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PubMed1990

Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter.

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the distal one of which controls synthesis of actin in a tissue- and developmental stage-specific manner. This very strong promoter has widely been used for expression of heterologous genes in cultured cells. To locate functional regulatory elements in this distal promoter, mutants of the promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. The results showed that the upstream end of the promoter extends to 522 bp from the transcription start site. In addition, there are two remote activating regions about 2 kb upstream. Between -522 and -379 are two regions that exert a strong negative effect. Downstream from these negative regions are at least six positive regions and a TATA element. The strongest positive determinant of the promoter was identified at -320 as AAAATGTG by footprinting and by a replacement experiment. When the relevant region was replaced by a synthetic sequence containing this element in a random context, the transient expression activity was restored. The sequence TGTATG located at -355 was also identified as a positive element by a similar replacement approach. Apparently the very high activity of this promoter is the result of the combined activities of multiple factors.

Actins↗

PubMed1997

Evidence for the presence of smooth muscle alpha-actin within pericytes of the renal medulla.

This study was designed to determine whether smooth muscle alpha-actin mRNA and smooth muscle alpha-actin contractile protein elements were present within the renal medullary pericytes. Extraction of total RNA from microdissected outer medullary descending vasa recta allowed for the detection of smooth muscle alpha-actin mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). Expression of smooth muscle alpha-actin was specific to the descending vasa recta and not a result of tubular contamination because RT-PCR amplification of the vasopressin V2 receptor, which is a specific tubular marker, did not occur. To determine the exact cell type(s) that translate the mRNA into protein, we performed immunohistochemistry on the renal outer and inner medulla using a monoclonal smooth muscle alpha-actin antibody, whose specificity was determined by immunoblot analysis. Smooth muscle alpha-actin protein was found selectively within the pericytes surrounding the descending vasa recta from the outer and inner medullary tissue sections. This study demonstrates that the pericytes alone that surround the descending vasa recta within the outer and inner medulla contain smooth muscle alpha-actin mRNA and protein and are therefore the site of the contractile elements that could play a vasomodulatory role in the control of renal medullary blood flow and its distribution within the renal medulla.

Actins↗

PubMed1996

Developmental and tissue-specific regulation of the murine cardiac actin gene in vivo depends on distinct skeletal and cardiac muscle-specific enhancer elements in addition to the proximal promoter.

Cardiac actin is an early marker of cardiac and skeletal muscle lineages in the mouse. After birth, the gene is down-regulated in skeletal muscle. High-level expression of the murine cardiac actin gene in skeletal myotubes in vitro involves distal (-7.8/-7.0 kb) and proximal (-5.4/-3.5 kb) enhancer sequences as well as the proximal promoter (-0.7/+0.1 kb). Transgenic mice carrying an nlacZ reporter gene under the control of different fragments of the upstream region of the cardiac actin gene were generated. This analysis led to the conclusions that (1) the proximal promoter is a weak but tissue specific element in vivo, (2) consistent high-level expression in skeletal muscle depends on the presence of at least one of the enhancers, (3) expression in adult cardiac muscle requires a cardiac enhancer located in the (-5.4/-0.7 kb) region, and (4) a construct containing these three elements gives a strong specific expression of the transgene in the heart throughout the life of the animal and in embryonic skeletal muscle. All transgenes tested reproduce the down-regulation observed in adult skeletal muscle for the cardiac actin gene. Nonuniform expression of these transgenes in the heart may mark cardiomyocytes derived from different cardiac progenitors.

Actins↗

PubMed1987

Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles.

Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin-based cytoskeletal elements.

Actins↗

PubMed2001

Actomyosin promotes cell plate alignment and late lateral expansion in Tradescantia stamen hair cells.

Cytokinesis in higher-plant cells involves the formation of a cell plate in the interzone between the separating chromatids. The process is directed by the phragmoplast, an array of microtubules, actin filaments, and membranous elements. To determine if the role of actin in cytokinesis is dependent on myosin, we treated stamen hair cells of Tradescantia virginiana L. with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase and ML-7, a specific inhibitor of myosin light-chain kinase. Treatment with BDM resulted in a tilted cytokinetic apparatus during early initiation and a wavy cell plate with curved phragmoplasts during late lateral expansion. Treatment with ML-7 also resulted in inefficient late lateral expansion of the cell plate, with effects ranging from slower expansion to complete inhibition. Taken together, these results implicate myosin in the control of cell plate expansion and alignment.

Actins↗

PubMed1998

Domain motions in actin.

Previous crystallographic investigations have shown that actin can undergo large conformational changes, even when complexed to the same actin binding protein. We have conducted a formal analysis of domain motions in actin, using the four available crystal structures, to classify the mechanism as either hinge or shear and to quantify the magnitude of these changes. We demonstrate that actin consists of two rigid cores, a semi-rigid domain and three conformationally variable extended loops. Confirming predictions about the nature of the domain rotation in actin based on its structural similarity to hexokinase, we show, using an algorithm previously used only to identify protein hinges, that residues at the interface between the two rigid cores undergo a shear between alternative conformations of actin. Rotations of less than 7 degrees in the torsion angles of five residues in the polypeptides that connect the rigid cores enable one actin conformation to be transformed into another. Because these torsion angle changes are small, the interface between the domains is maintained. In addition, we show that actin secondary structure elements, including those outside the rigid cores, are conformationally invariant among the four crystal structures, even when actin is complexed to different actin binding proteins. Finally, we demonstrate that the current F-actin models are inconsistent with the principles of actin conformational change identified here.

Actins↗

PubMed1997

A strong inhibitory element down-regulates SRE-stimulated transcription of the A3 cytoplasmic actin gene of Bombyx mori.

To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.

Actins↗

PubMed1983

Actin filaments, stereocilia, and hair cells of the bird cochlea. II. Packing of actin filaments in the stereocilia and in the cuticular plate and what happens to the organization when the stereocilia are bent.

A comparison of hair cells from different parts of the cochlea reveals the same organization of actin filaments; the elements that vary are the length and number of the filaments. Thin sections of stereocilia reveal that the actin filaments are hexagonally packed and from diffraction patterns of these sections we found that the actin filaments are aligned such that the crossover points of adjacent actin filaments are in register. As a result, the cross-bridges that connect adjacent actin filaments are easily seen in longitudinal sections. The cross-bridges appear as regularly spaced bands that are perpendicular to the axis of the stereocilium. Particularly interesting is that, unlike what one might predict, when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. In the images of bent bundles, the bands of cross-bridges are then tilted off perpendicular to the stereocilium axis. When the stereocilium is bent at its base, all cross-bridges in the stereocilium are affected. Thus, resistance to bending or displacement must be property of the number of bridges present, which in turn is a function of the number of actin filaments present and their respective lengths. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. Perhaps its physiological vulnerability may result from changes of stereociliary structure.

Animals↗

PubMed1994

Phenotypic rescue by a bovine transgene in a Cu/Zn superoxide dismutase-null mutant of Drosophila melanogaster.

Null mutants for Cu/Zn superoxide dismutase (CuZnSOD) in Drosophila melanogaster are male sterile, have a greatly reduced adult life span, and are hypersensitive to paraquat. We have introduced a synthetic bovine CuZnSOD transgene under the transcriptional control of the D. melanogaster 5C actin promoter into a CuZnSOD-null mutant of D. melanogaster. This was carried out by P-element-mediated transformation of the Drosophila-bovine CuZnSOD transgene into a CuZnSOD+ recipient strain followed by genetic crossing of the transgene into a strain carrying the CuZnSOD-null mutation, cSODn108. The resulting transformants express bovine CuZnSOD exclusively to about 30% of normal Drosophila CuZnSOD levels. Expression of the Drosophila-bovine CuZnSOD transgene in the CuZnSOD-null mutant rescues male fertility and resistance to paraquat to apparently normal levels. However, adult life span is restored to only 30% of normal, and resistance to hyperoxia is 90% of that found in control flies. This striking differential restoration of pleiotropic phenotypes could be the result of a threshhold of CuZnSOD expression necessary for normal male fertility and resistance to the toxicity of paraquat or hyperoxia which is lower than the threshold required to sustain a normal adult life span. Alternatively, the differential rescue of fertility, resistance to active oxygen, and life span might indicate different cell-specific transcriptional requirements for these functions which are normally provided by the control elements of the native CuZnSOD gene but are only partly compensated for by the transcriptional control elements of the actin 5C promoter.

Animals↗

PubMed1995

Serum response element associated transcription factors in mouse embryos: serum response factor, YY1, and PEA3 factor.

Many mammalian transcription factors, including human and mouse serum response factors (SRFs), are post-translationally modified with O-linked N-acetylglucosamine monosaccharides on multiple serine and/or threonine residues. Nuclear extracts were prepared from 9.5 to 19 days postcoitum mouse embryos and subsequently were fractionated by wheat germ agglutinin (WGA)-agarose affinity chromatography. SRF binds WGA-agarose and apparently is O-glycosylated. On the other hand, the low molecular weight serum response element (SRE)-binding proteins, including the previously named band I and band II factors, did not bind WGA-agarose. Furthermore, we showed that the fastest migrating complex contains the Yin-Yang 1 (YY1) factor. YY1 binds to the c-fos SRE and skeletal alpha-actin muscle regulatory element (MRE), but not the cardiac alpha-actin MRE. Nuclear extracts from NIH/3T3 fibroblasts contain similar, if not identical, SRE-binding complexes. Besides these SRE-binding factors, mouse PEA3-binding factor, presumably an ETS domain-containing protein, was found to bind SRF protein. This physical interaction, between SRF and ETS domain proteins, was shown to involve the DNA-binding domain-containing region of SRF and not the carboxyl-terminal transactivation domain.

Animals↗

PubMed1990

Effects of ionizing radiation on expression of genes encoding cytoskeletal elements: kinetics and dose effects.

We examined the modulation in expression of genes encoding three cytoskeletal elements (beta-actin, gamma-actin, and alpha-tubulin) in Syrian hamster embryo (SHE) cells following exposure to ionizing radiations. Early-passage SHE cells were irradiated in plateau phase with various low doses (12-200 cGy) of neutrons, gamma-rays, or x-rays. RNA samples were prepared from cells at different times postexposure and were analyzed for levels of specific transcripts by northern blots. The results revealed that alpha-tubulin was induced by both high-linear energy of transfer (LET) (neutrons) and low-LET (gamma-rays and x-rays) radiations with similar kinetics. The peak in alpha-tubulin mRNA accumulation occurred between 1 and 3 h postexposure; for gamma-actin mRNA, accumulation was similarly induced. For both gamma-actin and alpha-tubulin, the higher the dose during the first hour postexposure (up to 200 cGy gamma-rays), the greater the level of mRNA induction. In contrast, mRNA specific for beta-actin showed decreased accumulation during the first hour following radiation exposure, and remained low up to 3 h postexposure. These results document the differential modulation of genes specific for cytoskeletal elements following radiation exposure. In addition, they demonstrate a decrease in the ratio of beta-actin:gamma-actin mRNA within the first 3 h following gamma-ray exposure. These changes in mRNA accumulation are similar to those reported in some transformed cell lines and in cells treated with tumor promoters, which suggests a role for changes in actin- and tubulin-mRNA expression in radiation-mediated transformation.

Actins↗

PubMed1992

Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene.

The human salivary amylase genes are associated with two inserted elements, a gamma-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.

Actins↗

PubMed1989

In vitro activation of bovine adrenal tyrosine hydroxylase by rabbit skeletal muscle actin: evidence for a possible role of cytoskeletal elements as an activator for cytoplasmic enzymes.

Tyrosine hydroxylase prepared from the soluble fraction of bovine adrenal medulla was markedly activated by rabbit skeletal muscle G-actin, and this activation was accompanied by a decrease in the apparent Km of the enzyme for the pterin cofactor. The activating effect of G-actin on the soluble enzyme was still observed in the medium containing a high concentration of salt or excess amounts of proteinase inhibitors. Furthermore, this effect was not affected by either cytochalasin B or DNase I. These results therefore suggest that G-actin interacts with the enzyme molecule at the binding site(s) different from that involved in actin polymerization, and that it causes the activation of the soluble enzyme as a result of an allosteric alteration in the enzyme structure, thus giving rise to the possibility that cytoskeletal elements play an important role in the regulation of catecholamine synthesis as a factor modulating the activity of cytoplasmic tyrosine hydroxylase.

Actins↗

PubMed1991

Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties.

DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specific expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.

Actins↗

PubMed1998

[Association of phosphoinositide-specific phospholipase Cgamma1 with elements of cytoskeleton in A-431 cells].

It is shown that phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1), a substrate of growth factor receptors, is associated with cytoskeleton in A-431 cells. PLC gamma 1 is co-localized only with the cortical actin but not with actin stress fibers. Since EGF receptor is also co-localized with the cortical actin it is concluded that PLC gamma 1 co-localized with actin is mediated by the EGF receptor. After the treatment with cytochalasin B PLC gamma 1 is co-localized with actin aggregates and cytoskeleton elements other than actin. Using double immunofluorescence PLC gamma 1 is shown to be associated with cytokeratin intermediate filaments. The cross-talking of different cytoskeleton elements and their participation in cell signaling is discussed.

Actins↗

PubMed1994

Contractile units in stress fibers of fetal human astroglia in tissue culture.

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca(2+)-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed 'contractile units', suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.

Actins↗

PubMed1999

A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

Actins↗

PubMed2007

Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.

Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.

Actin Depolymerizing Factors↗