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‣ Competition between Na+ and Li+ for Unsealed and Cytoskeleton-Depleted Human Red Blood Cell Membrane: A 23Na Multiple Quantum Filtered and 7Li NMR Relaxation Study

Srinivasan, Chandra; Minadeo, Nicole; Toon, Jason; Graham, Daniel; Freitas, Duarte Mota de; Geraldes, Carlos F. G. C.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
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Evidence for competition between Li+ and Na+ for binding sites of human unsealed and cytoskeleton-depleted human red blood cell (csdRBC) membranes was obtained from the effect of added Li+ upon the 23Na double quantum filtered (DQF) and triple quantum filtered (TQF) NMR signals of Na+-containing red blood cell (RBC) membrane suspensions. We found that, at low ionic strength, the observed quenching effect of Li+ on the 23Na TQF and DQF signal intensity probed Li+/Na+ competition for isotropic binding sites only. Membrane cytoskeleton depletion significantly decreased the isotropic signal intensity, strongly affecting the binding of Na+ to isotropic membrane sites, but had no effect on Li+/Na+ competition for those sites. Through the observed 23Na DQF NMR spectra, which allow probing of both isotropic and anisotropic Na+ motion, we found anisotropic membrane binding sites for Na+ when the total ionic strength was higher than 40 mM. This is a consequence of ionic strength effects on the conformation of the cytoskeleton, in particular on the dimer-tetramer equilibrium of spectrin. The determinant involvement of the cytoskeleton in the anisotropy of Na+ motion at the membrane surface was demonstrated by the isotropy of the DQF spectra of csdRBC membranes even at high ionic strength. Li+ addition initially quenched the isotropic signal the most...

‣ Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

GOULART-SILVA, F.; SERRANO-NASCIMENTO, C.; NUNES, M.T.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica
Português
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358.61055%
The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation...

‣ Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia

MARTINS-DE-SOUZA, Daniel; GATTAZ, Wagner F.; SCHMITT, Andrea; MACCARRONE, Giuseppina; HUNYADI-GULYAS, Eva; EBERLIN, Marcos N.; SOUZA, Gustavo H. M. F.; MARANGONI, Sergio; NOVELLO, Jose C.; TURCK, Christoph W.; DIAS-NETO, Emmanuel
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
Português
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Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann`s Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients` dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia...

‣ Inibição da migração mediada pelo gene RECK em modelo de glioma humano através de alterações no citoesqueleto e adesão focal; RECK-mediated inhibition of glioma migration with changes in cytoskeleton and focal adhesion

Haga, Raquel Brandão
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 18/05/2012 Português
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Gliomas são tumores altamente invasivos, resistentes aos tratamentos disponíveis atualmente e com alta taxa de mortalidade. A superexpressão de RECK na linhagem de glioma humano T98G comprometeu a capacidade das células de migrar e invadir in vitro, com rearranjo do citoesqueleto e alteração na distribuição espacial de FAK fosforilado. Entretanto, o possível mecanismo envolvido na inibição da migração mediada por RECK não foi desvendado. Para estudarmos os mecanismos envolvidos nesta alteração da capacidade migratória, as células T98G foram transfectadas com o vetor plasmidial pCXN2-hRECK (RECK+). A via das integrinas, a atividade de alguns membros da família das RhoGTPases e elementos do citoesqueleto foram avaliados através de imunoblotting, imunomarcação e ensaios de pull-down para as células RECK+ em comparação com células T98G não-transfectadas (WT), células T98G transfectadas com vetor pCXN2 na ausência do gene RECK (vetor) e fibroblastos primários humanos (FF287). Nossos resultados mostram um aumento na expressão de integrina β1 e uma diminuição da fosforilação de FAK no sítio de auto-fosforilação Tyr397 que, juntamente com o aumento das fibras de estresse e a diminuição dos lamelipódios...

‣ Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia

MARTINS-DE-SOUZA, Daniel; GATTAZ, Wagner F.; SCHMITT, Andrea; MACCARRONE, Giuseppina; HUNYADI-GULYAS, Eva; EBERLIN, Marcos N.; SOUZA, Gustavo H. M. F.; MARANGONI, Sergio; NOVELLO, Jose C.; TURCK, Christoph W.; DIAS-NETO, Emmanuel
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
351.0964%
Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann`s Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients` dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia...

‣ Sulfation degree of glycosaminoglycans triggers distinct cytoskeleton organisation in mesenchymal stem cells

Costa, Diana Soares da; Yang, Y.; Moller, Stephanie; Groth, T.; Reis, R. L.; Pashkuleva, I.
Fonte: Journal of Tissue Engineering and Regenerative Medicine Publicador: Journal of Tissue Engineering and Regenerative Medicine
Tipo: Conferência ou Objeto de Conferência
Publicado em /10/2012 Português
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Glycosaminoglycans (GAGs) comprise the closest cellular environment: they are building elements of the ECM and can be also found on cells surface. Their biological activity depends on several parameters among which the negative charge is of prime importance[1]. This charge is generally associated with the presence of sulfate groups (-OSO3H). Sulfation is a dynamic modification: it can occur at various positions within the glycan and different sulfation patterns have been identified for the same organs and cells during their development. However, the mechanisms of coding and transferring information by these functionalities are not yet complete understood, mainly because of (i)the complex physiological microenvironment in which GAGs interactions occur and (ii)the inability to access homogeneous GAGs[2]. In this work, we propose model surfaces bearing GAGs with different sulfation degree as platform to investigate the pathways by which mesenchymal stem cells (MSCs) sense and respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this study: hyaluronic acid (HA) because it is the only non-sulfated glycan and heparin (HEP) as it is the GAG with the highest degree of sulfation. To obtain a larger range of sulfation degrees...

‣ Microtubule cytoskeleton behavior in the initial steps of host cell invasion by Besnoitia besnoiti

REIS, Y; CORTES, H; VISEUMELO, L; FAZENDEIRO, I; LEITAO, A; SOARES, H; Cortes, H
Fonte: Universidade de Évora Publicador: Universidade de Évora
Tipo: Artigo de Revista Científica
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Microtubule cytoskeleton behavior in the initial steps of host cell invasion by Besnoitia besnoiti Besnoitia besnoiti is a protozoan parasite responsible for bovine besnoitiosis. Indirect immunofluorescence showed that isolated B. besnoiti possesses a set of subpellicular microtubules, radiating from the apical end and extending for more than 2/3 of the cell body. Upon interaction with the host cell, B. besnoiti undergoes dramatic modifications of shape and surface, as revealed by atomic force microscopy, accompanied by a distinct tubulin labeling on the posterior region. In the host cell, the microtubule cytoskeleton shows a re-arrangement around the invading parasite suggesting a filamentous interaction with the parasite cytoskeleton during invasion.

‣ Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

Fernandes,Maria Cecília; Andrade,Leonardo Rodrigues de; Andrews,Norma Windsor; Mortara,Renato Arruda
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2011 Português
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It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.

‣ JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Díaz-Chiguer,Dylan L; Hernández-Luis,Francisco; Nogueda-Torres,Benjamín; Castillo,Rafael; Reynoso-Ducoing,Olivia; Hernández-Campos,Alicia; Ambrosio,Javier R
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2014 Português
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Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

‣ Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

Goulart-Silva,F.; Serrano-Nascimento,C.; Nunes,M.T.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2011 Português
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The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation...

‣ A Green Fluorescent Protein Fusion to Actin-Binding Domain 2 of Arabidopsis Fimbrin Highlights New Features of a Dynamic Actin Cytoskeleton in Live Plant Cells1[w]

Sheahan, Michael B.; Staiger, Chris J.; Rose, Ray J.; McCurdy, David W.
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /12/2004 Português
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The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

‣ Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

Uriarte, Silvia M.; Jog, Neelakshi R.; Luerman, Gregory C.; Bhimani, Samrath; Ward, Richard A.; McLeish, Kenneth R.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
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We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data...

‣ Actin cytoskeleton regulates pollen tube growth and tropism

Bou Daher, Firas
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
Português
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La fertilisation chez les plantes dépend de la livraison des cellules spermatiques contenues dans le pollen à l’ovule. Au contact du stigmate, le grain de pollen s’hydrate et forme une protubérance, le tube pollinique, chargé de livrer les noyaux spermatiques à l’ovule. Le tube pollinique est une cellule à croissance rapide, anisotrope et non autotrophe; ainsi tout au long de sa croissance à travers l’apoplaste du tissu pistillaire, le tube pollinique puise ses sources de carbohydrates et de minéraux du pistil. Ces éléments servent à la synthèse des constituants de la paroi qui seront acheminés par des vésicules de sécrétion jusqu’à l’apex du tube. Ce dernier doit aussi résister à des pressions mécaniques pour maintenir sa forme cylindrique et doit répondre à différents signaux directionnels pour pouvoir atteindre l’ovule. Mon projet de doctorat était de comprendre le rôle du cytosquelette dans la croissance anisotrope du tube pollinique et d’identifier les éléments responsables de sa croissance et de son guidage. Le cytosquelette du tube pollinique est composé des microfilaments d’actine et des microtubules. Pour assurer une bonne croissance des tubes polliniques in vitro, les carbohydrates et les éléments de croissance doivent être ajoutés au milieu à des concentrations bien spécifiques. J’ai donc optimisé les conditions de croissance du pollen d’Arabidopsis thaliana et de Camellia japonica qui ont été utilisés avec le pollen de Lilium longiflorum comme modèles pour mes expériences. J’ai développé une méthode rapide et efficace de fixation et de marquage du tube pollinique basée sur la technologie des microondes. J’ai aussi utilisé des outils pharmacologiques...

‣ The cytoskeleton as a regulator and target of biotic interactions in plants

Takemoto, Daigo; Hardham, Adrienne R
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica Formato: 13 pages
Português
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The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and actin microfilaments and plays an important role in many aspects of plant cell growth and development, including such fundamental processes as cell division, cell expansion, and intracellular organization and motility (Staiger, 2000; Wasteneys and Galway, 2003). During evolution, plants have developed mechanisms to exploit, survive, or minimize the negative impact of a diverse range of environmental factors, and in many cases the plant cytoskeleton is instrumental in mediating the plant’s response. Cytoskeletal elements, for example, translocate chloroplasts under high light conditions (Takagi, 2000), facilitate gravity sensing (Blancaflor, 2002), and direct cellular response to wounding (Foissner et al., 1996; Hush and Overall, 1996). In addition to these abiotic factors, plants also encounter and must deal with a range of other organisms that may be potential partners or pathogens. Once again, the plant cytoskeleton plays a key role. In many ways, biotic factors in the environment present a greater challenge to the plant than do abiotic stresses because living organisms, like their plant hosts, are continually evolving. Potential pathogens develop new ways of avoiding or overcoming existing plant defenses; symbionts may attain aggressive traits or lose beneficial ones. Plants must thus constantly refine existing defenses and develop new strategies to maintain an upper hand in their interactions with other organisms. Changes in the organization of the plant cytoskeleton during plant interactions with microbial and other organisms are complex and varied...

‣ p53-mediated transcriptional regulation and activation of the actin cytoskeleton regulatory RhoC to LIMK2 signaling pathway promotes cell survival

Croft, D.; Crighton, D.; Samuel, M.; Lourenco, F.; Munro, J.; Wood, J.; Bensaad, K.; Vousden, K.; Sansom, O.; Ryan, K.; Olson, M.
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.; Daniel R Croft, Diane Crighton, Michael S Samuel, Filipe C Lourenco, June Munro, Jenifer Wood, Karim Bensaad, Karen H Vousden, Owen J Sansom, Kevin M Ryan...

‣ Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures

Pagani, R.; Portolés, M.T.; De la Viña, S.; Melzner, I.; Vergani, G.
Fonte: Murcia : F. Hernández Publicador: Murcia : F. Hernández
Tipo: Artigo de Revista Científica Formato: application/pdf
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Endotoxins (lipopolysaccharide, LPS) from Gram-negative bacteria are considered as the agents responsible for the induction of endotoxic shock, producing severe cellular metabolic dishomeostasis. Cytotoxic lesions, as well as functional and metabolic disturbances, occur mainly in the liver, which is one of the target organs and exerts an LPS clearance function. In an attempt to approach the molecular basis of endotoxic shock, and to propose an experimental model, we have focused this study on cytoskeleton (microtubules and microfilaments) alterations induced by different doses of endotoxin in different target liver cells. Microfilaments and microtubules were studied by immunofluorescence and different microscopy techniques (optic fluorescence microscopy and confocal laser scanning microscopy) in order to improve the cytoskeleton study resolution. Parenchymal and sinusoidal cell morphology, severely damaged by the LPS action, is related to a disturbance on the cytoskeletal organisation, even more evident in a particular proliferating rat liver cell culture. The most relevant changes are seen in the microtubule patterns in all liver cells tested, which could be related, depending on cell type, either to a direct LPS action or to [Ca+2]i dishomeostasis as well as free radical and cytokine (IL-1ß and TNF-a) production. Due to their features...

‣ Dissecting the role of adducin in the axonal cytoskeleton; Identificação do papel da aducina no citosqueleto axonal

Rodrigues, Joana Nogueira
Fonte: Universidade de Aveiro Publicador: Universidade de Aveiro
Tipo: Dissertação de Mestrado
Português
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The neuronal cytoskeleton is an interconnected network of filamentous polymers, having in its constitution three major components: actin, microtubules and intermediate filaments. Up to the discovery of axon actin rings, the neuronal actin cytoskeleton has gained relevance. Still, the molecular details of the regulation of the actin cytoskeleton in neurons are largely unknown. Helping in the actin cytoskeleton regulation and maintenance, there is adducin. Adducin is organized in heterotetamers of heterodimers which comprises α/β and α/γ subunits. In the nervous system, the depletion of α subunit results in an almost complete absence of functional adducin. Given this, α-adducin KO mice arose as relevant models to study the role of this protein in actin cytoskeleton. Results from our group showed that α-adducin KO mice develop progressive axon enlargement and degeneration. As defects in axonal transport have been related to axon enlargement, we determined the importance of adducin in the axonal cytoskeleton and, more specifically, in axonal transport. Although no differences were found in the retrograde transport of CTB in the optic nerve, the lack of adducin resulted in a decreased speed of axonal transport of mitochondria and lysosomes. Several neurodegenerative disorders have been associated with axonal transport deficits and...

‣ The Role of Multifilament Structures and Lateral Interactions in Dynamics of Cytoskeleton Proteins and Assemblies

Li, Xin; Kolomeisky, Anatoly B.
Fonte: Universidade Rice Publicador: Universidade Rice
Tipo: Journal article; Text; post-print
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Microtubules and actin filaments are biopolymer molecules that are major components of cytoskeleton networks in biological cells. They play important roles in supporting fundamental cellular processes such as cell division, signaling, locomotion, and intracellular transport. In cells, cytoskeleton proteins function under nonequilibrium conditions that are powered by hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) molecules attached to them. Although these biopolymers are critically important for all cellular processes, the mechanisms that govern their complex dynamics and force generation remain not well explained. One of the most difficult fundamental issues is to understand how different components of cytoskeleton proteins interact together. We develop an approximate theoretical approach for analyzing complex processes in cytoskeleton proteins that takes into account the multifilament structure, lateral interactions between parallel protofilaments, and the most relevant biochemical transitions during the biopolymer growth. It allows us to fully evaluate collective dynamic properties of cytoskeleton filaments as well as the effect of external forces on them. It is found that for the case of strong lateral interactions the stall force of the multifilament protein is a linear function of the number of protofilaments. However...

‣ Implications of human LRRK2 protein on the regulation of cytoskeleton dynamics in Parkinson's disease; Implikationen der menschlichen LRRK2 Protein auf die Regulation der Zytoskelett Dynamik in der Parkinson-Krankheit

Garcia Miralles, Marta
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
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Parkinson’s disease (PD) is the most common neurodegenerative movement disorder in the ageing population. Although the vast majority of cases are considered sporadic, around 10-20% is caused by pathogenic missense mutations in PD-associated genes. Among these, mutations in the LRRK2 gene are the most frequent cause of late-onset autosomal-dominant familial but interestingly also sporadic PD. In recent years, over 40 missense variants have been reported in the LRRK2 gene. Of these, only seven have been demonstrated to be pathogenic based upon clear segregation with disease in LRRK2-linked families including N1437H, R1441C/G/H, Y1699C, G2019S, and I2020T. The most prevalent mutation is the G2019S contributing up to 1-2% of sporadic and 7% of familial PD case in Caucasians, and up to 20% of total PD cases in Ashkenazy Jews, or 40% in North African Berbers. Recently, mutations in the LRRK2 gene have been implicated in the dysfunction of several cellular pathways, but still the pathophysiological function of the gene remains unknown. In the present work, a novel LRRK2 transgenic mouse model was generated and characterized in an attempt to elucidate the pathophysiological function of LRRK2. The LRRK2 transgenic mouse model expressed either human wild-type or G2019S LRRK2 protein under the neuron specific Thy1.2 promoter resulting in almost physiological levels of LRRK2 (~2-fold increased protein levels). The characterization of Thy1-2-LRRK2 transgenic mouse model showed expression of LRRK2 protein in affected-PD in brain regions such as cortex...

‣ Remodelling the cytoskeleton for growth and form: An overview with some new views

Wasteneys, Geoffrey; Galway, M E
Fonte: Annual Reviews Inc Publicador: Annual Reviews Inc
Tipo: Artigo de Revista Científica
Português
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The cytoskeleton coordinates all aspects of growth in plant cells, including exocytosis of membrane and wall components during cell expansion. This review seeks to integrate current information about cytoskeletal components in plants and the role they play in generating cell form. Advances in genome analysis have fundamentally changed the nature of research strategies and generated an explosion of new information on the cytoskeleton-associated proteins, their regulation, and their role in signaling to the cytoskeleton. Some of these proteins appear novel to plants, but many have close homologues in other eukaryotic systems. It is becoming clear that the mechanisms behind cell growth are essentially similar across the growth continuum, which ranges from tip growth to diffuse expansion. Remodeling of the actin cytoskeleton at sites of exocytosis is an especially critical feature of polarized and may also contribute to axial growth. We evaluate the most recent work on the signaling mechanisms that continually remodel the actin cytoskeleton via the activation of actin-binding proteins (ABPs) and consider the role the microtubule cytoskeleton plays in this process.