct localiza-se, em sua maioria, na região periférica do taquizoíta de N. caninum e sua distribuição é alterada após incubação dos taquizoítas com 5 ?M de jasplakinolida (JAS) ou 2 ?M de citocalasina D (CytD). Por fim, por meio de ensaio de fracionamento de actina monomérica (actina-G) e filamentosa (actina-F), demonstramos que a JAS é capaz de aumentar a quantidade de actina-F em taquizoítas de N. caninum.Neospora caninum is an Apicomplexan protozoan that infects, among a whole range of intermediate hosts, bovine where it is emerging as a relevant cause of reproductive problems and abortion in dairy and beef cattle. As obligatory intracellular organisms, parasites from Apicomplexa Phylum use their own active locomotion system to move and invade host cells. This mechanism is driven by the actin/myosin motor known as gliding motility, localized between the plasma and the inner membrane complex. Studies involving this locomotion and invasion system have been conducted mainly in Toxoplasma gondii and Plasmodium spp. To our knowledge there is no publication involving actin in N. caninum, so this work was outlined and involved the cloning and expression of the sequence NcAct201-310, initiating the characterization of actin of N. caninum (NcAct). The sequence NcAct was obtained from the Database ToxoDB, and a comparison of actins from Apicomplexa-related revealed total identity of NcAct with TgACT1 (100% identity). With other species, NcAct has higher identity/similarity with Eimeria tenella actin (97%/99%), followed by Plasmodium falciparum actin PfACT1 (93%/97%), Babesia bovis actin (86%/94%) and PfACT2 (80%/92%). When localized with the antibody anti-?-actin C4, NcAct is presented as two bands of 43 and 45 kDa in 1D acrylamide gel and as nine isoforms in 2D acrylamide gel. All these findings were confirmed by mass spectrometry (MS/MS). Moreover, NcAct localizes predominantly in the peripheric region of N. caninum tachyzoites. This distribution is altered after incubation of the tachyzoites with 5 ?M of jasplakinolide (JAS) or 2 ?M of cytochalasin D (CytD). Finally through fractionating assay of monomeric (actin-G) and filamentous (actin-F), we demonstrated that JAS is capable of increasing the quantity of actin-F in N. caninum tachyzoites.
39
Wang, Hong. "Regulation of actin polymerization by cell-matrix adhesion complexes : a biochemical study of the talin-vinculin complex Integrin-bound talin head inhibits actin filament barbed-end elongation Talin and vinculin combine their individual activities to trigger actin assembly The C-terminal domain of EFA6A interacts directly with F-actin and assembles F-actin bundles." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS132.
Full textAbstract:
Pour migrer efficacement dans différents tissus, les cellules doivent détecter et s'adapter aux variations des propriétés mécaniques de leur environnement. Dans ce processus d’adaptation, les adhérences focales (AF) renforcent leur lien avec la matrice extracellulaire et le cytosquelette d'actine. En réponse à la force, l'association de la taline et de la vinculine pourrait renforcer l'ancrage de l'actine aux AF en contrôlant l'assemblage de l'actine par un mécanisme inconnu. Des études antérieures ont montré que la vinculine contient un seul domaine de liaison à l'actine (ABD) qui lie les filaments d’actine, coiffe les extrémités barbées des filaments d’actine et nuclée des filaments d'actine. La taline contient également trois ABD mais leur capacité à réguler l'assemblage de l'actine n'était pas connue avant ce projet. L'objectif global de ce projet de thèse était de déterminer les mécanismes précis par lesquels le complexe taline-vinculine contrôle l'assemblage de l'actine en réponse à la force. Dans une première partie de ce projet de thèse, préalable à l'étude du complexe taline-vinculine, j'ai terminé la caractérisation de la taline. J'ai démontré que le domaine ABD1 de la taline bloque l'allongement des extrémités barbées des filaments d'actine observés en microscopie à fluorescence (TIRFM), alors que ABD2 et ABD3 n'affectent pas la dynamique de l'actine. Dans la deuxième partie de ce projet, j'ai déterminé l'activité du complexe taline-vinculine. Parce que la force est nécessaire pour déclencher l'association de la vinculine à la taline, et parce que les deux protéines sont auto-inhibées, il a jusqu'à présent été difficile de déterminer la capacité du complexe taline-vinculine à réguler la polymérisation de l'actine. Par conséquent, nous avons d'abord conçu des mutants de taline et de vinculine qui s'associent de manière constitutive en un complexe stable. En combinant des études cinétiques en spectroscopie de fluorescence, des tests de liaison à l’actine et l’observation de filaments d’acine uniques en microscopie TIRF, nous avons déterminé les activités de ces mutants et de leurs complexes sur la dynamique de l'actine. Notre étude a d'abord révélé que les trois activités de la vinculine sont contrôlées par des contacts auto-inhibiteurs spécifiques. Nous montrons également que des suppressions d'hélice le long de la taline exposent les sites voisins de liaison à la vinculine, imitant ainsi l'étirement mécanique de la taline. L'association des mutants de taline aux mutants de vinculine forme un complexe qui nuclée des filaments coiffés à leur extrémité barbée. La caractérisation d'une série de complexes, dans lesquels la vinculine et la taline sont amputés de divers ABD, révèle la contribution de chaque protéine dans ce mécanisme. En conclusion, nos données suggèrent un mécanisme pour le renforcement de l'ancrage de l'actine dans les AF en réponse à la forceTo migrate efficiently in different tissues, cells must sense and adapt to variations of the mechanical properties of their environment. In this adaptive process, focal adhesions (FAs) strengthen their link with the extracellular matrix and the actin cytoskeleton. The force-dependent association of the actin binding proteins talin and vinculin could reinforce actin anchoring to FAs by controlling actin assembly though an unknown mechanism. Previous studies showed that vinculin contains a single actin-binding domain (ABD) which binds to actin filaments, caps actin filament barbed-ends and nucleates actin filaments. Talin also contains three ABDs but their ability to regulate actin assembly was not known before this project. The global objective of this PhD project was to determine the precise mechanisms by which the force-dependent talin-vinculin complex controls actin assembly. In a first part of this PhD project, as a prerequisite to the study of the talin-vinculin complex, I finished the characterization of talin. I demonstrated that the N-terminal ABD1 of talin blocks the elongation of actin filament barbed ends observed in fluorescent microscopy (TIRFM), whereas ABD2 and ABD3 do not affect actin dynamics. In the second and main part of this project, I determined the activity of the talin-vinculin complex. Because force is required to trigger vinculin association to talin, and because both proteins are autoinhibited, it has so far been difficult to determine the ability of the talin-vinculin complex to regulate actin polymerization. Therefore, we first designed talin and vinculin mutants that associate constitutively into a stable complex. By combining fluorescence spectroscopy, binding assays and single filament observation in TIRF microscopy, we determined the activities of these mutants and their complex on actin dynamics. Our study first revealed that the three activities of vinculin are controlled by specific auto-inhibitory contacts. We also show that helix deletions along the rod domain of talin expose neighboring vinculin-binding sites, mimicking the mechanical stretching of talin. The binding of these talin and vinculin mutants forms a complex that nucleates filaments capped at their barbed ends. The characterization of a series of complexes, in which vinculin and talin are deleted from various ABDs, reveals the contribution of each protein in this mechanism. Altogether our data suggest a mechanism for the force-dependent reinforcement of actin anchoring in FAs
40
Badaoui, Magid. "Multi-scale analysis of the mechanics of branched actin material." Electronic Thesis or Diss., Université Paris Cité, 2023. http://www.theses.fr/2023UNIP7074.
Full textAbstract:
La polymérisation de filaments d'actine contre une membrane peut générer des forces importantes entraînant l'endocytose chez la levure ou la formation de lamellipodes à l'extrémité de cellules motiles comme les kératocytes. Cette polymérisation est thermodynamiquement favorable car l'ajout d'un monomère s'accompagne d'une diminution du potentiel chimique. Cependant, la croissance du filament ralentie lorsqu'une contrainte lui est opposée, évaluant la force de décrochage à quelques pN. Si ce schéma est bien établi pour un filament unique, il n'est pas évident de le transposer à un réseau composé de centaines de filaments comme le lamellipodium. Plus généralement, comprendre l'émergence de caractéristiques à grande échelle à partir des propriétés moléculaires reste un défi majeur en biologie. Ainsi, l'objectif global de cette thèse est d'étudier l'émergence des propriétés mécaniques de l'actine ramifiée d'un point de vue numérique, théorique et statistique. Pour ce faire, nous nous appuyons sur des simulations numériques de réseaux à grande échelle dans lesquelles les caractéristiques macroscopiques peuvent être mesurées. Dans la première partie de notre travail, nous utilisons des simulations stochastiques (dynamique de Langevin) pour créer des réseaux ramifiés en croissance soumis à une contrainte mécanique externe, imitant ainsi la résistance de la matrice extracellulaire. Précisément, nous étudions comment les propriétés stationnaires du système sont déterminées à la fois par la force de décrochage et par la contrainte. Pour un réseau de filaments avec une force de décrochage infinie, la vitesse de croissance présente un maximum lorsque la contrainte tend vers zéro et diminue ensuite comme une loi de puissance de la contrainte. Une théorie mécanique des réseaux ramifiés fondée sur l'enchevêtrement des filaments s'accorde sur cette loi de puissance. La valeur maximale à faible contrainte peut être expliquée par la traînée du réseau, qui devient ici le principal facteur déterminant. Par l'étude de filaments plus réalistes nous montrons qu'il existe un seuil de force de décrochage à partir duquel le mouvement est possible. Ce seuil est proportionnel à la contrainte externe. Enfin, pour mieux comprendre le régime de faible contrainte, nous avons étudié les réseaux à croissance libre et avons montré qu'ils s'adaptent d'eux-mêmes en ralentissant et en devenant plus denses. Dans la deuxième partie, nous cherchons à quantifier l'information qui peut être obtenue à partir de statistiques réalisées sur de nombreuses simulations. Plus précisément, nous cherchons à identifier les combinaisons de paramètres (e.g. rigidité de l'actine, longueur du filament) qui influencent le plus les observables de notre système ramifié (e.g. densité, module d'Young). Afin de les identifier, nous appliquons des outils issus de la théorie de l'information aux statistiques générées par nos simulations, ces dernières ayant été répétées en appliquant de petites modifications aux paramètres. Sur la base de travaux antérieurs sur la dynamique des microtubules, nous avons calculé la matrice d'information de Fisher (MIF) associée qui permet de quantifier la dépendance observable-paramètre. En supposant que chaque observables suit localement une distribution normale, et grâce à une meilleure utilisation des statistiques de simulation, nous avons pu obtenir un calcul plus efficace de la MIF. L'analyse des vecteurs et valeurs propres de la MIF fournit une hiérarchie de modes de sensibilité dans l'espace des paramètres. Ces modes peuvent être interprétés géométriquement comme les directions pour lesquelles les caractéristiques du réseau sont le plus influencées par les paramètres. Ainsi, nous avons caractérisé notre système branché avec ses principaux modes de sensibilité, correspondant à une dimension effective de notre système, dont la valeur, deux, a été confirmée à l'aide de modèles analytiquesPolymerization of actin filaments against membranes can generate significant forces, leading to endocytosis in yeast or to the formation of lamellipodium protrusions at the leading edge of motiles cells like keratocytes. This polymerization is thermodynamically favorable, as the addition of a monomer is accompanied by a decrease in chemical potential. However, filament growth slows down when a stress opposes its growth, with a stall force of a few pN. While this picture is well established for a single filament, it is not clear how it translates to a network of hundreds of filaments like the lamellipodium. More generally, understanding the emergence of large-scale characteristics from molecular properties remains a major challenge in biology. Thus, the overall aim of this thesis is to understand the emergence of the mechanical properties of branched actin from a numerical, theoretical and statistical point of view. To achieve this, we rely on numerical simulations of large-scale networks in which macroscopic characteristics can be measured. In the first part of our work, we use stochastic simulations (Langevin dynamics) to create growing branched networks subjected to external mechanical stress, mimicking the resistance of the extracellular matrix. Specifically, we investigate on how stationary properties of the system are determined by both stall force and stress. For a network made of filaments with an infinite stall force, the growth velocity exhibits a maximum when the stress tends towards zero and then decreases as a power law of the stress. A mechanical theory of branched networks based on filaments entanglement agrees with this power law. The maximum value at low stress can be explained by the drag of the network, which becomes the main determining factor here. By studying more realistic filaments, we show that there is a stall force threshold above which movement is possible. This threshold is proportional to the external stress. Finally, to better understand the low-stress regime, we studied free-growing networks and showed that they self-adapt by slowing down and densifying. In the second part, we aim at quantify the information that can be obtained from statistics carried out on numerous simulations. More specifically, we seek to identify the combinations of parameters (e.g. actin stiffness, filament length) that most influence the observables of our branched system (e.g. density). To identify them, we apply tools from information theory to the statistics generated by our simulations, which have been repeated by applying small modifications to the parameters. Based on previous work on microtubule dynamics, we have calculated the associated Fisher information matrix (FIM), which quantifies the observable-parameter dependence. By assuming that each observable locally follows a normal distribution, and by a better use of simulation statistics, we were able to obtain a more efficient calculation of the FIM. Analysis of the eigenvectors and eigenvalues of the FIM provides a hierarchy of sensitivity modes in the parameter space, which can be interpreted geometrically as the direction in which network characteristics can be most influenced by the parameters. Thus, we characterize our branched system with its main sensitivity modes, corresponding to an effective dimension of our system. We find that this system has two effective dimensions, which has been confirmed using analytical models
41
Huber, Florian. "Emergent structure formation of the actin cytoskeleton." Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-86666.
Full textAbstract:
Anders als menschengemachte Maschinen verfügen Zellen über keinen festgeschriebenen Bauplan und die Positionen einzelner Elemente sind häufig nicht genau festgelegt, da die Moleküle diffusiven Zufallsbewegungen unterworfen sind. Darüber hinaus sind einzelne Bauteile auch nicht auf eine einzelne Funktion festgelegt, sondern können parallel in verschiedene Prozesse einbezogen sein. Basierend auf Selbstorganisation und Selbstassemblierung muß die Organisation von Anordnung und Funktion einer lebenden Zelle also bereits in ihren einzelnen Komponenten inhärent enthalten sein.
Die intrazelluläre Organisation wird zum großen Teil durch ein internes Biopolymergerüst reguliert, das Zytoskelett. Biopolymer-Netzwerke und –Fasern durchdringen die gesamte Zelle und sind verantworlich für mechanische Integrität und die funktionale Architektur. Unzählige essentielle biologische Prozesse hängen direkt von einem funktionierenden Zytoskelett ab.
Die vorliegende Arbeit zielt auf ein besser Verständnis und den Nachbau zweier verschiedener funktionaler Module lebender Zellen anhand stark reduzierter Modellsysteme. Als zentrales Element wurde Aktin gewählt, da dieses Biopolymer eine herausragende Rolle in nahezu allen eukaryotischen Zellen spielt.
Mit dem ersten Modellsystem wird der bewegliche Aktin-Polymerfilm an der Vorderkante migrierender Zellen betrachtet. Die wichtigsten Elemente dieser hochdynamischen Netzwerke sind bereits bekannt und wurden in dieser Arbeit benutzt um ein experimentelles Modellsystem zu etablieren. Vor allem aber lieferten detailierte Computersimulationen und ein mathematisches Modell neue Erkenntnisse über grundlegende Organisationsprinzipien dieser Aktinnetzwerke. Damit war es nicht nur möglich, experimentelle Daten erfolgreich zu reproduzieren, sondern das Entstehen von Substrukturen und deren Charakteristika auf proteinunabhängige, generelle Mechanismen zurückzuführen.
Das zweite studierte System betrachtet die Selbstassemblierung von Aktinnetzwerken durch entropische Kräfte. Aktinfilamente aggregieren hierbei durch Kondensation multivalenter Ionen oder durch Volumenausschluss hochkonzentrierter inerter Polymere. Ein neu entwickelter Experimentalaufbau bietet die Möglichkeit in gut definierten zellähnlichen Volumina, Konvektionseinflüsse zu umgehen und Aggregationseffekte gezielt einzuschalten. Hierbei wurden neuartige, regelmäßige Netzwerkstrukturen entdeckt, die bislang nur im Zusammenhang mit molekularen Motoren bekannt waren. Es konnte ferner gezeigt werden, dass die Physik der Flüssigkristalle entscheidend zu weiteren Variationen dieser Netzwerke beiträgt. Dabei wird ersichtlich, dass entstehende Netzwerke in ihrer Architektur direkt die zuvor herrschenden Anisotropien der Filamentlösung widerspiegeln.
42
Hsu, C. R. "Characterisation of a Salmonella actin-binding protein." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604683.
Full textAbstract:
Salmonella invasion protein C (SipC) and SipA bind actin directly. SipC inserts into the host cell plasma membrane from where it promotes effector delivery and is essential for Salmonella invasion. SipC-C (residues 200-409) directly nucleates actin polymerisation in vitro and induces cytoskeletal rearrangements at the leading edge when expressed in cells. By analysis a panel of SipC-C derivatives in transfected cells, a region spanning residues 377-409 was defined as necessary for actin reorganisation and leading-edge localisation. Residues 377-409 were sufficient to localise to zones of actin polymerisation in cells. SipC-N (residues 1-120) is sufficient to pair actin filaments in vitro and to induce elongated filopodia in cells. Two adjacent regions spanning residues 41-60 and 51-70 were identified as essential for inducing elongated filopodia. Corresponding proteins (SipC-NΔ41-60) and SipC-NΔ51-70) were expressed and purified. TEM of protein-F-actin mixtures revealed a deficiency in F-actin pairing. These data define the minimal N-terminal region required for filament pairing in vitro and corresponding filopodial elongation in cells. SipC derivatives containing equivalent deletions (SipC Δ41-60 and SipC Δ51-70) exhibited significantly reduced F-actin bundling activity. Derivatives with an extended deletion (SipC Δ31-70) exhibited an equivalent defect. TEM of a C-terminal truncate (SipC1-377) in complex with F-actin revealed an unanticipated defect in F-actin bundling, and a recombinant derivative containing a double deletion (SipC1-377 Δ31-70) was unable to bundle or pair F-actin. These data demonstrate that SipC-induced F-actin bundling requires unexpected interplay between the N-terminal and C-terminal domains. SipC is proposed to bundle F-actin via the N-terminal region to pair filaments and the C-terminal actin-binding domain to efficiently cluster filaments for formation of highly dense actin bundles.
43
Bai, Limiao, and 白利苗. "In silico simulation of actin-based motility." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B46429116.
Full text
44
Schnauß, Jörg. "Self-assembly effects of filamentous actin bundles." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-179722.
Full textAbstract:
Das Zytoskelett einer eukaryotischen Zelle besteht aus drei Hauptbestandteilen: Aktin, Intermediärfilamenten und Mikrotubuli. Die vorliegende Arbeit beschäftigt sich mit dem Protein Aktin, welches unter physiologischen Bedingungen dynamische Filamente durch Polymerisation ausbildet. Diese Filamente können sowohl in Netzwerken als auch Bündeln angeordnet werden. Diese Anordnungen bilden die Grundlage für eine Vielfalt von Strukturen zur Realisierung diverser zellulärer Funktionen. Konventionell wurde die Ausprägung solcher Strukturen durch zusätzliche Proteine erklärt, welche Aktin beispielsweise vernetzen oder sogar aktive, dissipative Prozesse durch ATP Hydrolyse ermöglichen. Durch diese Erklärungen prägte sich ein sehr komplexes Bild zellulärer Funktionen heraus. Die dissipative Natur der meisten Prozesse führte dazu, dass meist auf grundlegende physikalische Beschreibungen, welche auf nicht-dissipativen Gleichgewichtszuständen beruhen, verzichtet wurde. Diese Arbeit widmet sich solchen nicht-dissipativen Prozessen und beschreibt deren inhärente Bedeutung auch in aktiven, dissipativen Systemen.
Ein erstes Beispiel beschreibt die Generierung von kontraktilen Kräften in Aktinbündeln durch eine hohe makromolekulare Dichte der Umgebung. Diese hohe Dichte führt zu einem entropischen Effekt, welcher durch Volumenausschluss hochkonzentrierter inerter Polymere Aktinfilamente in Bündel ordnet. Werden diese Strukturen aus ihrem energetischen Minimum ausgelenkt, so entsteht eine rücktreibende Kraft, welche nach Ausschaltung der auslenkenden Kraft zu einer Kontraktion des gesamten Bündels führt. Dieses Bespiel zeigt klar, dass selbst in sehr einfachen Systemen äußerst komplexe Prozesse ablaufen können, welche konventionell mittels dissipativer Umwandlung von chemischer Energie in mechanische Arbeit beschrieben wurden.
Die Komplexität der Eigenschaften von Aktinbündeln nimmt zudem drastisch zu sobald zusätzliche Proteine mit eigenen mechanischen Eigenschaften das System beeinflussen. Zur Untersuchung eines solchen Mehrkomponentensystems wurden Aktinfilamente mittels transienter Vernetzungsproteine gebündelt. Versuche auf unterschiedlichen Zeitskalen zeigten klar differenzierbare mechanische Antworten auf induzierte, aktive Biegedeformationen. Im Falle kurzer Deformationen verhielt sich das System völlig elastisch, während für lange Deformationszeiten deutliche plastische Effekte auftraten. Als Ursprung dieser Plastizität wurde die dynamische Umordnung der Vernetzungsproteine identifiziert.
Jedoch führen nicht nur zusätzliche Proteine zu einer erhöhten Komplexität. Bereits die Anordnung von reinen Aktinbündeln in Netzwerke mittels entropischer Kräfte führt zu einer überraschenden Variabilität von entstehenden Mustern. Im besonderen Fokus dieser Untersuchung stehen Aster ähnliche Muster, welche regelmäßige Netzwerkstrukturen ausbilden und nur in Verbindung mit Aktin assoziierten Proteinen bekannt waren. Störungen der isotropen Ausgangssituation führen zu veränderter Musterbildung, welche die initiale Störung direkt widerspiegeln.
Mit den präsentierten Resultaten leistet die Arbeit einen wichtigen Beitrag zum Verständnis der Dynamik von Aktinbündeln sowie deren Interaktionen.
45
Förster, Florian. "Targeting the actin cytoskeleton with natural compounds." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-168914.
Full textAbstract:
Targeting the cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. Whereas drugs which address microtubule CSK such as vinca alkaloids or taxanes are well established in the clinic, compounds binding to the actin CSK are still far away from their therapeutical application. One reason might be the lacking knowledge on their mode of cytotoxicity and moreover their tumor specific mechanism of action.
We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity by actin targeting in different breast cancer cells, namely MCF7 and MDA-MB-231. Chondramide inhibits actin filament assembly and dynamics shown by a fluorescence-based analysis (FRAP) in whole cells and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases (-9 and -3). Detailed analysis revealed, that Chondramide induces apoptosis by enhancing the occurrence of mitochondrial permeability transition (MPT). Known MPT-modulators were found to be affected by Chondramide: Hexokinase II (HkII) bound to the voltage dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad was recruited to the mitochondria. Importantly, PKCε, a prosurvival serine/threonine kinase possessing an actin-binding site and known to regulate the HkII/VDAC interaction as well as Bad phosphoylation was identified as the link between actin CSK and apoptosis induction. PKCε which was found overexpressed in breast cancer cells accumulated in actin bundles induced by Chondramide and lost its activity.
The second goal of our work was to inform on a potential tumor specific action of actin binding agents such as Chondramide. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide induced apoptosis and notably express very low level of PKCε we claim that trapping PKCε via Chondramide induced actin hyperpolymerization displays tumor cell specificity.
Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCε, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCε signaling shown in a xenograft breast cancer model.
For the actin targeting compound Doliculide we could show that Doliculide impairs the dynamics of the actin CSK similar to Chondramide. Moreover, it reduces the proliferation rate and migration of cancer cells and also leads to the induction of apoptosis, thus Doliculide is also an interesting lead structure for further preclinical investigations.
46
McAnulty, Ciaron. "Actin gene variability in different porcine breeds." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250527.
Full text
47
Murtagh, Michael Stephen. "Electron microscopy of actin and thin filaments." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421969.
Full text
48
Gedge, Lucinda J. E. "Actin in the nuclei of mammalian cells." Thesis, University of Leeds, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400260.
Full text
49
Drouin, Guy. "The evolution of actin genes in potato." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330216.
Full text
50
Fox, Helen Mary. "Toca-1 driven actin polymerisation at membranes." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275610.
Full textAbstract:
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.