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At least 811 records · Page 45Linked to original sources
PubMed1987

A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract.

The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.

Animals↗
PubMed1988

Inhibition of deoxyribonuclease I activity by actin covalently cross-linked to chick brain actin depolymerizing factor through exposed sulfhydryls.

All but one of the six free sulfhydryl groups of chick brain actin depolymerizing factor (ADF) are protected from modification when ADF forms a 1:1 complex with actin. This exposed sulfhydryl can be cross-linked to cys 374 of actin with N,N'-phenylenedimaleimide. The cross-linked complex inhibits the hydrolytic activity of pancreatic deoxyribonuclease (DNase I) to an identical extent as both the untreated complex and an equivalent amount of free actin. These data indicate that ADF binds to actin at a site which does not overlap with the DNase I binding site.

Actin Depolymerizing Factors↗
PubMed1990

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.

Actin Depolymerizing Factors↗
PubMed2000

Scinderin, a Ca2+-dependent actin filament severing protein that controls cortical actin network dynamics during secretion.

Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca2+, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca2+-dependent activity of scinderin together with its Ca2+-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.

Actin Cytoskeleton↗
PubMed1999

Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion.

Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases. An important virulence factor of A. actinomycetemcomitans is the ability to invade human oral epithelial cells. A clinical isolate, A. actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism. The internalized bacteria are surrounded by an actin halo upon entry. These data are consistent with the mode of entry associated with many enteric pathogens. We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A. actinomycetemcomitans clinical isolates. Cytochalasin D was added prior to infection. A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D. Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A. actinomycetemcomitans SUNY 523. Inhibitors of receptor-mediated endocytosis inhibited invasion of A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065. Microtubule effectors did not inhibit invasion of A. actinomycetemcomitans. A. actinomycetemcomitans SUNY 523, but not A. actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium. These data suggest that A. actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A. actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread.

Actin Cytoskeleton↗
PubMed1999

Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

Actin Cytoskeleton↗
PubMed1985

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence.

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.

Actin Depolymerizing Factors↗
PubMed1992

Movement along actin filaments of the perijunctional area and de novo polymerization of cellular actin are required for Shigella flexneri colonization of epithelial Caco-2 cell monolayers.

Shigella flexneri invades eucaryotic cells and grows in the cytoplasm. Lysis of the phagosomal membrane is a prerequisite for both intracellular multiplication and movement of the bacteria that gain direct access to the host cell actin. In HeLa cells, bacteria generate their own movement essentially by inducing actin polymerization. Polymerization of actin enables them to move rapidly and randomly in the cytoplasm and to spread from one cell to another through protrusions of the host cell membrane. This movement was designated the Ics phenotype. In contrast, in chicken embryo fibroblasts, bacteria move along actin filaments in a very organized manner, following the cytoskeletal architecture; this movement was designated the Olm phenotype. Bacterial movement is a major virulence factor in that it is necessary for efficient colonization of the intestinal epithelium of infected macaque monkeys. Further characterization of the cellular events that lead to colonization of the colonic intestinal epithelium was needed. In order to characterize the movement in vitro in a cell assay system more closely related to the intestinal epithelium, we used human colonic epithelial Caco-2 cells. The movement of bacteria as observed by using immunofluorescence and confocal microscopy appeared to result from the expression of both the Olm and Ics phenotypes. The former allowed colonization of cells along the actin filament ring of the perijunctional area. The latter promoted passage from one cell to adjacent cells. This in vitro pattern of movement and multiplication gives S. flexneri, once it has entered an epithelial cell, the unique capacity to spread through the entire epithelial layer without having further contact with the extracellular compartment.

Actin Cytoskeleton↗
PubMed1997

Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis.

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.

Actin Cytoskeleton↗
PubMed2001

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.

Actin Depolymerizing Factors↗
PubMed2005

Rapid downregulation of beta-actin-based CAG promoter and filamentous actin in injured podocytes.

Glomerular visceral epithelial cells or podocytes are located on the outer surface of the glomerular basement membrane and play an indispensable role as a filtration barrier. The core cytoskeleton of the foot processes is actin filaments, which play an important role in maintaining the unique structure of podocytes. We previously established a transgenic mouse line (NEP25), which expresses human (h)CD25 selectively on podocytes. By injecting an hCD25-targeted recombinant immunotoxin (LMB2), podocyte injury can be induced on demand. After LMB2 injection, NEP25 mice develop nephrotic syndrome with downregulation of podocyte-specific proteins. In the present study, we genetically labeled podocytes with lacZ linked with beta-actin-based CAG promoter. Utilizing the Cre-loxP system, this labeling was confined to the podocyte lineage. Without LMB2, all podocytes were positive for lacZ. After LMB2 injection, lacZ expression was rapidly downregulated, before podocytes showed any discernible morphological changes. Confocal imaging of filamentous (F)-actin-binding Alexa 488-phalloidin revealed that the normal continuous pattern of F-actin distribution in podocytes was punctuated after LMB2 injection. These collectively suggest that disturbance of actin filaments may be one of the key initial events leading to subsequent podocyte damage.

Actin Cytoskeleton↗
PubMed1989

Changes in the association of actin-binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum.

Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.

Actins↗
PubMed1986

A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.

Actins↗
PubMed1993

The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34.

During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. To investigate the protein-protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N-terminal 42 kDa ATPase region and the C-terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1996

Isolation of 88F actin mutants of Drosophila melanogaster and possible alterations in the mutant actin structures.

The 88F actin (act88F) gene of Drosophila, melanogaster encodes an actin isoform that is expressed exclusively in the indirect flight muscle. In order to isolate a large number of act88F mutants, an efficient screening method was used to obtain dominant flightless mutants. Genetic analyses revealed that 25 mutations were located near or at the act88F locus. From each mutant strain, the DNA fragments including the coding region of the act88F gene were asymmetrically amplified by the polymerase chain reaction method, and the amplified fragments were directly sequenced. Eighteen of them were found to have point mutations within their coding regions. Of these, 13 were novel alleles of this gene. We have characterised these mutations in detail. First, their flight abilities were tested after introducing two normal alleles of this gene. Second, two-dimensional gel electrophoresis was used to examine actin isoforms and whole thorax proteins. Third, morphological anomalies of indirect flight muscle fibres and myofibrils were examined with an optical microscope. On the basis of these phenotypes and the known atomic structure of actin, possible alterations in the structure of actin brought about by these mutations are discussed.

Actins↗
PubMed1994

Lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility.

Consideration of the arrangement of actin filaments in the lamellipodia of crawling cells indicates that, in addition to a rearward flow of the actin cytoskeleton due to treadmilling, there is a lateral flow of filaments in both directions. The existence of such a lateral flow of actin filaments is supported by observation of the lateral movement of actin filament bundles in fibroblasts and by fluorescence photoactivation data. In addition to explaining the formation, dispersal and lateral movement of filament bundles, lateral filament flow could add a further velocity component to the centripetal flow of cytoplasm. As such, lateral filament flow may explain the discrepancies in the rates of the rearward flow of actin, cytoplasmic materials and dorsal particles.

Actins↗
PubMed1993

Identification of actin surface interacting with myosin during the actin-myosin sliding.

We constructed several mutant actin genes from the Dictyostelium actin 15 gene by the site-directed mutagenesis. Mutations were designed to change acidic residues in actin subdomain 1 to histidine residues. Amino acid replacements were: D1H (single replacement of Asp1 to His), D4H, D1H/D4H (double replacements of Asp1 and Asp4 to histidine), D1H/E3H/D4H (triple replacements of Asp1, Glu3 and Asp4 to histidine), D24H/D25H, E99H/E100H, E360H/E361H, and D363H/E364H. Mutant genes were then expressed in Dictyostelium cells. In vitro motility assays were carried out for purified actins to see whether the mutations affect sliding motion of actin filaments driven by HMM. The assays showed that replacement of N-terminal acidic residues inhibited the sliding. Replacement of D24/D25 and E99/E100 also resulted in inhibition of the sliding motion. However, replacement of acidic residues at the C-terminal cluster E360/E361/D363/E363 did not resulted in loss of motility.

Actins↗
PubMed1981

Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation.

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [3H]actin in a time- and Mg2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their 'barbed' ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.

Actins↗