Academic literature on the topic 'Actin cytoskeleton'
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Journal articles on the topic "Actin cytoskeleton"
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Liu, Yi, Keyvan Mollaeian, and Juan Ren. "An Image Recognition-Based Approach to Actin Cytoskeleton Quantification." Electronics 7, no. 12 (December 17, 2018): 443. http://dx.doi.org/10.3390/electronics7120443.
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Quantification of the actin cytoskeleton is of prime importance to unveil the cellular force sensing and transduction mechanism. Although fluorescence imaging provides a convenient tool for observing the morphology of the actin cytoskeleton, due to the lack of approaches to accurate actin cytoskeleton quantification, the dynamics of mechanotransduction is still poorly understood. Currently, the existing image-based actin cytoskeleton analysis tools are either incapable of quantifying both the orientation and the quantity of the actin cytoskeleton simultaneously or the quantified results are subject to analysis artifacts. In this study, we propose an image recognition-based actin cytoskeleton quantification (IRAQ) approach, which quantifies both the actin cytoskeleton orientation and quantity by using edge, line, and brightness detection algorithms. The actin cytoskeleton is quantified through three parameters: the partial actin-cytoskeletal deviation (PAD), the total actin-cytoskeletal deviation (TAD), and the average actin-cytoskeletal intensity (AAI). First, Canny and Sobel edge detectors are applied to skeletonize the actin cytoskeleton images, then PAD and TAD are quantified using the line directions detected by Hough transform, and AAI is calculated through the summational brightness over the detected cell area. To verify the quantification accuracy, the proposed IRAQ was applied to six artificially-generated actin cytoskeleton mesh work models. The average error for both the quantified PAD and TAD was less than 1.22 ∘ . Then, IRAQ was implemented to quantify the actin cytoskeleton of NIH/3T3 cells treated with an F-actin inhibitor (latrunculin B). The quantification results suggest that the local and total actin-cytoskeletal organization became more disordered with the increase of latrunculin B dosage, and the quantity of the actin cytoskeleton showed a monotonically decreasing relation with latrunculin B dosage.
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Breuer, David, Alexander Ivakov, Arun Sampathkumar, Florian Hollandt, Staffan Persson, and Zoran Nikoloski. "Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles." Journal of The Royal Society Interface 11, no. 97 (August 6, 2014): 20140362. http://dx.doi.org/10.1098/rsif.2014.0362.
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The actin and microtubule (MT) cytoskeletons are vital structures for cell growth and development across all species. While individual molecular mechanisms underpinning actin and MT dynamics have been intensively studied, principles that govern the cytoskeleton organization remain largely unexplored. Here, we captured biologically relevant characteristics of the plant cytoskeleton through a network-driven imaging-based approach allowing us to quantitatively assess dynamic features of the cytoskeleton. By introducing suitable null models, we demonstrate that the plant cytoskeletal networks exhibit properties required for efficient transport, namely, short average path lengths and high robustness. We further show that these advantageous features are maintained during temporal cytoskeletal rearrangements. Interestingly, man-made transportation networks exhibit similar properties, suggesting general laws of network organization supporting diverse transport processes. The proposed network-driven analysis can be readily used to identify organizational principles of cytoskeletons in other organisms.
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Jack, R. M., R. M. Ezzell, J. Hartwig, and D. T. Fearon. "Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils." Journal of Immunology 137, no. 12 (December 15, 1986): 3996–4003. http://dx.doi.org/10.4049/jimmunol.137.12.3996.
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Abstract As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.
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Vaduva, Gabriela, Nancy C. Martin, and Anita K. Hopper. "Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton." Journal of Cell Biology 139, no. 7 (December 29, 1997): 1821–33. http://dx.doi.org/10.1083/jcb.139.7.1821.
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Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.
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Bezanilla, Magdalena, Amy S. Gladfelter, David R. Kovar, and Wei-Lih Lee. "Cytoskeletal dynamics: A view from the membrane." Journal of Cell Biology 209, no. 3 (May 11, 2015): 329–37. http://dx.doi.org/10.1083/jcb.201502062.
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Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane’s central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization.
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SAUMET, Anne, Nando de JESUS, Chantal LEGRAND, and Véronique DUBERNARD. "Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an αIIbβ3- or CD36-independent mechanism." Biochemical Journal 363, no. 3 (April 24, 2002): 473–82. http://dx.doi.org/10.1042/bj3630473.
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Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from α-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted α-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. αIIbβ3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking αIIbβ3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of αIIbβ3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
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Ballestrem, C., B. Wehrle-Haller, and B. A. Imhof. "Actin dynamics in living mammalian cells." Journal of Cell Science 111, no. 12 (June 15, 1998): 1649–58. http://dx.doi.org/10.1242/jcs.111.12.1649.
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The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.
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Holly, Stephen P., and Kendall J. Blumer. "Pak-Family Kinases Regulate Cell and Actin Polarization Throughout the Cell Cycle of Saccharomyces cerevisiae." Journal of Cell Biology 147, no. 4 (November 15, 1999): 845–56. http://dx.doi.org/10.1083/jcb.147.4.845.
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During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho–guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4–green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.
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Uray, Karen, Evelin Major, and Beata Lontay. "MicroRNA Regulatory Pathways in the Control of the Actin–Myosin Cytoskeleton." Cells 9, no. 7 (July 9, 2020): 1649. http://dx.doi.org/10.3390/cells9071649.
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MicroRNAs (miRNAs) are key modulators of post-transcriptional gene regulation in a plethora of processes, including actin–myosin cytoskeleton dynamics. Recent evidence points to the widespread effects of miRNAs on actin–myosin cytoskeleton dynamics, either directly on the expression of actin and myosin genes or indirectly on the diverse signaling cascades modulating cytoskeletal arrangement. Furthermore, studies from various human models indicate that miRNAs contribute to the development of various human disorders. The potentially huge impact of miRNA-based mechanisms on cytoskeletal elements is just starting to be recognized. In this review, we summarize recent knowledge about the importance of microRNA modulation of the actin–myosin cytoskeleton affecting physiological processes, including cardiovascular function, hematopoiesis, podocyte physiology, and osteogenesis.
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Vindin, Howard, Leanne Bischof, Peter Gunning, and Justine Stehn. "Validation of an Algorithm to Quantify Changes in Actin Cytoskeletal Organization." Journal of Biomolecular Screening 19, no. 3 (September 9, 2013): 354–68. http://dx.doi.org/10.1177/1087057113503494.
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The actin cytoskeleton plays an important role in most, if not all, processes necessary for cell survival. Given the fundamental role that the actin cytoskeleton plays in the progression of cancer, it is an ideal target for chemotherapy. Although it is possible to image the actin cytoskeleton in a high-throughput manner, there is currently no validated method to quantify changes in the cytoskeleton in the same capacity, which makes research into its organization and the development of anticytoskeletal drugs difficult. We have validated the use of a linear feature detection algorithm, allowing us to measure changes in actin filament organization. Its ability to quantify changes associated with cytoskeletal disruption will make it a valuable tool in the development of compounds that target the cytoskeleton in cancer. Our results show that this algorithm can quantify cytoskeletal changes in a cell-based system after addition of both well-established and novel anticytoskeletal agents using either fluorescence microscopy or a high-content imaging approach. This novel method gives us the potential to screen compounds in a high-throughput manner for cancer and other diseases in which the cytoskeleton plays a key role.
Dissertations / Theses on the topic "Actin cytoskeleton"
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Brown, Jennifer. "Investigating the actin cytoskeleton in cancer." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7266/.
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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.
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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.
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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.
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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.
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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.
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Kim, Taeyoon Ph D. Massachusetts Institute of Technology. "Simulation of actin cytoskeleton structure and rheology." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39875.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.Includes bibliographical references (p. 81-87).
Structures consisting of G-actin or other filament-forming monomers show a variety of morphologies with widely different properties in regard to pore size, degree of isotropy, and extent of cross-linking. These characteristics are primarily determined by the concentration and feature of proteins which cross-link filaments, but little is known how the filament-forming monomers and cross-linking proteins are organized in order to produce various network morphologies. In addition, it's generally known that mechanical force plays an important role in the physiology of eukaryote cells whose major structural component in cortex is actin cytoskeleton. Thus, understanding the origin of viscoelasticity of cross-linked networks should be crucial to figure out the exact role of cytoskeletal behaviors in many cellular functions. Here, we introduce a Brownian dynamics (BD) simulation model in three dimensions in which actin monomers polymerize into a filament and become cross-linked by two types of cross-linking molecules that constitute either perpendicular or parallel cross-links. We evaluate the influences of system parameters on the morphology of resultant networks. Some scaling behaviors that are independent of the specific choice of most parameters appear.
(cont.) Additionally, the modified model is employed to investigate the viscoelastic property of actin-like network by tracking the trajectories of filaments. This method is theoretically more direct and more precise than micro-bead rheology used in experiments. The viscoelastic property appears to be highly affected by characteristics of cross-linking molecules, average filament length, and concentration of actin monomers. Our model has the high potential as a BD model that can be applied for investigating a variety of actin-related phenomena after further refinement and modification.
by Taeyoon Kim.
S.M.
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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.
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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.
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Speldewinde, Shaun. "Prions, autophagy, ageing and actin cytoskeleton in yeast." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/prions-autophagy-ageing-and-actin-cytoskeleton-in-yeast(03085d7f-283a-40e1-bcf7-d9533ff2e2fc).html.
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Prions are infectious protein entities capable of self-replication. Prions are the causal agents behind the transmissible spongiform encephalopathies causing neurodegeneration and death in affected organisms. Prions have been identified in yeast with the best-characterized prions being [PSI+] and [PIN+], whose respective native proteins are the Sup35 translation termination factor and Rnq1 (function unknown). Autophagy is a cellular housekeeping mechanism mediating the degradation of damaged proteins and superfluous organelles. It is a highly sequential process regulated by autophagy related genes (ATGs). Autophagy has also been implicated in the clearance of amyloidogenic proteins including prions. However, the mechanistic basis underlying this activity is poorly understood, and a key objective of this project was to characterize how autophagy prevents spontaneous prion formation. Our study found that the deletion of core ATGs correlated with an increase in de novo [PSI+] and [PIN+] formation as well as Sup35 aggregation. Enhancement of autophagic flux through spermidine treatment attenuated the increased levels of de novo [PSI+] formation in mutants that normally show elevated levels of [PSI+] formation. Defective autophagy correlated with increased oxidatively damaged Sup35 in an atg1 mutant whereas anaerobic growth abrogated the increased [PSI+] formation in the atg1 mutant to wild-type levels. Our data suggest that autophagy serves a protective role in the clearance of oxidatively damaged Sup35 proteins that otherwise has a higher propensity towards [PSI+] prion formation. We also investigated the role of prion formation and autophagy during yeast chronological ageing which is the time that non-dividing cells remain viable. Prion diseases are associated with advanced age which correlates with a decline in cellular protective mechanisms including autophagy. Our study found an age dependent increase in the frequency of de novo [PSI+] formation with chronological age of yeast cells, more so in an atg1 mutant relative to the wild-type. Autophagy competent cells carrying the [PSI+] and [PIN+] prions also had improved chronological lifespan relative to prion free cells and atg1 cells. Cells carrying the [PSI+] prion elicited elevated autophagic flux that may promote improved lifespan thus suggesting a beneficial role of the [PSI+] prion during chronological ageing. The actin cytoskeleton provides the structural framework essential for a multitude of cellular processes to occur. We investigated the role of the Arp2/3 complex responsible for branching of actin filaments towards prion formation. Knockout mutants of the nucleation promoting factors of the Arp2/3 complex, in particular the abp1 mutant, showed reduced de novo [PSI+] formation and Sup35 aggregation under basal and oxidative stress conditions. Similarly, treatment with latrunclin A, an actin monomer-sequestering drug also abrogated de novo [PSI+] formation. Colocalization studies revealed that Sup35 often does not colocalize with Rnq1, a marker for the insoluble protein deposit (IPOD) in an abp1 mutant. This suggests a role for the Abp1 protein in the efficient transport of Sup35 molecules to the IPOD that may facilitate de novo [PSI+] prion formation under vegetative states and oxidant challenges.
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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.
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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
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Thodey, Catherine. "Actin cytoskeleton dynamics mediate sugar response in Arabidopsis thaliana." Thesis, University of East Anglia, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518364.
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Price, Leo Sebastian. "Secretion and the actin cytoskeleton in rat mast cells." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307776.
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Filippi, Beatrice Maria. "Cellular effects of phosphoinositide derivatives on the actin cytoskeleton." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424620.
Full textBooks on the topic "Actin cytoskeleton"
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Jockusch, Brigitte M., ed. The Actin Cytoskeleton. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-46371-1.
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Mannherz, Hans Georg, ed. The Actin Cytoskeleton and Bacterial Infection. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-50047-8.
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D, Lappalainen Pekka Ph, ed. Actin-monomer-binding proteins. Austin, Tex: Landes Bioscience, 2007.
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E, Estes James, Higgins Paul J, and International Conference on the Biophysics, Biochemistry, and Cell Biology of Actin (1992 : Troy, N.Y.), eds. Actin: Biophysics, biochemistry, and cell biology. New York: Plenum Press, 1994.
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G, Dos Remedios Cristobal, and Chhabra Deepak, eds. Actin-binding proteins and disease. New York: Springer, 2008.
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G, Dos Remedios Cristobal, and Chhabra Deepak, eds. Actin-binding proteins and disease. New York: Springer, 2008.
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J, Staiger C., ed. Actin: A dynamic framework for multiple plant cell functions. Dordrecht: Kluwer Academic Publishers, 2000.
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Sing, Cierra Nicole. Aging Actin' Up: A novel aging determinant regulates the actin cytoskeleton, nutrient sensing, and lifespan in Saccharomyces cerevisiae. [New York, N.Y.?]: [publisher not identified], 2021.
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1933-, Sugi Haruo, and Pollack Gerald H, eds. Mechanism of myofilament sliding in muscle contraction. New York: Plenum Press, 1993.
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Malapitan, Irish Ann. Mapping an F-actin and cytoskeletal binding region in the basic domain of the mouse LSP1 protein. Ottawa: National Library of Canada, 1994.
Find full textBook chapters on the topic "Actin cytoskeleton"
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Isenberg, Gerhard. "Actin and Actin-Associated Proteins." In Cytoskeleton Proteins, 25–149. Berlin, Heidelberg: Springer Berlin Heidelberg, 1995. http://dx.doi.org/10.1007/978-3-642-79632-6_8.
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Wang, Juan, Ruihui Zhang, Ming Chang, Xiaolu Qu, Min Diao, Meng Zhang, and Shanjin Huang. "Actin Cytoskeleton." In Cell Biology, 1–28. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4614-7881-2_6-1.
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Bershadsky, Alexander D., and Juri M. Vasiliev. "Systems of Actin Filaments." In Cytoskeleton, 13–78. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4684-5278-5_2.
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Baluška, F., and S. Mancuso. "Actin Cytoskeleton and Action Potentials: Forgotten Connections." In The Cytoskeleton, 63–83. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-33528-1_5.
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Dugina, Vera, Richard Arnoldi, Paul A. Janmey, and Christine Chaponnier. "ACTIN." In Cytoskeleton and Human Disease, 3–28. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-788-0_1.
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Noegel, A. A., B. Köppel, U. Gottwald, W. Witke, R. Albrecht, and M. Schleicher. "Actin and Actin-Binding Proteins in the Motility of Dictyostelium." In The Cytoskeleton, 117–26. Berlin, Heidelberg: Springer Berlin Heidelberg, 1995. http://dx.doi.org/10.1007/978-3-642-79482-7_13.
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Kelber, Jonathan A., and Richard L. Klemke. "The Actin Cytoskeleton." In Cellular Domains, 197–212. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118015759.ch12.
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Amos, Linda A., and W. Bradshaw Amos. "Actin Filaments." In Molecules of the Cytoskeleton, 42–55. London: Macmillan Education UK, 1991. http://dx.doi.org/10.1007/978-1-349-21739-7_3.
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Aktories, Klaus, Carsten Schwan, and Alexander E. Lang. "ADP-Ribosylation and Cross-Linking of Actin by Bacterial Protein Toxins." In The Actin Cytoskeleton, 179–206. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/164_2016_26.
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Viita, Tiina, and Maria K. Vartiainen. "From Cytoskeleton to Gene Expression: Actin in the Nucleus." In The Actin Cytoskeleton, 311–29. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/164_2016_27.
Full textConference papers on the topic "Actin cytoskeleton"
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Liu, Yi, and Juan Ren. "Modeling and Control of Dynamic Cellular Mechanotransduction: Part I — Actin Cytoskeleton Quantification." In ASME 2018 Dynamic Systems and Control Conference. American Society of Mechanical Engineers, 2018. http://dx.doi.org/10.1115/dscc2018-9180.
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Living cells respond to external stimuli through the reorganization of the actin cytoskeleton, and the actin cytoskeleton significantly affects the cellular mechanical behavior. However, due to the lack of approaches to actin cytoskeleton quantification, the dynamics of mechanotransduction is still poorly understood. In this study, we propose an image recognition-based quantification (IRQ) approach to actin cytoskeleton quantification. IRQ quantifies the actin cytoskeleton through three parameters: the partial actin-cytoskeletal deviation (PAD), the total actin-cytoskeletal deviation (TAD) and the average actin-cytoskeletal intensity (AAI). First, Canny and Sobel edge detectors are applied to skeletonize the actin cytoskeleton images, then PAD and TAD are quantified using the direction of lines detected by Hough transform, and AAI is calculated through the summational brightness over the detected cell area. For validation, six different actin cytoskeleton meshwork models were generated to verify the quantification accuracy of IRQ. The average error for both the quantified PAD and TAD was less than 1.22°. Then IRQ was implemented to quantify the actin cytoskeleton of NIH/3T3 cells treated with an F-actin inhibitor. The quantification results suggest that the local and total actin-cytoskeletal organization of treated cells were more disordered than untreated cells, and the quantity of the actin cytoskeleton decreased significantly after the F-actin treatment.
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Wen, Shin-Min, and Pen-hsiu Grace Chao. "Spatial Actin Structure Does Not Correlate With Nuclear Organization." In ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14167.
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Cells in situ exhibit a great variety of morphologies that intimately relates to phenotypic controls. Cell morphology regulates cytoskeletal organization, which in turn influences nuclear shape and organization [1–4]. The actomyosin cytoskeleton is connected to a structure known as the linker of nucleoskeleton and cytoskeleton (LINC) complex located on the nuclear membrane. LINC is believed to transmit deformation of the actin cytoskeleton into the nucleus and nucleoskeleton, change nuclear shape as well as chromatin conformation, and modulate gene expression [5, 6]. Khatau and coworkers reported a structure of apical actin dome, called the actin cap, that controls nuclear deformation through LINC [7]. In addition, actin stress fibers hves been shown to compress the nucleus laterally and increase chromatin condensation [4]. Based on these findings, we hypothesize that there is a spatial correlation between the actin cytoskeleton and chromatin density. In the current study, we investigated the role of actin cytoskeleton in nuclear deformation with respect to the z-axis. We found no spatial relationships between actin structure and nuclear deformation or chromatin condensation, suggesting that the actomyosin cytoskeleton acts globally to influence nuclear structure and additional structural components may contribute to the actin-nucleus mechanical coupling.
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Dutta, Surjendu Bikash, Anders Kokkvoll Engdahl, Stefan Belle, Wolfgang Hübner, Mark Schüttpelz, Thomas Huser, and Francesco Dell'Olio. "Waveguide chip based super-resolution microscopy for T cell imaging." In Integrated Photonics Research, Silicon and Nanophotonics. Washington, D.C.: Optica Publishing Group, 2022. http://dx.doi.org/10.1364/iprsn.2022.itu1b.6.
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Imaging and the quantitative estimation of T cell actin cytoskeletal dynamics are important to describe immunological processes. This study presents waveguide chip based super-resolution imaging of the filamentous actin cytoskeleton of Jurkat T cells.
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Kiran, Kranthi, Sanjay Govindjee, and Mohammad R. K. Mofrad. "On the Cytoskeleton and Soft Glassy Rheology." In ASME 2007 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2007. http://dx.doi.org/10.1115/sbc2007-176736.
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Cytoskeleton is an integrated system of biomolecules, providing the cell with shape, integrity, and internal spatial organization. Cytoskeleton is a three-dimensional (3-D) network consisting of a complex mixture of actin filaments, intermediate filaments and microtubules that are collectively responsible for the main structural properties and motilities of the cell. A wide range of theoretical models have been proposed for cytoskeletal mechanics, ranging from continuum models for cell deformation to actin filament-based models for cell motility [1]. Numerous experimental techniques have also been developed to quantify cytoskeletal mechanics, typically involving a mechanical perturbation to the cell in the form of either an imposed deformation or force and observation of the static and dynamic response of the cell. These experimental measurements along with new theoretical approaches have given rise to several theories for describing the mechanics of living cells, modeling the cytoskeleton as a simple mechanical elastic, viscoelastic, or poro-viscoelastic continuum, tensegrity (tension integrity) network incorporating discrete structural elements that bear compression, porous gel or most recently soft glassy material. In this paper, we will revisit cytoskeleton as a soft glassy material and give insights in to new dynamic relationships for cytoskeleton.
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Allen, Kathleen B., and Bradley Layton. "A Mechanical Model for Cytoskeleton and Membrane Interactions in Neuronal Growth Cones." In ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-42008.
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Revealing the molecular events of neuronal growth is critical to obtaining a deeper understanding of nervous system development, neural injury response, and neural tissue engineering. Central to this is the need to understand the mechanical interactions among the cytoskeleton and the cell membrane, and how these interactions affect the overall growth mechanics of neurons. Using ANSYS, the force produced by a cytoskeletal protein acting against a deformable membrane was modeled, and the deformation, stress, and strain were computed for the membrane. Parameters to represent the flexural rigidities of the well-studied actin and tubulin cytoskeletal proteins as well as the mechanical properties of neuronal growth cones were used in the simulations. Our model predicts that while a single actin filament is able to produce a force sufficient to cause membrane deformation and thus growth, it is also possible that the actin filament may cause the membrane to rupture, if a dilatational strain of more than 3–4% occurs. Additionally, neurotoxins or pharmaceuticals that alter the mechanical properties of either the cell membrane or cytoskeletal proteins could disrupt the balance of forces required for neurons to not only push out and grow correctly, but also to sustain their shapes as high-aspect-ratio structures once growth is complete. Understanding how cytoskeletal elements have coevolved mechanically with their respective cell membranes will yield insights into the events that gave rise to the sequences and quaternary structures of the major cytoskeletal elements.
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Dangaria, Jhanvi H., and Peter J. Butler. "Interaction of Shear Stress, Myosin II, and Actin in Dynamic Modulation of Endothelial Cell Microrheology." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192947.
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The endothelial cell (EC) cytoskeleton mediates several biological functions such as adhesion, migration, phagocytosis, cell division, and mechanosensitivity. These functions are carried out in part through dynamic cytoskeletal polymerization, modulation of crosslinking, and development of tension between intracellular organelles and the extracellular matrix via focal adhesion plaques. One important component of the cytoskeleton is actin which polymerizes into filaments and is thought to be prestressed by virtue of crosslinking proteins such as α-actinin, filamin and myosin II molecular motors. Additionally, actomyosin interaction has been hypothesized to act as a stress dissipation mechanism by virtue of dynamic crossbridging which facilitates actin diffusion through the polymer network of the cytoplasm (Humphrey et al., 2002).
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Shibatay, N., K. Tanaka, K. Okamoto, and T. Onji. "REORGANIZATION OF ACTIN AND MYOSIN IN THE ACTIVATED PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643539.
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This study was done to clarify the intracellular dynamic arrangements of myosin(My) and actin(Ac) in activation process of human platelets (PLs) from unactivated to activated stage (clot retraction) in electron microscopy. The observation of unactivated PLs was done either in the fresh whole blood fixed directly with 0.1 % glutaraldehyde or in PLs isolated by gel filtration of platelet rich plasma(PRP) containing prostaglandin I2 (10 ng/ml). The isolated PLs mounted on a glass cover slip were used as activated PLs (adrerent ones). The contracted PLs were prepared in PRP incubated with thrombin (0.5 u/ml) and 20 mM CaCl- for 10-60 min. Treating PLs with 0.15 % Triton X-100 containing 0.05 % glutaraldehyde produced cytoskeleton. My and F-Ac were identified by an indirect immuno-cytochemical method using the specific antibody (rabbit IgG) against PL-My and protein A-gold and by demonstration of in “arrow-head” decoration by Ishikawa's method using skeletal meromyosin (HMM), respectively. [Results] (1) Unactivated PLs. Mys in monomer or oligomer distributed homogenously in scare association with cytoskeleton. Cytoskeletons were exclusively composed of F-Ac networks of crossolinked short filaments which were thinly distributed in the cytoplasm with partial connection to the cell membrance. (2) Surface activated spreading PLs. PLs adhered to the glass cover slip in dendritic forms. Mys were densely located around granulomere and formed linear arrays associated with F-Ac filaments of the cytoskeleton surrounding the granulomere and running straightly in cytoplasm. (3) Contracted PLs. Activated PLs protruded several filopodia in which networks or bundles of F-Ac filaments were found connecting to extracellular fibrin strand through cell membrene. Microfilaments formed arrow-head decoration with HMM pointing toward the cell body. The cytoskeleton in contracted PLs contained thick filaments of My-polymers attaching to F-Ac filaments end by end. It is concluded that the reorganization of Ac-My is the basis for the shape change, secretion and clot retraction of activated PLs.
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Bidone, Tamara C., Marco A. Deriu, Francesco Mastrangelo, Giacomo Di Benedetto, Monica Soncini, and Umberto Morbiducci. "Elastic Network Modeling of Actin Filaments." In ASME 2010 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2010. http://dx.doi.org/10.1115/sbc2010-19074.
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Cell mechanics depends on the mechanical properties of actin microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs), that build the cytoskeleton. Actin microfilaments are the most abundant components and play significant roles in various cellular processes [1]. Among them, the mechanical properties of MFs are essential for the functions of the cytoskeleton and are directly related to their molecular architecture.
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Horiguchi, Atsushi, and Toshihiko Shiraishi. "Study on a Cell Mechanosensing System by Measuring Structural Deformation and Biochemical Response." In ASME 2015 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2015. http://dx.doi.org/10.1115/imece2015-51456.
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Mechanical stimulation induces new bone formation in vivo and promotes the metabolic activity and the gene expression of osteoblasts in vitro. It was reported that biochemical signals of osteoblasts to sense mechanical stimulation are activated according to their actin cytoskeletal deformation. However, there have been not so many researches on the relationship between cytoskeletal deformation and biochemical response. Here we show an original method to investigate a cell mechanosensing system and the quantitative relationship between the deformation of cytoskeletal structure and the change of intracellular calcium ion concentration as biochemical response in a living cell stimulated by a micropipette. Gene transfection of green fluorescent protein to osteoblastic cells enabled visualization of actin in cells. When local deformation was applied to a single osteoblastic cell by a micropipette, the displacement distribution of cytoskeletal structure in the whole cell was automatically obtained from the two images of the cell before and after deformation by using Kanade-Lucas-Tomasi (KLT) method. Intracellular calcium ion response to mechanical stimulation was measured as the spatial and temporal changes of intensity of Fura Red loaded to a cell. As a result, we obtained the quantitative relationship between structural deformation and biochemical response of a cell and found that the change of calcium ion concentration increases with increasing the displacement of actin cytoskeleton. It indicates that the deformation of actin cytoskeleton is highly related to the cell mechanosensing system.
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Oswald, Elizabeth S., Pen-hsiu Grace Chao, J. Chloe Bulinski, Gerard A. Ateshian, and Clark T. Hung. "The Role of Microtubule Organization in Chondrocyte Response to Osmotic Loading." In ASME 2007 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2007. http://dx.doi.org/10.1115/sbc2007-176634.
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The cytoskeleton, including actin filaments and microtubules, provides chondrocytes with structure, cytoplasmic organization, and intracellular transport. The cytoskeleton is known to be involved in cellular responses to physiologic mechanical and osmotic loading signals, including morphological changes and mechanostransduction [1, 2]. Here, we examine microtubule (MT) involvement in volume response of chondrocytes to osmotic loading, as well as organization of stable MT with hypoosmotic loading. We also explore the hypothesis that chondrocytes from different zones of cartilage possess cytoskeletons with different properties, which help explain variations in their volume response to osmotic loading in situ and in vitro [3].
Reports on the topic "Actin cytoskeleton"
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Sadot, Einat, Christopher Staiger, and Zvi Kam Weizmann. functional genomic screen for new plant cytoskeletal proteins and the determination of their role in actin mediated functions and guard cells regulation. United States Department of Agriculture, January 2003. http://dx.doi.org/10.32747/2003.7587725.bard.
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The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids sequence). In addition, several publications demonstrated the recognition of mammalian cytoskeleton by plant cytoskeletal binding proteins and vice versa. The major achievements described here are: 1. The development of an automated microscope equipped with fast laser auto-focusing for high magnification and a software controlling 6 dimensions; X, Y position, auto focus, time, color, and the distribution and density of the fields acquired. This system is essential for the high throughput screen. 2. The construction of an extremely competent YFP library efficiently cloned (tens of thousands of clones collected, no empty vectors detected) with all inserts oriented 5't03'. These parameters render it well representative of the whole transcriptome and efficient in "in-frame" fusion to YFP. 3. The strategy developed for the screen allowing the isolation of individual positive cDNA clones following three rounds of microscopic scans. The major conclusion accomplished from the work described here is that the concept of using mammalian host cells for fishing new plant cytoskeletal proteins is feasible and that screening system developed is complete for addressing one of the major bottlenecks of the plant cytoskeleton field: the need for high throughput identification of functionally active cytoskeletal proteins. The new identified plant cytoskeletal proteins isolated in the pilot screen and additional new proteins which will be isolated in a comprehensive screen will shed light on cytoskeletal mediated processes playing a major role in cellular activities such as cell division, morphogenesis, and functioning such as chloroplast positioning, pollen tube and root hair elongation and the movement of guard cells. Therefore, in the long run the screen described here has clear agricultural implications.
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Ramesh, Vijaya. Neurofibromatosis 2 Tumor Suppressor Protein, Merlin, in Cellular Signaling to Actin Cytoskeleton. Fort Belvoir, VA: Defense Technical Information Center, October 2000. http://dx.doi.org/10.21236/ada395581.
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Philosoph-Hadas, Sonia, Peter B. Kaufman, Shimon Meir, and Abraham H. Halevy. Inhibition of the Gravitropic Shoot Bending in Stored Cut Flowers Through Control of Their Graviperception: Involvement of the Cytoskeleton and Cytosolic Calcium. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7586533.bard.
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Original objectives: The basic goal of the present project was to study the mechanism involved in shoot graviperception and early transduction, in order to determine the sequence of events operating in this process. This will enable to control the entire process of gravity-induced differential growth without affecting vertical growth processes essential for development. Thus, several new postulated interactions, operating at the perception and early transduction stages of the signaling cascade leading to auxin-mediated bending, were proposed to be examined in snapdragon spikes and oat shoot pulvini, according to the following research goals: 1) Establish the role of amyloplasts as gravireceptors in shoots; 2) Investigate gravity-induced changes in the integrity of shoot actin cytoskeleton (CK); 3) Study the cellular interactions among actin CK, statoliths and cell membranes (endoplasmic reticulum - ER, plasma membrane - PM) during shoot graviperception; 4) Examine mediation of graviperception by modulations of cytosolic calcium - [Ca2+]cyt, and other second messengers (protein phosphorylation, inositol 1,4,5-trisphosphate - IP3). Revisions: 1) Model system: in addition to snapdragon (Antirrhinum majus L.) spikes and oat (Avena sativa) shoot pulvini, the model system of maize (Zea mays) primary roots was targeted to confirm a more general mechanism for graviperception. 2) Research topic: brassinolide, which were not included in the original plan, were examined for their regulatory role in gravity perception and signal transduction in roots, in relation to auxin and ethylene. Background to the topic: The negative gravitropic response of shoots is a complex multi-step process that requires the participation of various cellular components acting in succession or in parallel. Most of the long-lasting studies regarding the link between graviperception and cellular components were focused mainly on roots, and there are relatively few reports on shoot graviperception. Our previous project has successfully characterized several key events occurring during shoot bending of cut flowers and oat pulvini, including amyloplast displacement, hormonal interactions and differential growth analysis. Based on this evidence, the present project has focused on studying the initial graviperception process in flowering stems and cereal shoots. Major conclusions and achievements: 1) The actin and not the microtubule (MT) CK is involved in the graviperception of snapdragon shoots. 2) Gravisensing, exhibited by amyloplast displacement, and early transduction events (auxin redistribution) in the gravitropic response of snapdragon spikes are mediated by the acto-myosin complex. 3) MTs are involved in stem directional growth, which occurs during gravitropism of cut snapdragon spikes, but they are not necessary for the gravity-induced differential growth. 4) The role of amyloplasts as gravisensors in the shoot endodermis was demonstrated for both plant systems. 5) A gravity-induced increase in IP.
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Syed, Aleem. Spatial and temporal dynamics of receptor for advanced glycation endproducts, integrins, and actin cytoskeleton as probed with fluorescence-based imaging techniques. Office of Scientific and Technical Information (OSTI), January 2016. http://dx.doi.org/10.2172/1342583.
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Stoyanova, Tihomira, Veselina Uzunova, Albena Momchilova, Rumiana Tzoneva, and Iva Ugrinova. The Treatment of Breast Cancer Cells with Erufosine Leads to Actin Cytoskeleton Reorganization, Inhibition of Cell Motility, Cell Cycle Arrest and Apoptosis. "Prof. Marin Drinov" Publishing House of Bulgarian Academy of Sciences, January 2021. http://dx.doi.org/10.7546/crabs.2021.01.11.
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Philosoph-Hadas, Sonia, Peter Kaufman, Shimon Meir, and Abraham Halevy. Signal Transduction Pathway of Hormonal Action in Control and Regulation of the Gravitropic Response of Cut Flowering Stems during Storage and Transport. United States Department of Agriculture, October 1999. http://dx.doi.org/10.32747/1999.7695838.bard.
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Original objectives: The basic goal of the present project was to increase our understanding of the cellular mechanisms operating during the gravitropic response of cut flowers, for solving their bending problem without affecting flower quality. Thus, several elements operating at the 3 levels o the gravity-induced signal transduction pathway, were proposed to be examined in snapdragon stems according to the following research goals: 1) Signaling: characterize the signal transduction pathway leading to the gravitropic response, regarding the involvement of [Ca2+]cyt as a mediator of IAA movement and sensitivity to auxin. 2) Transduction by plant hormones: a) Examine the involvement of auxin in the gravitropic response of flower stems with regard to: possible participation of auxin binding protein (ABP), auxin redistribution, auxin mechanism of action (activation of H+-ATPase) mediation by changes in [Ca2+]cyt and possible regulation of auxin-induced Ca2+ action b: calmodulin-activated or Ca2+-activated protein kinases (PK). b) Examine the involvement of ethylene in the gravitropic response of flower stems with regard to auxin-induced ethylene production and sensitivity of the tissue to ethylene. 3) Response: examine the effect of gravistimulation on invertase (associated with growth and elongation) activity and invertase gene expression. 4) Commercial practice: develop practical and simple treatments to prevent bending of cut flowers grown for export. Revisions: 1) Model systems: in addition to snapdragon (Antirrhinum majus L.), 3 other model shoe systems, consisting of oat (Avena sativa) pulvini, Ornithogalun 'Nova' cut flowers and Arabidopsis thaliana inflorescence, were targeted to confirm a more general mechanism for shoot gravitropism. 2 Research topics: the involvement of ABP, auxin action, PK and invertase in the gravitropic response of snapdragon stems could not be demonstrated. Alternatively, the involvement in the gravity signaling cascade of several other physiological mediators apart of [Ca2+]cyt such as: IP3, protein phosphorylation and actin cytoskeleton, was shown. Additional topics introduced: starch statolith reorientation, differential expression of early auxin responsive genes, and differential shoot growth. Background to the topic: The gravitropic bending response of flowering shoots occurring upon their horizontal placement during shipment exhibits a major horticultural problem. In spite of extensive studies in various aboveground organs, the gravitropic response was hardly investigated in flowering shoots. Being a complex multistep process that requires the participation of various cellular components acting in succession or in parallel, analysis of the negative gravitropic response of shoot includes investigation of signal transduction elements and various regulatory physiological mediators. Major achievements: 1) A correlative role for starch statoliths as gravireceptors in flowering shoot was initially established. 2) Differentially phosphorylated proteins and IP3 levels across the oat shoe pulvini, as well as a differential appearance of 2 early auxin-responsive genes in snapdragon stems were all detected within 5-30 minutes following gravistimulation. 3) Unlike in roots, involvement of actin cytoskeleton in early events of the gravitropic response of snapdragon shoots was established. 4) An asymmetric IAA distribution, followed by an asymmetric ethylene production across snapdragon stems was found following gravistimulation. 5) The gravity-induced differential growth in shoots of snapdragon was derived from initial shrinkage of the upper stem side and a subsequent elongation o the lower stem side. 6) Shoot bending could be successfully inhibited by Ca2+ antagonists (that serve as a basis for practical treatments), kinase and phosphatase inhibitors and actin-cytoskeleton modulators. All these agents did not affect vertical growth. The essential characterization of these key events and their sequence led us to the conclusion that blocking gravity perception may be the most powerful means to inhibit bending without hampering shoot and flower growth after harvest. Implications, scientific and agriculture: The innovative results of this project have provided some new insight in the basic understanding of gravitropism in flower stalks, that partially filled the gap in our knowledge, and established useful means for its control. Additionally, our analysis has advanced the understanding of important and fundamental physiological processes involved, thereby leading to new ideas for agriculture. Gravitropism has an important impact on agriculture, particularly for controlling the bending of various important agricultural products with economic value. So far, no safe control of the undesired bending problem of flower stalks has been established. Our results show for the first time that shoot bending of cut flowers can be inhibited without adverse effects by controlling the gravity perception step with Ca2+ antagonists and cytoskeleton modulators. Such a practical benefit resulting from this project is of great economic value for the floriculture industry.
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Sadot, Einat, Christopher Staiger, and Mohamad Abu-Abied. Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7592652.bard.
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In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.
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Chew, Teng-Leong. Regulation of Actin-Myosin Cytoskeletal Changes Involved in Cancer Metastasis. Fort Belvoir, VA: Defense Technical Information Center, July 2001. http://dx.doi.org/10.21236/ada396798.
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Friedman, Haya, Julia Vrebalov, and James Giovannoni. Elucidating the ripening signaling pathway in banana for improved fruit quality, shelf-life and food security. United States Department of Agriculture, October 2014. http://dx.doi.org/10.32747/2014.7594401.bard.
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Background : Banana being a monocot and having distinct peel and pulp tissues is unique among the fleshy fruits and hence can provide a more comprehensive understanding of fruit ripening. Our previous research which translated ripening discoveries from tomato, led to the identification of six banana fruit-associated MADS-box genes, and we confirmed the positive role of MaMADS1/2 in banana ripening. The overall goal was to further elucidate the banana ripening signaling pathway as mediated by MADS-boxtranscriptional regulators. Specific objectives were: 1) characterize transcriptional profiles and quality of MaMADS1/2 repressed fruit; 2) reveal the role of additional MaMADSgenes in ripening; 3) develop a model of fruit MaMADS-box mode of action; and 4) isolate new components of the banana ripening signaling pathway. Major conclusion: The functions of the banana MaMADS1-5 have been examined by complimenting the rinor the TAGL1-suppressed lines of tomato. Only MaMADS5 exhibited partial complementation of TAGL1-suppressed and rinlines, suggesting that while similar genes play corresponding roles in ripening, evolutionary divergence makes heterologous complementation studies challenging. Nevertheless, the partial complementation of tomato TAGL1-surpessed and rinlines with MaMADS5 suggests this gene is likely an important ripening regulator in banana, worthy of further study. RNA-seqtranscriptome analysis during ripening was performed on WT and MaMADS2-suppressed lines revealing additional candidate genes contributing to ripening control mechanisms. In summary, we discovered 39 MaMADS-box genes in addition to homologues of CNR, NOR and HB-1 expressed in banana fruits, and which were shown in tomato to play necessary roles in ripening. For most of these genes the expression in peel and pulp was similar. However, a number of key genes were differentially expressed between these tissues indicating that the regulatory components which are active in peel and pulp include both common and tissue-specific regulatory systems, a distinction as compared to the more uniform tomato fruit pericarp. Because plant hormones are well documented to affect fruit ripening, the expressions of genes within the auxin, gibberellin, abscisic acid, jasmonic acid, salicylic and ethylene signal transduction and synthesis pathways were targeted in our transcriptome analysis. Genes’ expression associated with these pathways generally declined during normal ripening in both peel and pulp, excluding cytokinin and ethylene, and this decline was delayed in MaMADS2-suppressed banana lines. Hence, we suggest that normal MaMADS2 activity promotes the observed downward expression within these non-ethylene pathways (especially in the pulp), thus enabling ripening progression. In contrast, the expressions of ACSand ACOof the ethylene biosynthesis pathway increase in peel and pulp during ripening and are delayed/inhibited in the transgenic bananas, explaining the reduced ethylene production of MaMADS2-suppressed lines. Inferred by the different genes’ expression in peel and pulp of the gibberellins, salicylic acid and cytokinins pathways, it is suggested that hormonal regulation in these tissues is diverse. These results provide important insights into possible avenues of ripening control in the diverse fruit tissues of banana which was not previously revealed in other ripening systems. As such, our transcriptome analysis of WT and ripening delayed banana mutants provides a starting point for further characterization of ripening. In this study we also developed novel evidence that the cytoskeleton may have a positive role in ripening as components of this pathway were down-regulated by MaMADS2 suppression. The mode of cytoskeleton involvement in fruit ripening remains unclear but presents a novel new frontier in ripening investigations. In summary, this project yielded functional understanding of the role and mode of action of MaMADS2 during ripening, pointing to both induction of ethylene and suppression of non-ethylene hormonal singling pathways. Furthermore, our data suggest important roles for cytoskeleton components and MaMADS5 in the overall banana ripening control network. Implications: The project revealed new molecular components/genes involved in banana ripening and refines our understanding of ripening responses in the peel and pulp tissues of this important species. This information is novel as compared to that derived from the more uniform carpel tissues of other highly studied ripening systems including tomato and grape. The work provides specific target genes for potential modification through genetic engineering or for exploration of useful genetic diversity in traditional breeding. The results from the project might point toward improved methods or new treatments to improve banana fruit storage and quality.
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Hansen, Peter J., and Amir Arav. Embryo transfer as a tool for improving fertility of heat-stressed dairy cattle. United States Department of Agriculture, September 2007. http://dx.doi.org/10.32747/2007.7587730.bard.
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The overall objective of the current proposal is to develop procedures to improve the pregnancy rate achieved following transfer of fresh or cryopreserved embryos produced in the laboratory into heat-stress recipients. The overall hypothesis is that pregnancy rate in heat-stressed lactating cows can be improved by use of embryo transfer and that additional gains in pregnancy rate can be achieved through development of procedures to cryopreserve embryos, select embryos most likely to establish and maintain pregnancy after transfer, and to enhance embryo competence for post-transfer survival through manipulation of culture conditions. The original specific objectives were to 1) optimize procedures for cryopreservation (Israel/US), 2) develop procedures for identifying embryos with the greatest potential for development and survival using the remote monitoring system called EmbryoGuard (Israel), 3) perform field trials to test the efficacy of cryopreservation and the EmbryoGuard selection system for improving pregnancy rates in heat-stressed, lactating cows (US/Israel), 4) test whether selection of fresh or frozen-thawed blastocysts based on measurement of group II caspase activity is an effective means of increasing survival after cryopreservation and post-transfer pregnancy rate (US), and 5) identify genes in blastocysts induced by insulin-like growth factor-1 (IGF-1) (US). In addition to these objectives, additional work was carried out to determine additional cellular determinants of embryonic resistance to heat shock. There were several major achievements. Results of one experiment indicated that survival of embryos to freezing could be improved by treating embryos with cytochalasin B to disrupt the cytoskeleton. An additional improvement in the efficacy of embryo transfer for achieving pregnancy in heat-stressed cows follows from the finding that IGF-1 can improve post-transfer survival of in vitro produced embryos in the summer but not winter. Expression of several genes in the blastocyst was regulated by IGF-1 including IGF binding protein-3, desmocollin II, Na/K ATPase, Bax, heat shock protein 70 and IGF-1 receptor. These genes are likely candidates 1) for developing assays for selection of embryos for transfer and 2) as marker genes for improving culture conditions for embryo production. The fact that IGF-1 improved survival of embryos in heat-stressed recipients only is consistent with the hypothesis that IGF-1 confers cellular thermotolerance to bovine embryos. Other experiments confirmed this action of IGF-1. One action of IGF-1, the ability to block heat-shock induced apoptosis, was shown to be mediated through activation of the phosphatidylinositol 3-kinase pathway. Other cellular determinants of resistance of embryos to elevated temperature were identified including redox status of the embryo and the ceramide signaling pathway. Developmental changes in embryonic apoptosis responses in response to heat shock were described and found to include alterations in the capacity of the embryo to undergo caspase-9 and caspase-3 activation as well as events downstream from caspase-3 activation. With the exception of IGF-1, other possible treatments to improve pregnancy rate to embryo transfer were not effective including selection of embryos for caspase activity, treatment of recipients with GnRH.and bilateral transfer of twin embryos. In conclusion, accomplishments achieved during the grant period have resulted in methods for improving post-transfer survival of in vitro produced embryos transferred into heat-stressed cows and have lead to additional avenues for research to increase embryo resistance to elevated temperature and improve survival to cryopreservation. In addition, embryo transfer of vitrified IVF embryos increased significantly the pregnancy rate in repeated breeder cows.