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At least 541 records · Page 30Linked to original sources
PubMed1981

A new 220,000 dalton protein located in the Z lines of vertebrate skeletal muscle.

A new protein with a chain weight of approximately 220,000 was isolated from 0.6 M KI extracts of I-Z-I brushes of rabbit and chicken skeletal muscles, using (NH4)2SO4 precipitation and three column chromatographic procedures in succession. It was only possible to separate the high molecular weight protein from actin and alpha-actinin in the presence of 6 M urea or 0.1% sodium dodecyl sulfate (SDS). The purified protein migrated as a single band on SDS gel electrophoresis. The amino acid composition of the 220,000 dalton protein was distinct from any known proteins found in myofibrils, e.g., alpha-actinin and actin binding protein (ABP; filamin). An indirect immunofluorescence technique revealed that the new protein was exclusively located in the Z lines of myofibrils of chicken breast muscle. There is, however, a possibility that the 220,000 dalton protein is identical with synemin recently isolated from chicken gizzard (Granger, B.L. and Lazarides, E. (1980) Cell 22, 727).

Amino Acids↗
PubMed1981

Stimulation of actomyosin Mg2+-ATPase activity by a brain microtubule-associated protein fraction. High-molecular-weight actin-binding protein is the stimulating factor.

Actomyosin Mg2+-ATPase activity was stimulated by a brain microtubule-associated protein (MAP) fraction. The stimulating activity of the MAP fraction was abolished by boiling and trypsin treatment, suggesting the presence of a protein factor. The factor stimulated actomyosin Mg2+-ATPase activity stoichiometrically by about four times in the optimum conditions (50--75 mM KCl, pH 6.6). The stimulating factor was coprecipitable with actomyosin and was found to be a pair of high-molecular-weight polypeptides (mol wts, 240,000 and 235,000). The polypeptides were not associated with microtubules or myosin, but with fibrous actin. In column chromatographies used for purifying the stimulating factor, the amount of polypeptides coincided with the stimulating activity. Increases in both specific activity and the amount of the paired polypeptides were nearly parallel in the process of the purification. A purified fraction (65% pure with respect to the paired polypeptides) showed a 56-fold increase of the specific stimulating activity as compared with the initial brain supernatant. The two peptides were similar but not identical with filamin and spectrin in terms of electrophoretic mobility. Hence, the pair of polypeptides was identified as an actin-binding protein newly found in brain.

Actomyosin↗
PubMed1983

Further characterization of a brain high molecular weight actin-binding protein (BABP): interaction with brain actin and ultrastructural studies.

Crude actomyosin fraction from porcine brain contained a large amount of high molecular weight actin-binding protein (BABP). The molar ratio of BABP to actin (BABP/actin) in the fraction was estimated to be 0.22. From this fraction, BABP and actin were solubilized with a molar ratio of 0.25, suggesting the existence of an interaction between BABP and brain actin. BABP was finally purified to 90% purity. The purified BABP was negatively stained and observed by electron microscopy; it appeared to be a slender, flexible, two-stranded molecule whose contour length was about 200 nm. The structure was very similar to those of fodrin and other high molecular weight actin-binding proteins such as filamin, spectrin, and ABP. Lattice cage-like structures composed of BABP molecules were occasionally observed at high BABP concentrations. The addition of BABP to actin filaments resulted in the appearance of many branching, filamentous bundles. The electron microscopic observations suggested that a single BABP molecule could crosslink actin filaments, that is, one BABP molecule has two actin binding sites.

Actins↗
PubMed1985

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon.

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.

Actins↗
PubMed1997

Paraquat induces actin assembly in depolymerizing conditions.

The molecular mechanism (or mechanisms) at the basis of paraquat (PQ) (a widely used herbicide) toxicity is far from being fully understood. Until now, two main points of view have emerged: 1) PQ-related cell injuries could be mediated by toxic oxygen free radicals coming from the metabolism of the herbicide by the microsomal enzyme system, and/or 2) PQ, by inducing mitochondrial swelling and breakage, could cause troubles in cell energy charge, then driving the cell to death. Recently, some of cytoskeletal structures (microtubules and microfilaments) have been proposed as further PQ cell targets. The microfilament system in particular seems to be markedly affected by the herbicide, but so far no direct evidence associates PQ to actin damage. In this study, experimental data are presented concerning the direct effect of PQ on actin dynamics in solution. We demonstrate that actin selectively binds PQ; moreover, PQ induces the formation of actin sopramolecular structures in depolymerizing medium (G-buffer). Furthermore, by the interactions with F-actin cross-linking proteins (alpha-actinin and filamin), FITC-phalloidin, and myosin subfragment 1 (S1), it is demonstrated that PQ-induced actin aggregates are undoubtedly built up by F-actin. Electron micrographs showed that PQ-induced actin polymers are very short and tend to aggregate one to another. This mutual cohesion leads to the steric blockage of polymer growing ends as suggested by nucleated actin polymerization assays. Sonication, by releasing F-actin fragments from short polymer aggregates, allows actin polymer ends to regain their growing ability.

Actinin↗
PubMed2000

Presenilin I interaction with cytoskeleton and association with actin filaments.

Presenilin I (PSI) has been shown to interact with microfilament-associated proteins of the filamin family. Here, we investigated a possible association of PSI with the cytoskeleton. Immunoblotting of detergent-insoluble fractions of rat brain homogenate revealed enrichment of neuron-specific 36 and 14 kDa proteolytic fragments of PSI, whereas 30 and 20 kDa fragments were found in the detergent-soluble fraction. Specific severing of microfilaments with gelsolin in the detergent-insoluble pellet and subsequent centrifugation led to the detection of both actin and PSI fragments in the supernatant. In addition, in vitro translated PSI cosedimented with actin filaments. Our findings provide biochemical evidence for the association of PSI fragments with actin filaments.

Actin Cytoskeleton↗
PubMed1981

An immunoperoxidase study of senile cerebral amyloidosis with pathogenetic considerations.

Samples of human cerebral cortex were obtained from twelve autopsied patients with Alzheimer's disease or "normal" aging. Rabbit or goat anti-human antisera to the following plasma proteins: IgG, F(ab')2, Fc, kappa and lambda light chains, IgM, IgA, fibrinogen, albumin, C3, lysozyme, haptoglobin, macroglobulin, and microglobulin; antibodies to the following intracellular proteins: glial fibrillary acidic (GFA) protein, filamin, actin, non-muscle myosin, tubulin, cholinergic vesicle proteins, and neurofilament (NF) proteins were utilized in the immunoglobulin peroxidase bridge. Amyloid cores of classical or perivascular plaques and dyshoric angiopathy exhibited a strong reaction for intact IgG and for both of its light chains, moderate reactions for lysozyme, fibrinogen, albumin and IgA, and weak reactions for IgM, C3, Fc, F(ab')2, haptoglobin, macroglobulin and microglobulin. Antibodies to all three NF proteins, individually and pooled, stained dyshoric and plaque amyloid, while antibodies to other intracellular proteins did not. The coronae of classical plaques and many primitive plaques stained for GFA, but inconsistently for IgG, both light chains, lysozyme, actin, tubulin, and NF proteins. Affected vessels of three patients with Congophilic angiopathy were reactive for all plasma proteins (especially IgG, fibrinogen, and albumin) and for NF proteins. NF staining in Congophilic blood vessels, although variable, revealed a peripheral or adventitial distribution, whereas plasma proteins tended to be localized in the media of the vessel wall. The distributions of Congo red and NF positivity were often identical. Both NF and Congo red staining was sensitive to oxidation. Isolated NF proteins were Congophilic and capable of displaying apple-green birefringence. A hypothesis concerning the role of NF proteins in senile cerebral amyloid is presented.

Amyloid↗
PubMed2006

Pathogenesis of migration disorders.

PURPOSE OF REVIEW: Neocortical neurons have to migrate from their site of production in the periventricular germinative zone or in the ganglionic eminence towards the cortical plate. Our understanding of the underlying molecular mechanisms has advanced considerably in recent years due to the identification of genes involved in human migration disorders and experimental studies. This review will highlight some of the most recent findings in the deciphering of the molecular machinery controlling neuronal migration. RECENT FINDINGS: Neuronal migration is a complex process which involves cytoskeletal molecules controlling the initiation of migration, leading edge extension and nucleokinesis; signalling molecules (the reelin pathway playing a central role) integrating external signals and linking them to the cytoskeleton; stop signals; and other molecular players including neurotrophins, glutamate receptors and peroxisome-derived factors. Emerging evidence supports the existence of cross-talk between these pathways. SUMMARY: Identifying these mechanisms has shed light on typical human neuronal migration disorders such as periventricular heterotopias (disorder of migration initiation linked to filamin), type I lissencephaly (cytoskeletal abnormality linked to Lis1, a microtubule-associated protein), double cortex syndrome (cytoskeletal abnormality linked to doublecortin, a microtubule-associated protein), or lissencephaly plus cerebellar hypoplasia (reelin defect).

Animals↗
PubMed2006

Antipsychotic-induced alterations in D2 dopamine receptor interacting proteins within the cortex.

Current antipsychotic treatment involves the regulation of D2 dopamine receptor activity in the brain. Here, we examined the effects of chronic haloperidol and clozapine on cortical D2 dopamine receptors and six different dopamine receptor interacting proteins. Using comparative immunoblot analysis, we found that treatment with either haloperidol or clozapine increased D2 dopamine receptors, calcium activator protein for secretion, protein 4.1N, and neuronal calcium sensor-1 expression. Treatment with clozapine increased calmodulin and spinophilin expression, while treatment with haloperidol decreased expression of these two dopamine receptor interacting proteins. Neither antipsychotic drug was found to have an effect on filamin-A expression. These findings underscore a role for cortical D2 dopamine receptor in the mechanism of antipsychotic drug action, and suggest dopamine receptor interacting proteins as novel targets in antipsychotic drug development.

Animals↗
PubMed2005

Genetic malformations of the cerebral cortex and epilepsy.

We reviewed the epileptogenic cortical malformations for which a causative gene has been cloned or a linkage obtained. X-linked bilateral periventricular nodular heterotopia (BPNH) consists of typical BPNH with epilepsy in female patients and prenatal lethality in most males. About 90% of patients have focal epilepsy. Filamin A mutations have been reported in all families and in approximately 20% of sporadic patients. A rare recessive form of BPNH also has been reported. Most cases of lissencephaly-pachygyria are caused by mutations of LIS1 and XLIS genes. LIS1 mutations cause a more severe malformation posteriorly. Most children have isolated lissencephaly, with severe developmental delay and infantile spasms, but milder phenotypes have been recorded. XLIS usually causes anteriorly predominant lissencephaly in male patients and subcortical band heterotopia (SBH) in female patients. Thickness of the band and severity of pachygyria correlate with the likelihood of developing Lennox-Gastaut syndrome. Mutations of the coding region of XLIS are found in all reported pedigrees and in 50% of sporadic female patients with SBH. Autosomal recessive lissencephaly with cerebellar hypoplasia; accompanied by severe delay, hypotonia, and seizures, has been associated with mutations of the RELN gene. Schizencephaly has a wide anatomoclinical spectrum, including focal epilepsy in most patients. Familial occurrence is rare. Initial reports of heterozygous mutations in the EMX2 gene need confirmation. Among several syndromes featuring polymicrogyria, bilateral perisylvian polymicrogyria shows genetic heterogeneity, including linkage to Xq28 in some pedigrees, autosomal recessive inheritance in others, and association with 22q11.2 deletion in some patients. About 65% of patients have severe epilepsy, often Lennox-Gastaut syndrome. Recessive bilateral frontal polymicrogyria has been linked to chromosome 16q12.2-21.

Adolescent↗
PubMed2005

Transforming growth factor-beta and malignant melanoma: molecular mechanisms.

Transforming growth factor family members (TGF-beta) are secretory polypeptides that have dual tumor-suppressor and oncogenic effects. They signal through kinase receptor complexes on the cell surface, which phosphorylate cytoplasmic mediators (SMADs). Upon phosphorylation, SMADs march to the nucleus and interact with coactivators or corepressors to mediate the transcriptional regulation of several genes resulting in diverse effects. In tumorigenesis, malignant cells escape from the tumor-suppressive effects of TGF-beta by mutational inactivation or dysregulated expression of the molecular components in TGF-beta signaling pathway. Although melanoma cells are resistant to the tumor-suppressive effects of TGF-beta, there are no detectable defects at the receptor/SMAD level. Therefore, in these lesions, it is possible that TGF-beta effects occur independently of TGF-beta receptor/SMAD pathway. This review seeks to examine the present knowledge about TGF-beta receptor/SMAD signaling pathway and its related genes (SMADs, SKI, Filamin, endoglin, Follistatin, and other molecules) in melanomas.

Animals↗
PubMed1992

Intestinal pseudo-obstruction with deficient smooth muscle alpha-actin.

We describe a 48-year-old woman with chronic constipation since early childhood who has an intestinal myopathy associated with a hitherto undescribed absence by immunostaining of smooth muscle alpha-actin confined to the intestinal circular muscle. There were no abnormalities in other contractile proteins (myosin, tropomyosin, filamin, caldesmon or desmin) and despite the abnormality of a contractile protein isoform in the circular muscle, no significant morphological changes were identified by light microscopy or ultrastructural examination. A possible developmental mechanism for the observed change is proposed. The use of specific antibodies to isoforms of contractile proteins may have potential value in the study of intestinal myopathies.

Actins↗
PubMed1982

Isolation from thyroid cells or purified plasma membranes with associated actin microfilaments. Proteins bound to actin.

Plasma membranes of thyroid cells were purified from hog thyroid glands following two procedures. Their homogeneity was tested by electron microscopy and by measurements of the activity of membrane-bound enzyme markers. According to the procedure used the membrane fractions obtained present some differences in their morphological features as well as in the repartition of the activities of the membrane-bound enzyme markers. However, whatever the composition of the membrane fraction examined (membrane vesicles, single membrane sheets with junctional complexes), decoration with heavy meromyosin clearly shows the presence of actin filaments attached to these fragments. Analysis of proteins by polyacrylamide gel electrophoresis indicates the presence of about twelve major components with actin. Treatment of membranes with Triton X-100 results in an insoluble core which contains all the actin and most of the major proteins. The selective extraction of these components by buffers differing in their ionic strength, pH, or the presence or absence of ATP X Mg has been used to characterize some of the proteins associated to actin; among them are filamin, myosin, alpha-actinin, tropomyosin.

Actins↗
PubMed1987

Cytoplasmic gels from macrophages. Evidence for the involvement of four proteins in the calcium-dependent solation of actin networks.

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca2+ cytosolic extracts (less than 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, alpha-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521-530]. Increasing concentrations of free Ca2+ to 1-2 microM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 microM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or alpha-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I-calmodulin revealed that a high-Mr calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.

Actins↗
PubMed1999

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on int 407 and HeLa cells.

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Calcium↗
PubMed2000

Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins.

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi, to have no actin tails and short (approximately 1- to 3-micrometer) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii, displayed long actin tails (>10 micrometer) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 micrometer long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria. The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.

Actins↗
PubMed1996

The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.

Actins↗
PubMed1998

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule.

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.

Actinin↗