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At least 775 records · Page 43Linked to original sources
PubMed1974

Actin antibody: the specific visualization of actin filaments in non-muscle cells.

Actin purified from mouse fibroblasts by sodium dodecyl sulfate gel electrophoresis was used as antigen to obtain an antibody in rabbits. The elicited antibody was shown to be specific for actin as judged by immunodiffusion and complement fixation against partially purified mouse fibroblast actin and highly purified chicken muscle actin. The antibody was used in indirect immunofluorescence to demonstrate by fluorescence light microscopy the distribution and pattern of actin-containing filaments in a variety of cell types. Actin filaments were shown to span the cell length or to concentrate in "focal points" in patterns characteristic for each individual cell.

Actins↗
PubMed1980

Cloning of sea urchin actin gene sequences for use in studying the regulation of actin gene transcription.

In order to investigate the regulation of actin gene transcription during early sea urchin development, a specific hybridization probe for actin sequences is required. Such a probe was produced by cloning cDNA transcribed from a sea urchin poly(A)-containing mRNA preparation enriched for actin message. Double-stranded DNA was ligated into the BamHI restriction site of plasmid pBR322, and the resulting hybrid molecules were used to transform the Escherichia coli strain ML100. After preliminary screening of bacterial colonies by antibiotic sensitivity and hybridization back to the original cDNA, clones containing sea urchin DNA were further characterized by a positive translation assay in which total sea urchin mRNA was hybridized to plasmid, and the hybridized message then was eluted and translated in a reticulocyte cell-free protein-synthesizing system. In this way, one clone (pSA38) was found to hybridize selectively to sea urchin mRNA coding for a protein of 43,000 daltons. This protein was identified as actin by three criteria: electrophoretic migration in two-dimensional polyacrylamide gels, affinity for DNase I, and peptide mapping. Restriction endonuclease and heteroduplex mapping of pSA38 indicate that it contains a 1.5-kilobase-pair insert and is therefore likely to contain a large portion of the actin coding sequence. By using pSA38 as a hybridization probe, it has been found that the level of actin-specific RNA sequences increases dramatically during early sea urchin development.

Actins↗
PubMed1989

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.

Actins↗
PubMed1999

Histidine-tagged wild-type yeast actin: its properties and use in an approach for obtaining yeast actin mutants.

Wild-type and an N-terminal 6-histidine-tagged actin have each been expressed by using a yeast strain that contains the actin gene on a plasmid and not on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild-type protein and the other the 6-histidine-tagged protein. Yeast cells can be grown with either plasmid alone or with both plasmids together and appear to be normal in that the growth rates of all the yeast strains are quite similar, as is the morphology of the yeast cells. The polymerization properties of the 6-histidine-tagged actin appear almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be purified by standard techniques and then separated using nickel-nitrilotriacetate chromatography. The method can be used to prepare actin mutants including those that are nonfunctional or might not support yeast growth for other reasons.

Actins↗
PubMed1995

Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

3T3 Cells↗
PubMed2001

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

3T3 Cells↗
PubMed2003

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport.

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein.

Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.

Actins↗
PubMed2006

Adenovirus E4orf4 hijacks rho GTPase-dependent actin dynamics to kill cells: a role for endosome-associated actin assembly.

The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin-myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate.

Actins↗
PubMed1986

Nucleotide sequence of the human gamma cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes.

Two distinct, but iso-coding, gamma non-muscle actin cDNAs were isolated from an SV40-transformed human fibroblast library. The complete nucleotide sequence of the human gamma non-muscle actin cDNAs indicates that they may have arisen from polymorphic alleles. By using genomic DNA and cellular RNA transfer blots, we demonstrate that the 3' untranslated region (UTR) of the gamma actin mRNA consists of an evolutionarily conserved 5' and more divergent 3' segments. In fact, the conserved segment of the 3' UTR detects a single-copy sequence in the chicken genome and a 20S RNA transcript in chicken non-muscle tissues. The coding regions of these cDNAs were compared with those of other vertebrate non-muscle actin genes. Surprisingly, the percentage of silent base substitutions between the human beta and gamma actin coding regions is anomalously low and indicates greater sequence conservation than would be expected for a gene pair which arose during pre-avian evolution. We discuss gene conversion and recent selective pressure as possible explanations of the apparently anomalous evolution of the gamma non-muscle actin gene.

Actins↗
PubMed1986

The 45K molecular weight actin-modulating protein from sea urchin eggs forms a complex with actin in the presence of calcium ions.

The 45K protein from sea urchin eggs forms a complex with actin in the presence of Ca2+. The fraction was not readily dissociated on depletion of Ca2+ by gel filtration, but was dissociated on dialysis against an EGTA-containing solution. The free 45K protein and the 45K protein-actin complex affect F-actin in different manners in the presence of Ca2+. The former severs F-actin in the presence of Ca2+ but not in its absence, while the latter caps the barbed end of F-actin in a Ca2+-independent manner thereby lowering the viscosity of F-actin slowly.

Actins↗
PubMed1983

Cross-linking study on skeletal muscle actin: interaction of suberimidate-treated actin with deoxyribonuclease I.

We have recently reported that actin modified with dimethyl suberimidate takes a filamentous form even under depolymerizing conditions, and this phenomenon is accounted for by the conformational fixation caused by the introduction of an intramolecular cross-link (Ohara, O., Takahashi, S., Ooi, T., & Fujiyoshi, Y. (1982) J. Biochem. 91, 1999-2012). The suberimidate-treated actin (SA) is not immediately depolymerized by deoxyribonuclease I (DNase I) but is depolymerized after incubation for one day, i.e., depolymerization is much slower than that for intact F-actin. The results on circular dichroic spectra of a mixture of SA and DNase I suggest that DNase I flips the conformation of SA into a G-actin-like state from the F-actin-like one when a tight SA-DNase I complex is formed. The suberimidate cross-link introduced in an SA molecule does not completely prevent the conformational change from the F-state to the G-state but stabilizes the actin conformation very greatly in the F-state.

Actins↗
PubMed1983

Interaction of alkali light chain 1 with actin: effect of ionic strength on the cross-linking of alkali light chain 1 with actin.

To determine the spatial relationship between alkali light chain and actin in the actosubfragment-1 complex, we studied the cross-linking of actin and subfragment-1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We found that (a) alkali light chain 1 was cross-linked to actin at two sites in the extrapeptide region, and (b) cross-linking of these two sites, especially the one which was very close to the NH2 terminal of the alkali light chain, to actin was inhibited drastically when the KCl concentration was increased from 0 to 100 mM. Since the inhibition of cross-linking with carbodiimide reagent means separation of amino and carboxyl groups in alkali light chain and actin, we suggest that this decrease in electrostatic attraction is the reason why subfragment-1 with alkali light chain 1 has higher affinity to actin than subfragment-1 with alkali light chain 2 at low ionic strength but has almost the same affinity at moderate ionic strength.

Actins↗
PubMed2000

Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin.

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K(d) = approximately 5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.

Actins↗
PubMed1981

Actin in Xenopus development: indirect immunofluorescence study of actin localization.

Actin was studied in Xenopus unfertilized eggs and early developmental stages. Immunochemical proof is given of structural differences between Xenopus laevis muscle actin and nonmuscle cell actin. Actin localization and changes of actin aggregation during Xenopus development were observed using indirect immunofluorescence. We have also tried to explain the presence of an actin shell around the yolk platelets that appeared in our experiments.

Actins↗
PubMed1992

The mammalian anti-alpha-smooth muscle actin monoclonal antibody recognizes an alpha-actin-like protein in planaria (Dugesia lugubris s.l.).

The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.

Actins↗
PubMed1977

Human platelet actin. Evidence of beta and gamma forms and similarity of properties with sarcomeric actin.

Human blood platelet actin was purified using 30% sucrose to extract actomyosin and potassium iodide to dissociate actomyosin and to depolymerize actin. Pure actin thus obtained resembles skeletic muscle actin in its polymerization properties, CD spectra and ability to activate myosin myosin Mg2+-ATPase. Isoelectric focusing gel analysis shows that human blood platelet actin exists in beta and gamma forms. The ratio of beta to gamma forms is of 5 in purified actin, in whole cell extract and in all the fractions studied.

Actins↗
PubMed1986

Interactions of plasma gelsolin with actin. Isolation and characterization of binary and ternary plasma-gelsolin-actin complexes.

We have studied the interactions between plasma gelsolin and actin: firstly the complex formation between both proteins, secondly the effects of gelsolin and its complexes on G-actin polymerization and F-actin fragmentation. Complex formation has been studied by high-performance gel permeation chromatography; plasma gelsolin alone elutes at an Mr of about 77000 and a Stokes radius of 3.7 nm; complex formation occurs in the presence of Ca2+: by chromatography in the presence of EGTA, a binary complex is obtained with an Mr of 134000 and a Stokes radius of 4.7 nm; and by chromatography in the presence of Ca2+, a ternary complex is obtained with an Mr of 173000 and a Stokes radius of 5.2 nm. The binary complex is EGTA-stable. In relation to this stability of the binary complex, the depolymerizing function of gelsolin is not reversed upon chelation of Ca2+. The effects of plasma gelsolin and its complexes on both G-actin polymerization and F-actin fragmentation, and their Ca2+ dependence have been examined by viscometry and electron microscopy. The main conclusions of these studies are the following: the fast processes are the formation of ternary complex, which acts as a heteronucleus for G-actin polymerization, and the severing function of gelsolin, these fast processes are Ca2+-dependent; the slow processes are related to the capping ability of gelsolin or its complexes and are Ca2+-independent.

Actins↗