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PubMed2005

Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions.

Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.

Actin-Related Protein 2↗

PubMed1999

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Actins↗

PubMed1983

[Structure of the cytoskeleton of embryonic muscle fibroblasts. Electron microscopic study of platinum replicas].

Cultured mouse embryo fibroblasts were extracted with 1% Triton X-100 at pH 6.7 in buffer containing EGTA and stabilizing supplements. Exposed during this extraction cytoskeletons were fixed, dried with the critical point technique, and shadowed with platinum. The platinum replicas of cytoskeleton were used for electron microscopic characterization of the three-dimensional distribution of cytoskeletal fibrils in cytoplasm. Four cytoplasmic regions are revealed with different cytoskeletal structure; these being a "mesh-work zone", a "loose zone", which together formed an active edge of cell lamelloplasm, the "lamella proper" and endoplasm. The two former zones are occupied with dense three-dimensional or loose planar actin network, respectively. The "lamella proper" contains two-dimensional network of different fibrils: microfilaments, microtubules, intermediate filaments and thin connective filaments. The central perinuclear area has a thick microfilament sheath at dorsal cell surface. The most important cytoplasmic elements of fibroblasts are actin microfilament bundles. The fine structure of these bundles, their junctions with each other ("organizing centers") and their terminal parts corresponding to cell-substrate focal contacts are described.

Animals↗

PubMed2003

A mutation in the gamma actin 1 (ACTG1) gene causes autosomal dominant hearing loss (DFNA20/26).

Linkage analysis in a multigenerational family with autosomal dominant hearing loss yielded a chromosomal localisation of the underlying genetic defect in the DFNA20/26 locus at 17q25-qter. The 6-cM critical region harboured the gamma-1-actin (ACTG1) gene, which was considered an attractive candidate gene because actins are important structural elements of the inner ear hair cells. In this study, a Thr278Ile mutation was identified in helix 9 of the modelled protein structure. The alteration of residue Thr278 is predicted to have a small but significant effect on the gamma 1 actin structure owing to its close proximity to a methionine residue at position 313 in helix 11. Met313 has no space in the structure to move away. Moreover, the Thr278 residue is highly conserved throughout eukaryotic evolution. Using a known actin structure the mutation could be predicted to impair actin polymerisation. These findings strongly suggest that the Thr278Ile mutation in ACTG1 represents the first disease causing germline mutation in a cytoplasmic actin isoform.

Actins↗

PubMed2005

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.

Animals↗

PubMed1988

Actin depolymerization and inhibition of capping induced by pentoxifylline in human lymphocytes and neutrophils.

Pentoxifylline is used clinically for the treatment of intermittent claudication. It is believed to exert its effect by altering the rheologic properties of blood. The cytoskeleton plays an important role in the maintenance of cell structure and function. In particular, alterations in the state of actin seem to play an important role in cell motility. Therefore, we examined the effect of pentoxifylline on the actin state in human polymorphonuclear leukocytes (PMN) and mononuclear cells. Pentoxifylline (10 mM final concentration) decreased F-actin content in both PMN and mononuclear cells. Pentoxifylline also inhibited concanavalin A-induced capping in PMN and mononuclear cells. Similarly, surface immunoglobulin capping in B lymphocytes was also inhibited. Pretreatment of cells with pertussis toxin did not inhibit pentoxifylline-induced decrease in F-actin, suggesting pentoxifylline does not act through pertussis toxin-sensitive G-proteins. Dibutyryl cyclic AMP failed to show any significant effect on the F-actin content in PMN. Therefore, the effect of pentoxifylline cannot be attributed to changes in cyclic AMP levels. Chemotactic peptide-induced actin polymerization was unaffected in PMN when expressed as percent changes in F-actin. The observations reported here suggest that the rheological effects of pentoxifylline might be due to its effects on the actin state in the cellular elements of the blood. Further studies on the mechanism of action of pentoxifylline on actin state in leukocytes will prove useful in delineating the physiological mechanisms regulating actin state in leukocytes.

Actins↗

PubMed2006

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism.

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.

Adenosine Triphosphate↗

PubMed2006

A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function.

To localize the different actin paralogs found in Paramecium and to disclose functional implications, we used overexpression of GFP-fusion proteins and antibody labeling, as well as gene silencing. Several isoforms are associated with food vacuoles of different stages. GFP-actin either forms a tail at the lee side of the organelle, or it is vesicle bound in a homogenous or in a speckled arrangement, thus reflecting an actin-based mosaic of the phagosome surface appropriate for association and/or dissociation of other vesicles upon travel through the cell. Several paralogs occur in cilia. A set of actins is found in the cell cortex where actin outlines the regular surface pattern. Labeling of defined structures of the oral cavity is due to other types of actin, whereas yet more types are distributed in a pattern suggesting association with the numerous Golgi fields. A substantial fraction of actins is associated with cytoskeletal elements that are known to be composed of other proteins. Silencing of the respective actin genes or gene subfamilies entails inhibitory effects on organelles compatible with localization studies. Knock down of the actin found in the cleavage furrow abolishes cell division, whereas silencing of other actin genes alters vitality, cell shape and swimming behavior.

Actin Cytoskeleton↗

PubMed1989

Muscle-specific (CArG) and serum-responsive (SRE) promoter elements are functionally interchangeable in Xenopus embryos and mouse fibroblasts.

The Xenopus cardiac actin gene contains four copies of a promoter element, the CArG box, which is conserved amongst striated muscle actin genes and is essential for tissue-specific expression in the developing Xenopus embryo. Our aim is to identify embryo and muscle proteins that interact with the CArG box as a step towards understanding the molecular basis of this developmentally regulated gene expression. The CArG box shares some sequence similarity with the Serum Response Element (SRE), which mediates the transcriptional activation by serum of genes such as c-fos and cytoskeletal actin. We show here that the most proximal cardiac actin CArG box is recognized by the same binding activity as the cytoskeletal actin SRE in nuclear extracts from both Xenopus embryos and mammalian muscle cells. This activity is indistinguishable from the previously characterized HeLa cell SRE-binding activity, Serum Response Factor (SRF). Importantly, we extend these in vitro studies to demonstrate that the CArG box and SRE are functionally interchangeable, both in Xenopus embryos and mouse fibroblasts. This implies that the CArG box and SRE can bind the same protein in vivo, as well as in vitro. Our results identify an SRF-like protein as a CArG box-binding factor and we discuss the implication that a common mechanism may be utilized in both muscle-specific gene expression and serum-responsive transcription.

Actins↗

PubMed1985

An examination of the soluble oligomeric complexes extracted from the red cell membrane and their relation to the membrane cytoskeleton.

A part of the spectrin extracted from red cell membranes at low ionic strength occurs in the form of a high-molecular weight oligomeric complex with actin and proteins 4.1 and 4.9. When the extraction is performed at 35 degrees, the spectrin is present in this complex as the dimer, all higher forms being dissociated. We have been unable to establish any correlation between the fraction of the spectrin thus complexed and the metabolic state of the cell. At least a large part of the complex appears to be a defined monodisperse species, sedimenting at 31S. The actin is present as short protofilaments. The average number of spectrin molecules associated with each molecule of complex has been studied by cytochalasin binding and electron microscopy. The complexes present the appearance in the electron microscope of spiders, in which the legs are spectrin dimers, attached to a globular element, containing by inference, actin and proteins 4.1 and 4.9; they are active in nucleating the polymerization of G-actin. The complexes are extremely stable, being resistant to dissociation under the conditions of the deoxyribonuclease assay, even after treatment with trypsin to degrade the actin-associated proteins. It is suggested that the complexes represent intact junctions of the membrane cytoskeletal network. Relevant structural features of the network are revealed by electron microscopy. The results lead to inferences concerning the mechanism of dissociation of the network from the membrane.

Actins↗

PubMed1980

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

Actins↗

PubMed1994

Retrotransposition of a marked Drosophila line-like I element in cells in culture.

We have marked a Drosophila transposable element--the LINE-like I element--with an intron-containing indicator gene inserted in place of a large deletion in the I element second ORF encompassing the reverse transcriptase domain, and this marked element was placed downstream to a potent actin promoter. An expression vector for the I element ORFs was also constructed, under the same heterologous promoter. The indicator gene contains a lacZ reporter gene the expression of which is conditioned by retrotransposition of the marked element, thus allowing detection of transposition events by testing for either beta-galactosidase expression or occurrence of spliced DNA molecules. The marked I element was introduced into Drosophila melanogaster cells in culture by transfection. Spliced DNA copies of the marked element and specifically stained beta-galactosidase-expressing cells were detected only upon co-transfection with the I expression vector, thus indicating that an ORF2-deleted element can be complemented in trans for transposition. This simple assay for retrotransposition in Drosophila cells in culture provides a tool for the rapid analysis of the mechanism of I transposition in its cis and trans sequence requirements.

Animals↗

PubMed2005

Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-beta1 control element.

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-SMA expression in smooth muscle cells. A Lac Z transgene construct from -2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous alpha-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of alpha-SMA expression in myofibroblasts in granulation tissue.

Actins↗

PubMed1988

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.

Acetyltransferases↗

PubMed2004

Ionic wave propagation along actin filaments.

We investigate the conditions enabling actin filaments to act as electrical transmission lines for ion flows along their lengths. We propose a model in which each actin monomer is an electric element with a capacitive, inductive, and resistive property due to the molecular structure of the actin filament and viscosity of the solution. Based on Kirchhoff's laws taken in the continuum limit, a nonlinear partial differential equation is derived for the propagation of ionic waves. We solve this equation in two different regimes. In the first, the maximum propagation velocity wave is found in terms of Jacobi elliptic functions. In the general case, we analyze the equation in terms of Fisher-Kolmogoroff modes with both localized and extended wave characteristics. We propose a new signaling mechanism in the cell, especially in neurons.

Actin Cytoskeleton↗

PubMed2001

Clustering and anchoring mechanisms of molecular constituents of postsynaptic scaffolds in dendritic spines.

Recent technological progress has yielded great amounts of information about the molecular constituents of postsynaptic scaffolds in the dendritic spine. Actin filaments are major cytoskeletal elements in the dendritic spine, and they functionally interact with neurotransmitter receptors via regulatory actin-binding proteins. Drebrin A and alpha-actinin-2 are two major actin-binding proteins in dendritic spines. In adult brains, they are characteristically concentrated in spines, but not in dendritic shafts or cell bodies. Thus, they are part of a unique postsynaptic scaffold consisting of actin filaments, PSD protein family, and neurotransmitter receptors. Localization of NMDA receptors, actin filaments, and actin-binding proteins in spines changes in parallel with development, and in response to synaptic activity. This raises the possibility that clustering and anchoring of these characteristic molecular constituents at postsynaptic scaffolds play important roles in spine function. This article focuses on the clustering and anchoring mechanisms of NMDA receptors and actin filaments, and the involvement of actin-binding proteins, in dendritic spines, and the way in which characteristic postsynaptic scaffolds are built up.

Actin Cytoskeleton↗

PubMed1998

Cell-specific transcription of the smooth muscle gamma-actin gene requires both positive- and negative-acting cis elements.

We have characterized the function of putative regulatory sequences upon the smooth muscle transcription of the SMGA gene, using promoter deletion analyses. We demonstrate that the SMGA promoter contains four domains: a basal promoter (-1 to -100), a smooth muscle specifier sequence (-100 to -400), a negative regulator (-400 to -1000), and a smooth muscle-specific modulator (-1000 to -2000). The basal or core promoter supports equivalent transcription in both smooth and skeletal muscle cells. Addition of sequences containing a CArG motif juxtaposed to an E-box element stimulates smooth muscle transcription by five- to sixfold compared to skeletal muscle. This smooth muscle-specific segment is maintained for about 200 bp, after which is a segment of DNA that appears to inhibit the transcriptional capacity of the SMGA promoter in smooth muscle cells. Within the boundary between the smooth muscle specifier and negative regulatory sequences (-400 to -500) are three E-box elements. The smooth muscle modulator domain contains two CArG elements and multiple E-boxes. When added to the SMGA promoter it causes an additional three- to fivefold increase in smooth muscle-specific transcription over that stimulated by the smooth muscle specifier domain. Thus, our studies show that the appropriate cell-specific transcription of the SMGA gene involves complex interactions directed by multiple cis-acting elements. Moreover, our characterization of a cell culture system employing embryonic gizzard smooth muscle cells lays the foundation for further molecular analyses of factors that regulate or control SMGA and other smooth muscle genes during differentiation.

Actins↗

PubMed2004

Fructose-1,6-diphosphate suppresses T-lymphocyte proliferation, promotes apoptosis and inhibits interleukins-1, 6, beta-actin mRNAs, and transcription factors expression.

The overall objective of this study was to determine the role fructose 1,6-diphosphate (FDP), a naturally occurring glycolytic intermediate, plays in activated T-lymphocytes. The hypothesis is twofold. First, we propose that FDP inhibits T cell proliferation to a greater extent than fructose-1-phosphate (F1P), fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P); second, we argue that FDP suppresses immune activation by inhibiting inflammatory cytokine expression, inhibiting expression of key transcription factors, and by inducing apoptosis in immune cells. Rat spleen cells were incubated with concanavalin A (ConA) and increasing concentrations of FDP. Proliferation was determined by tritiated thymidine uptake. FDP inhibited splenocyte proliferation in a dose-related manner while F1P, F6P, M6P demonstrated inhibition only at high concentrations (5000 microg/ml). RNA was harvested from FDP and ConA-treated cells and IL-1 and IL-6 gene expression was analyzed by RT-PCR. IL-1 and IL-6 mRNA expression was completely inhibited at 500-5000 microg/ml FDP. Apoptosis in FDP-treated lymphocytes was determined by DNA fragmentation and flow cytometry. Propidium iodide (PI) staining demonstrated a 39% rate of apoptosis in splenocytes treated with ConA and 5000 microg/ml FDP. Extensive DNA fragmentation was present at 250-5000 microg/ml FDP, and maximal inhibition occurred at 5 microg/ml. F1P, F6P and M6P showed maximal inhibition only at 5000 microg/ml. Nuclear extracts from FDP-treated splenocytes were analyzed by electrophoretic mobility shift assay. ConA activation of NF-kappaB and AP-1 was dramatically inhibited by FDP. Interestingly, beta-actin showed extensive inhibition with FDP and ConA, thus suggesting new possibilities of its being used as a therapeutic modality in arterial injury where the beta-actin, an important cytoskeleton element, plays a very important role. These data indicate that FDP may be a useful immunosuppressive agent. In conclusion, FDP is not only an immunosuppressant but also an anti-inflammatory agent.

Actins↗