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A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction

Cornachione, Anabelle S.; Rassier, Dilson E.
Fonte: AMER PHYSIOLOGICAL SOC; BETHESDA Publicador: AMER PHYSIOLOGICAL SOC; BETHESDA
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
36.64%
Cornachione AS, Rassier DE. A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction. Am J Physiol Cell Physiol 302: C566-C574, 2012. First published November 16, 2011; doi: 10.1152/ajpcell.00355.2011.-When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the " static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca2+ but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca2+-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 mu m at a speed of 40 L-o/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement...

Caracterização molecular da actina do Apicomplexa Neospora caninum; Molecular characterization of the actin from the Apicomplexan Neospora caninum

Baroni, Luciana
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 22/10/2012 PT
Relevância na Pesquisa
36.67%
Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies...

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis nildticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Poletto, Andreia B.; Wasko, Adriane Pinto; Oliveira, Claudio; Azevedo, Alexandre; Carvalho, Robson F.; Silva, Maeli Dal Pai; Foresti, Fausto; Martins, Cesar
Fonte: Soc Brasil Genetica Publicador: Soc Brasil Genetica
Tipo: Artigo de Revista Científica Formato: 352-356
ENG
Relevância na Pesquisa
36.64%
Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus) were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa) sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleoticle resemblance was observed between O. niloticus and O. mossambicus a-actin and P-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Poletto, Andréia B.; Wasko, Adriane Pinto; Oliveira, Claudio; Azevedo, Alexandre; Carvalho, Robson F.; Silva, Maeli Dal Pai; Foresti, Fausto; Martins, Cesar
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: 325-356
ENG
Relevância na Pesquisa
36.64%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus) were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa) sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Tiago, Teresa; Ramos, Susana; Aureliano, M.; Gutiérrez-Merino, Carlos
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2006 ENG
Relevância na Pesquisa
36.64%
Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by ‘decomposed SIN-1’, and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50–100 nM peroxynitrite leads to nearly 50% depolymerization in about 1 h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Ramos, Susana; Manuel, Miguel; Tiago, Teresa; Duarte, Rui O.; Martins, Jorge; Gutiérrez-Merino, Carlos; Moura, José J. G.; Aureliano, M.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2006 ENG
Relevância na Pesquisa
36.67%
Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ‘‘metavanadate’’ solution that contains ortho and metavanadate species, as observed by combining kinetic with 51V NMR spectroscopy studies. Although at 25 C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ‘‘decavanadate’’ interaction with G-actin, favored by the G-actin polymerization...

Recent advances into vanadyl, vanadate and decavanadate interactions with actin

Ramos, Susana; Moura, José J. G.; Aureliano, M.
Fonte: The Royal Society of Chemistry Publicador: The Royal Society of Chemistry
Tipo: Artigo de Revista Científica
Publicado em //2012 ENG
Relevância na Pesquisa
36.71%
Although the number of papers about ‘‘vanadium’’ has doubled in the last decade, the studies about ‘‘vanadium and actin’’ are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate 51V NMR signals, at 516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at 560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1 : 1 binding stoichiometry and a Kd of 7.5 mM 1. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC50 of 68 and 300 mM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 mM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface...

A comparison between Vanadyl, Vanadate, and decavanadate effects in actin structure and function: combination of several spectroscopic studies

Aureliano, M.
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em //2012 ENG
Relevância na Pesquisa
36.7%
The studies about the interaction of actin with vanadium are seldom. In the present paper the effects of vanadyl, vanadate, and decavanadate in the actin structure and function were compared. Decavanadate clearly interacts with actin, as shown by 51V-NMR spectroscopy. Decavanadate interaction with actin induces protein cysteine oxidation and vanadyl formation, being both prevented by the natural ligand of the protein, ATP. Monomeric actin (G-actin) titration with vanadyl, as analysed by EPR spectroscopy, indicates a 1 : 1 binding stoichiometry and a kd of 7.5 μM. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC50 of 68 and 300 μM, respectively, as analysed by light-scattering assays. However, only vanadyl induces G-actin intrinsic fluorescence quenching, which suggests the presence of vanadyl high-affinity actin-binding sites. Decavanadate increases (2.6-fold) actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Finally, both vanadium species increased ε-ATP exchange rate (k = 6.5 × 10−3 and 4.47 × 10−3 s−1 for decavanadate and vanadyl, resp.). Putting it all together, it is suggested that actin, which is involved in many cellular processes...

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Poletto,Andréia B.; Wasko,Adriane P.; Oliveira,Claudio; Azevedo,Alexandre; Carvalho,Robson F.; Silva,Maeli Dal Pai; Foresti,Fausto; Martins,Cesar
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2008 EN
Relevância na Pesquisa
36.64%
Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus) were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa) sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

AQP2 is Necessary for Vasopressin- and Forskolin-Mediated Filamentous Actin Depolymerization in Renal Epithelial Cells

Yui, Naofumi; Lu, Hua A Jenny; Bouley, Richard; Brown, Dennis
Fonte: The Company of Biologists Publicador: The Company of Biologists
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
36.66%
Remodeling of the actin cytoskeleton is required for vasopressin (VP)‐induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)‐mediated F‐actin depolymerization depends on AQP2 expression. Using various MDCK and LLC‐PK1 cell lines with different AQP2 expression levels, we performed F‐actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F‐actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F‐actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC‐PK1 cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F‐actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane‐associated F‐actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F‐actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells...

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites

Olshina, Maya A; Angrisano, Fiona; Marapana, Danushka S; Riglar, David T; Bane, Kartik; Wong, Wilson; Catimel, Bruno; Yin, Meng-Xin; Holmes, Andrew B; Frischknecht, Friedrich; Kovar, David R; Baum, Jake
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
36.67%
Background: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Methods: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. Results: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites...

Simultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells

McGrath, J. L.; Tardy, Y.; Dewey, C. F. Jr; Meister, J. J.; Hartwig, J. H.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica Formato: 428604 bytes; application/pdf
EN_US
Relevância na Pesquisa
36.69%
The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied previously to the study of actin dynamics in living cells. Traditionally, separate experiments estimate the mobility of actin monomer or the lifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fluorescence in PAF and FRAP experiments depends simultaneously on the diffusion coefficient of actin monomer, D, the fraction of actin in filaments, FF, and the lifetime of actin filaments, t (Tardy et al., 1995, Biophys. J. 69:1674–1682). Here we report the application of this mathematical model to the interpretation of PAF and FRAP experiments in subconfluent bovine aortic endothelial cells (BAECs). The following parameters apply for actin in the bulk cytoskeleton of subconfluent BAECs. PAF: D 5 3.1 6 0.4 3 1028 cm2/s, FF 5 0.36 6 0.04, t 5 7.5 6 2.0 min. FRAP: D 5 5.8 6 1.2 3 1028 cm2/s, FF 5 0.5 6 0.04, t 5 4.8 6 0.97 min. Differences in the parameters are attributed to differences in the actin derivatives employed in the two studies and not to inherent differences in the PAF and FRAP techniques. Control experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer...

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

Shannon, William R.
Fonte: Brock University Publicador: Brock University
Tipo: Electronic Thesis or Dissertation
ENG
Relevância na Pesquisa
36.66%
The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones...

Differential effect of wounding on actin and its associated proteins, paxillin and gelsolin, in fetal skin explants

Cowin, A.; Hatzirodos, N.; Teusner, J.; Belford, D.
Fonte: Blackwell Publishing Inc Publicador: Blackwell Publishing Inc
Tipo: Artigo de Revista Científica
Publicado em //2003 EN
Relevância na Pesquisa
36.66%
Skin from the embryonic day 17 rat retains the ability to epithelialize an excisional wound when isolated in serum-supplemented suspension culture. This ability is lost by embryonic day 19. We have investigated this effect of gestational age on fetal epithelial wound closure by correlating the involvement of filamentous actin (F-actin) and its associated proteins, paxillin and gelsolin, in the wound margins of embryonic day 17 and 19 rat skins, with the ability to close a full thickness excisional wound. Using fluorescent-phalloidin histochemistry and scanning confocal microscopy, actin polymerization was observed some five to six cells back from the margin of wounds in the embryonic day 17 skin as early as 3 h postwounding. As the wounds closed over the following 48–72 h, the actin further condensed around the epithelial margin before dispersing after wound closure. In contrast, no organization of actin was seen in the epithelial margin of wounds in skin from the embryonic day 19 embryos. Instead, actin filaments were observed surrounding the dermal wound margins. Chemical or mechanical disruption of the actin in wounded embryonic day 17 skins prevented epithelial closure, although wound repair was independent of cell division. In particular...

Serum Response Factor and Actin Treadmilling Influence Neuronal Mitochondrial Dynamics; Der Transkriptionsfaktor SRF und das Aktin-Zytoskelett regulieren die Dynamik von Mitochondrien in Neuronen

Beck, Henning
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
EN
Relevância na Pesquisa
36.67%
This work addresses the influences of the transcription factor SRF, actin treadmilling and cofilin activity on mitochondrial dynamics with regard to size, (ultra)structure and trafficking. In vivo experiments showed that SRF loss-of-function (LOF) resulted in multi-vesiculated mitochondria in corpus-callosal cross-sections and impairment of mitochondrial distribution in cortical neurons. Furthermore, ATP-content of Srf mutant brain was reduced as well as ATP-production capacity. In vitro Srf mutant neurons displayed mitochondrial fragmentation, impaired mitochondrial occupancy and ultrastructural membrane disorganization. Furthermore, fewer mitochondria moved at slower velocities as compared to wild-type neurons. Of note, all parameters could be rescued upon overexpression of constitutively active SRF-VP16 in an Srf mutant background. Furthermore, in wild-type neurons SRF-VP16 overexpression resulted in the formation of large mitochondrial networks and increased movement velocity. These effects were SRF- as well as mitochondria-specific. As SRF is a major regulator of actin treadmilling, the contribution of actin microfilament dynamics to mitochondria was investigated. It turned out that shifting the G/F-actin ratio towards monomeric G-actin led to mitochondrial fragmentation (similar to SRF LOF)...

Immunohistochemical change of actin in exprerimental myocardial ischemia. Its usefulness to detect very early myocardial damages

Shozo Nishida; Shingo Hiruma; Shigeo Hashimoto
Fonte: Murcia : F. Hernández Publicador: Murcia : F. Hernández
Tipo: Artigo de Revista Científica Formato: application/pdf
ENG
Relevância na Pesquisa
36.66%
Pathomorphological diagnosis of acute myocardial infarction has many problems in human autopsy materials less than 4 to 6 hours after clinical onset and in rats 2 to 3 hours after experimental coronary occlusion. Since immunohistochemical reaction deuends - ~ on the antigei determinant site of the material, chañges in the reaction may reflect alterations at the molecular level in myocardial fibers. With this consideration in mind, the effectiveness of diagnosing infarction at the earliest (possible) stage, and the changes of actin filaments were investigated through experiments, using immunohistochemical methods involving anti-actin antibodies produced from chicken gizzards in our laboratory. The left coronary arteries of rats were ligated to produce ischemia. Dehydrogenases were shown to be still present by triphenyltetrazolium chloride (TTC) reaction, but the anti-actin antibody reaction had disappeared in areas corresponding to the ischemic sites. However, on electron microscopic examination of these sites, actin fibers were clearly revealed. In the case of ischemia lasting for more than 6 hours, the anti-actin antibody reaction had disappeared, corresponding to the disappearance of the TTC reaction. At this stage...

Molecular characterisation of Shigella flexneri IcsA and the role of lipopolysaccharide O-antigen in actin-based motility.

May, Kerrie Leanne
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Tese de Doutorado
Publicado em //2007
Relevância na Pesquisa
36.66%
Shigella spp. cause bacillary dysentery through invasion of the colonic epithelium. Shigella flexneri IcsA (VirG) is a polarly localised, outer membrane (OM) protein that is essential for virulence. IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in-turn recruits the Arp2/3 complex that polymerises host actin. The resultant F-actin comet tails initiate bacterial actin-based motility (ABM) and intercellular spread. The N-terminal surface-exposed region of IcsA, referred to as the passenger domain (aa 53-758), is responsible for IcsA activity in ABM. A glycine-rich region (aa 140-307) within this passenger domain is involved in mediating N-WASP binding. This thesis sought to conduct a comprehensive study of IcsA structure-function. Linker-insertion mutagenesis was undertaken to randomly introduce in-frame insertions of 5 aa within the IcsA passenger domain. Forty-seven linker-insertion mutants (IcsA [subscript i]) mutants were isolated and expressed in S. flexneri ∆icsA. The resultant strains were characterised for IcsA protein production, cell surface-expression and localisation, as well as intercellular spreading, F-actin comet tail formation, and the recruitment of N-WASP. Linker-insertions between aa 595-716 of IcsA affected production and lie in a region homologous to the putative auto-chaperone domain of Bordetella pertussis BrkA. Two mutant proteins (IcsA [subscript i532] and IcsA [subscript i563]) exhibited disrupted polar targeting...

Rice actin-binding protein RMD is a key link in the auxin-actin regulatory loop that controls cell growth

Li, G.; Liang, W.; Zhang, X.; Ren, H.; Hu, J.; Bennett, M.J.; Zhang, D.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em //2014 EN
Relevância na Pesquisa
36.66%
The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression...

Evolution of the gelsolin family of actin-binding proteins as novel transcriptional coactivators

Archer, Stuart; Claudianos, Charles; Campbell, Hugh
Fonte: The Company of Biologists Ltd Publicador: The Company of Biologists Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
36.69%
The gelsolin gene family encodes a number of higher eukaryotic actin-binding proteins that are thought to function in the cytoplasm by severing, capping, nucleating or bundling actin filaments. Recent evidence, however, suggests that several members of the gelsolin family may have adopted unexpected nuclear functions including a role in regulating transcription. In particular, flightless I, supervillin and gelsolin itself have roles as coactivators for nuclear receptors, despite the fact that their divergence appears to predate the evolutionary appearance of nuclear receptors. Flightless I has been shown to bind both actin and the actin-related BAF53a protein, which are subunits of SWI/SNF-like chromatin remodelling complexes. The primary sequences of some actin-related proteins such as BAF53a exhibit conservation of residues that, in actin itself, are known to interact with gelsolin-related proteins. In summary, there is a growing body of evidence supporting a biological role in the nucleus for actin, Arps and actin-binding proteins and, in particular, the gelsolin family of actin-binding proteins.

Role of the p70S6K pathway in regulating the actin cytoskeleton and cell migration

Berven, Leise; Willard, Francis; Crouch, Michael F
Fonte: Academic Press Publicador: Academic Press
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
36.69%
We have examined the role of endogenous 70-kDa S6 kinase (p70 S6K) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70S6K with the actin cytoskeleton was demonstrated by cosedimentation of p70S6K with F-actin and by subcellular fractionation in which p70S6K activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70S6K, Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70S6K signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70 S6K. Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70S6K. Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632...