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PubMed1994

Identification and characterization of multiple A/T-rich cis-acting elements that control expression from Dictyostelium actin promoters: the Dictyostelium actin upstream activating sequence confers growth phase expression and has enhancer-like properties.

The promoter elements in the Dictyostelium actin 15 and actin 6 genes required for full growth phase expression were identified by assaying promoter/luciferase reporter constructs. We find that these promoters contain common cis-acting elements, an actin upstream activating sequence (UAS) and sequences proximal to the transcription start site that overlap with a poly(dT) region. The actin 15 promoter has two additional cis-acting elements not present in the actin 6 promoter that may account for the higher level of expression from the actin 15 promoter. All of the identified promoter elements are unusual for Dictyostelium in that they are all A/T-rich. Two cis-acting elements, the actin UAS and the poly(dT) domain were studied in greater detail. The actin UAS was tested on a heterologous promoter from the prespore-specific gene SP60 and shown to have the ability to confer growth phase expression. The actin UAS also exhibited the ability to function in a distance- and orientation-independent manner and activate expression synergistically when present in two copies. The poly(dT) domain of the actin 15 promoter was studied in greater detail by using a genetic selection scheme to define parameters that effect the strength of this element. This element is comprised of 45 consecutive dT residues immediately upstream of the putative TATA box. We show that the length of the homopolymer dT region correlates with the expression level of the promoter. The poly(dT) element is also shown to function to promote wild-type levels of expression with small deviations in the sequence, indicating that the element is not required to be homopolymeric to function.

Actins↗
PubMed1995

Actin cortical cytoskeleton and cell wall synthesis in regenerating protoplasts of the Saccharomyces cerevisiae actin mutant DBY 1693.

The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells.

Actins↗
PubMed2000

Calcium-calmodulin suppresses the filamentous actin-binding activity of a 135-kilodalton actin-bundling protein isolated from lily pollen tubes.

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421-1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca(2+) have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca(2+) and calmodulin (CaM), although neither Ca(2+) alone nor CaM in the presence of low concentrations of Ca(2+) affects the activity of P-135-ABP. A micromolar order of Ca(2+) and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca(2+)-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca(2+)-CaM. In the presence of both Ca(2+) and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca(2+)-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.

Actins↗
PubMedDate not supplied

Actin mRNA levels and actin synthesis during the encystation of Entamoeba invadens.

Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate the encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeletion occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.

Actins↗
PubMedDate not supplied

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma.

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.

Actins↗
PubMed1999

Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin beta4 in migrating IEC-6 cells.

The cause of reduced migration ability in polyamine-deficient cells is not known, but their actin cytoskeleton is clearly abnormal. We depleted polyamines with alpha-difluoromethylornithine (DFMO) in migrating cells with or without stimulation by epidermal growth factor (EGF) and investigated filamentous (F-) actin, monomeric (G-) actin, and thymosin beta4 (Tbeta4), using immunofluorescent confocal microscopy, DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cell interior, increased it in the cell cortex, redistributed G-actin, and increased nuclear staining of Tbeta4. However, DFMO did not affect the amount of Tbeta4 mRNA. EGF caused a rapid increase in the staining of F-actin in control cells, but DFMO prevented this response to EGF. Despite the visible changes shown by immunocytochemistry, statistically significant changes in the amount of either actin isoform or of total actin did not occur. We propose that DFMO reduces migration by interfering with the sequestration of G-actin by Tbeta4 and the association of F-actin with activated EGF receptors.

Actins↗
PubMed1992

Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.

Actin and actin-binding proteins form a peripheral network on the cytosolic side of the plasma membrane. These cytoskeleton proteins are involved in functions that require cellular movement and may also have a role in modulating signal transduction during cellular proliferation and differentiation. To measure changes in F-actin and actin-binding proteins during HL-60 differentiation, cells were induced to mature along the granulocytic pathway by exposure to 1 microM retinoic acid (RA) for 5 days and were analyzed for F-actin and actin-binding proteins by flow cytometry. The amounts of F-actin and spectrin in untreated HL-60 cells and in those undergoing differentiation by treatment with the retinoid did not differ. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin was used to measure F-actin content and a monoclonal antibody followed by fluorescence isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody was used to measure the content of spectrin; cells were analyzed by flow cytometry. In contrast, cells exposed to RA contained larger amounts of alpha-actinin, vinculin, talin, lipocortin I, and lipocortin II, as determined with their respective antibodies followed by flow cytometric analysis as described above. An RA-supersensitive clone of HL-60, designated HL-60/S4, exhibited lower constitutive levels of alpha-actinin, vinculin, and talin but a higher constitutive level of lipocortin II than parental cells. Treatment of HL-60/S4 with RA led to increases in vinculin, talin, lipocortin I, and lipocortin II. An RA-resistant clone, designated HL-60/R3, constitutively expressed larger amounts of alpha-actinin, vinculin, lipocortin I, and lipocortin II than parental HL-60 cells. Treatment of HL-60/R3 with RA resulted in decreases in the amounts of these actin-binding proteins. Changes in actin-binding proteins that occur during the differentiation of HL-60 cells suggest that these proteins may be of importance to the expression of the mature phenotype.

Actinin↗
PubMed1992

An actin footprint on villin. Single site substitutions in a cluster of basic residues inhibit the actin severing but not capping activity of villin.

Villin is a multidomain protein that severs, caps, and bundles actin filaments. We employed a chemical modification/cleavage strategy to identify residues whose chemical reactivities are reduced when villin is complexed with actin. We found that actin protects 3 methionine residues, Met125, Met379, and Met711 from oxidation by N-chlorosuccinimide. Because Met125 lies within the actin-severing domain of villin (44T), we probed this region for actin binding sites using a series of overlapping peptides each with an additional cysteine residue at their C terminus. Each peptide, as a disulfide-bonded dimer, was examined for actin cross-linking activity by electron microscopy and light scattering. Our results with M3R suggest this region contains an F-actin binding site and are consistent with proteolysis and deletion mutagenesis studies of gelsolin. Single substitution of the basic residues modulated actin severing but not capping activity of 44T. Circular dichroism and protease digestions did not detect alterations in secondary structure or conformational changes in the mutants, although some are cleaved more rapidly, thereby suggesting a change in the packing of the domains. Our results highlight that basic residues comprise part of the F-actin binding site that is involved in the actin severing activity of villin.

Actins↗
PubMed1983

Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. II. Ca2+-dependent f-actin-capping activity of subunit a and its regulation by phosphorylation of subunit b.

Cap 42 (a + b), a Ca2+-dependent, actin-binding and phosphorylatable protein consisting of two distinct subunits a and b of 42,000 Da in Physarum polycephalum, has been identified as a new F-actin-capping protein. It capped or bound to the fast growing ends of actin filaments and blocked actin polymerization at this end. The capping activity residing in subunit a and its Ca2+-dependency were regulated by phosphorylation of subunit b; subunit a required Ca2+ for its capping activity when subunit b was phosphorylated, whereas this activity became Ca2+ independent when subunit b was dephosphorylated. Subunit b contained at least two phosphorylatable threonine residues and probably three additional phosphorylation sites. Like cytochalasins and other F-actin-capping proteins, Cap 42 (a + b) was able to induce a rapid depolymerization of actin filaments at the slow growing end, and also to nucleate actin polymerization. However, unlike Physarum fragmin, Cap 42 (a + b) had no severing activity leading to the fragmentation of actin filaments. Our results indicate that Cap 42 (a + b) is the first Ca2+-dependent F-actin-capping phosphoprotein whose phosphorylation regulates its actin-binding and vice versa. A possible mechanism of the capping action of Cap 42 (a + b) in vitro and also its conceivable role in the regulation of the Ca2+/actin-dependent cytoplasmic streaming in plasmodia are discussed.

Actins↗
PubMed1998

Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover.

The mechanism of control of the steady state of actin assembly by actin depolymerizing factor (ADF)/cofilin and profilin has been investigated. Using Tbeta4 as an indicator of the concentration of ATP-G-actin, we show that ADF increases the concentration of ATP-G-actin at steady state. The measured higher concentration of ATP-G-actin is quantitatively consistent with the increase in treadmilling, caused by the large increase in the rate of depolymerization from the pointed ends induced by ADF (Carlier, M.-F. , Laurent, V., Santolini, J., Didry, D., Melki, R., Xia, G.-X., Hong, Y., Chua, N.-H., and Pantaloni, D. (1997) J. Cell Biol. 136, 1307-1322). Experiments demonstrate that profilin synergizes with ADF to further enhance the turnover of actin filaments up to a value 125-fold higher than in pure F-actin solutions. Profilin and ADF act at the two ends of filaments in a complementary fashion to increase the processivity of treadmilling. Using the capping protein CapZ, we show that ADF increases the number of filaments at steady state by 1. 3-fold, which cannot account for the 25-fold increase in turnover rate. Computer modeling of the combined actions of ADF and profilin on the dynamics of actin filaments using experimentally determined rate constants generates a distribution of the different actin species at steady state, which is in quantitative agreement with the data.

Actin Depolymerizing Factors↗
PubMed2000

Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

Actin-Related Protein 2↗
PubMed2004

Yeast actin patches are networks of branched actin filaments.

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.

Actin Cytoskeleton↗
PubMed1999

Involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type 3: possible role of actin in other viruses.

Multifunctional involvement of actin microfilaments during viral infection has been documented in many studies. The molecular mechanism underlying this important host-virus interaction, however, remains poorly understood. We have investigated the role of actin microfilaments in the life cycle of human parainfluenza virus type 3 (HPIV3), a paramyxovirus that causes severe respiratory illness in children. In vitro transcription with purified viral ribonucleoprotein (RNP) complex showed a requirement of cellular actin, in the polymeric form, for mRNA synthesis in vitro. This was further confirmed by using recombinant actin, which interacted with the viral RNP and also activated mRNA synthesis in vitro. Consistent with the role of the polymeric form of actin, the actin microfilaments of the cytoskeletal framework participate in the virus replication in vivo. Biochemical and immunological analyses revealed the association of viral RNPs with cytoskeletal framework during early stages of infection, and involvement of these RNPs in the synthesis of mRNAs and genome-length RNA. Immunofluorescent labeling and confocal microscopy showed that the viral nucleocapsids colocalize with the actin microfilaments. Treatment of cells with cytochalasin D, which depolymerizes actin microfilaments, inhibited viral RNA synthesis and RNP accumulation. These data indicate that actin microfilaments play a critical role in HPIV3 life cycle, specifically at the level of viral transcription and replication. Involvement of the cytoskeletal framework in the life cycle of several viruses containing RNA and DNA genomes is reviewed.

Actin Cytoskeleton↗
PubMed2005

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

Actin Cytoskeleton↗
PubMed1993

A conformational change in the actin subunit can change the flexibility of the actin filament.

The mechanical properties of F-actin are very significant, given the central structural role played by actin filaments within muscle and the cytoskeleton. We have determined that actin can exist in a state that has a fourfold increase in flexibility over normal F-actin, and nucleotide. Three-dimensional reconstructions from electron micrographs suggest that this increased flexibility arises from a rotation of subdomain-2, the smallest subdomain, of the actin subunit. The modulation of actin's flexibility by Ca2+ and Mg2+ may have important physiological consequences within the cell. Further, since it has been shown that myosin-decorated actin filaments are more flexible than pure F-actin, it is possible that myosin induces this more flexible state in actin.

Actin Cytoskeleton↗
PubMed1989

Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments.

The effect of nonmuscle actin ADP-ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP-ribosylation. ADP-ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP-ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP-ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.

Actin Depolymerizing Factors↗
PubMed1993

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

Actin Cytoskeleton↗
PubMed1986

Arrangement of actin filaments and myosin-like filaments in the contractile ring and of actin-like filaments in the mitotic spindle of dividing HeLa cells.

We used a glutaraldehyde-tannic acid-saponin fixative to improve the preservation of actin filaments in dividing HeLa cells during preparation for thin sectioning. The contractile ring in the cleavage furrow is composed of a parallel array of actin filaments that circle the equator. We show that many of these actin filaments are arranged in small bundles. These bundles consist of about 25 filaments throughout cytokinesis. For comparison, filopodia on these cells have about 23 actin filaments packed at a higher density than the filaments in the contractile ring bundles. Some of the contractile ring actin filaments appear to radiate out from electron-dense sites on the plasma membrane. The contractile ring also has a large number of short filaments 13 nm in diameter that closely resemble filaments formed from purified human cytoplasmic myosin. These thick filaments are aligned circumferentially and interdigitate with the actin filaments, as expected for a sliding filament mechanism of tension generation. There are no long actin filaments in the mitotic spindle, but there are a large number (400 to 1000 per micron 3) of very short filaments identical in appearance to actin filaments in other parts of these cells. These short filaments may account for the reported staining of the mitotic spindle with fluorescent antibodies to actin and with fluorescent myosin fragments.

Actin Cytoskeleton↗