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At least 379 records · Page 21Linked to original sources
PubMed2006

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Actins↗
PubMed2001

Role of actin filament organization in CFTR activation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion-selective channel whose dysfunction leads to the onset of cystic fibrosis. CFTR activation is normally elicited by stimulation of the cAMP pathway, which effects protein kinase A activation. However, previous studies from our laboratory indicate that the actin cytoskeleton is also required for a proper CFTR function. In this report, the regulatory role of actin filament organization in the activation of CFTR was explored. Maneuvers to modify the steady-state organization of actin filaments elicit the activation of CFTR in the absence of a functional cAMP pathway. Partial disruption of the actin cytoskeleton of CFTR-expressing cells with cytochalasin D (CD) induced CFTR activation in the absence of an activated PKA. Similar findings were obtained by intracellular dialysis with the actin-severing protein gelsolin. However, extended treatment with CD leading to the collapse of the actin cytoskeleton rendered CFTR completely insensitive to direct PKA activation. cAMP activation of CFTR was also found to be dysfunctional in cells lacking the actin-crosslinking protein ABP-280, which was recovered after dialysis of the cells with filamin, a homologue of ABP-280. The present data indicate that an organized actin network is required for the proper cAMP-dependent activation of CFTR. The possibility is also explored that actin must be directly associated with CFTR to elicit its activation, further suggesting that this channel protein may bind actin as well.

Actin Cytoskeleton↗
PubMed1984

Ca2+-activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins.

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.

Actinin↗
PubMed1990

Association of cytoskeletal proteins with newly formed acetylcholine receptor aggregates induced by embryonic brain extract.

Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.

Actinin↗
PubMed1986

Assembly and characterisation of a multi-component cytoskeletal gel from adrenal medulla.

Incubation of bovine adrenal medullary cytoplasmic extracts results in the formation of three-dimensional supramolecular gels. Ultrastructurally, the gels display a network of fibres similar in appearance to the cytoskeleton within intact chromaffin cells. Analysis of the protein composition using both electrophoretic and immunoblotting techniques indicates that the gels are composed exclusively of cytoskeletal elements; microfilaments, microtubules and intermediate filament proteins have been identified as having a number of actin-associated proteins. Among the latter class of components the following polypeptides have been identified: filamin (300 kDa), fodrin (240 kDa), a 235 kDa polypeptide, myosin (200 kDa), caldesmon (70 kDa) and tropomyosins (39 kDa). All of these polypeptides co-sedimented with F-actin when gels were assembled in the absence of Ca2+. When gelation was performed in the presence of 10 microM Ca2+ actin, the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosin were all absent from the gels. These results may suggest that the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosins could act either individually or as a functional regulatory unit in controlling the Ca2+-activated reorganisation of the actin network in the cytoplasmic gels.

Actins↗
PubMed2004

Etiological heterogeneity of familial periventricular heterotopia and hydrocephalus.

Periventricular heterotopia (PH) represents a neuronal migration disorder that results in gray matter nodules along the lateral ventricles beneath an otherwise normal appearing cortex. While prior reports have shown that mutations in the filamin A (FLNA) gene can cause X-linked dominant PH, an increasing number of studies suggest the existence of additional PH syndromes. Further classification of these cortical malformation syndromes associated with PH allows for determination of the causal genes. Here we report three familial cases of PH with hydrocephalus. One pedigree has a known FLNA mutation with hydrocephalus occurring in the setting of valproic acid exposure. Another pedigree demonstrated possible linkage to the Xq28 locus including FLNA, although uncharacteristically a male was affected and sequencing of the FLNA gene in this individual revealed no mutation. However, in the third family with an autosomal mode of inheritance, microsatellite analysis ruled out linkage with the FLNA gene. Routine karyotyping and fluorescent in situ hybridization using BAC probes localized to FLNA also showed no evidence of genomic rearrangement. Western blot analysis of one of the affected individuals demonstrated normal expression of the FLNA protein. Lastly, sequencing of greater than 95% of the FLNA gene in an affected member failed to demonstrate a mutation. In conclusion, these findings demonstrate the etiological heterogeneity of PH with hydrocephalus. Furthermore, there likely exists an autosomal PH gene, distinct from the previously described X-linked and autosomal recessive forms. Affected individuals have severe developmental delay and may have radiographic findings of hydrocephalus.

Adult↗
PubMed2006

A phototaxis signalling complex in Dictyostelium discoideum.

Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains - the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.

Animals↗
PubMed2006

Functional dissection of human protease mu-calpain in cell migration using RNAi.

Calpains are a family of calcium-dependent cysteine proteases involved in a variety of cellular functions. Two isoforms, m-calpain and mu-calpain, have been implicated in cell migration. However, since conventional inhibitors used for the studies of the functions of these enzymes lack specificity, the individual physiological function and biochemical mechanism of these two isoforms, especially mu-calpain, are not clear. In contrast, RNA interference has the potential to allow a sequence-specific destruction of target RNA for functional assay of gene of interest. In the present study, we found that small interfering RNAs-mediated knockdown of mu-calpain expression in MCF-7 cells that do not express m-Calpain led to a reduction of cell migration. This isoform-specific function of mu-calpain was further confirmed by the rescue experiment as overexpression of mu-calpain but not m-calpain could restore the cell migration rate. Knockdown of mu-calpain also altered cell morphology with increased filopodial projections and a highly elongated tail that seemed to prevent cell spreading and migration with reduced rear detachment ability. Furthermore, knockdown of mu-calpain decreased the proteolytic products of filamin and talin, which were specifically rescued by overexpression of mu-calpain but not m-calpain, suggesting that their proteolysis could be one of the key mechanisms by which mu-calpain regulates cell migration.

Calpain↗
PubMed2005

Temperature softening of a protein in single-molecule experiments.

Mechanical flexibility is crucial for the function of proteins. However, such material properties are not easily accessible experimentally. We used single-molecule force spectroscopy to study the stiffness of a single domain of Dictyostelium discoideum filamin (ddFLN4) in a temperature range from 5 degrees C to 37 degrees C. Analyzing the distributions of unfolding forces allowed us to extract transition barrier heights and positions of the underlying energy landscape. We found a marked narrowing of unfolding force distributions with increasing temperature. This narrowing reflects an increase in transition state position from 2.7 A to 7.8 A and thus a reduction of the molecular spring constant of the protein by a factor of 7. We suggest this temperature softening reflects a shift in the nature of the interactions responsible for mechanical stability from hydrogen bonds to hydrophobic interactions. This result has important consequences for all interpretations of protein mechanical studies if experimental results obtained at room temperature are to be transferred to physiological temperatures.

Animals↗
PubMed2004

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells.

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.

Actins↗
PubMed2005

Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.

During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin-ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion.

Actin Cytoskeleton↗
PubMed2005

Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity.

Guanine nucleotide exchange factor (GEF) domains of the Dbl family occur in a variety of proteins that include multiple protein-protein and protein-lipid interaction domains. We used an epithelial-derived cell line to investigate the mechanisms by which the two GEF domains of Kalirin, a neuronal Rho GEF, influence morphology. As expected, Kal-GEF1, an efficient GEF for Rac1 and RhoG, induced the formation of lamellipodia resembling those induced by active Rac1. Although Kal-GEF1 activated Rac and Pak, its ability to induce formation of lamellipodia was not blocked by dominant negative Rho GTPases or by catalytically inactive Pak. Consistent with this, a catalytically inactive mutant of Kal-GEF1 induced formation of lamellipodia and activated Pak. Active Pak was required for the GEF-activity independent effect of Kal-GEF1 and the lamellipodia produced were filled with ribs of filamentous actin. Kal-GEF1 and a GEF-dead mutant co-immunoprecipitated with Pak. The interaction of Kal-GEF1 with Pak is indirect and requires the regulatory protein binding domain of Pak. Filamin A, which is known to interact with and activate Pak, binds to both catalytically active and inactive Kal-GEF1, providing a link by which catalytically inactive Kal-GEF1 can activate Pak and induce lamellipodia. Together, our results indicate that Kal-GEF1 induces lamellipodia through activation of Pak, where GEF activity is not required. GEF-activity-independent effects on downstream targets may be a general property of RhoGEFs.

Actins↗
PubMed1995

H2O2-treated actin: assembly and polymer interactions with cross-linking proteins.

During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin.

Actinin↗
PubMed1999

Application of fluorescence polarization to the steady-state enzyme kinetic analysis of calpain II.

This paper presents the application of fluorescence polarization to the determination of dissociation constants for competitive inhibitors that bind to enzymes. This steady-state enzyme kinetic study measures the inhibition of the conversion of a fluorescently tagged substrate to a lower molecular weight fluorescent product by calpain II. It relies on the measurement of a parameter proportional to velocity, which is sufficient for this type of analysis. The strengths and limitations of the method are discussed. Inhibition constants for filamin and spectrin determined by this method are 125 nM and 13 nM respectively.

Calpain↗
PubMed2000

Potential mechanisms of mutations that affect neuronal migration in man and mouse.

Mutations in the genes that encode filamin-1, Lis1 and doublecortin are responsible for X-linked lissencephaly in man, whereas mutations in the genes that encode Cdk5, its activator p35 and the reelin-signaling pathway disturb migration and architectonic development in mice. To understand the action of genes that control neuronal migration and the phenotype of corresponding defects, it might be as important to consider the positioning of the nucleus as it is to consider the guidance of the leading process.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2002

The spectrum of pathology in central core disease.

Central core disease is a congenital myopathy with muscle weakness defined pathologically by the presence of extensive areas in muscle fibres that are devoid of oxidative enzyme activity. The gene responsible has been shown to be the ryanodine receptor 1 on chromosome 19q13 and mutations have now been identified in several patients. Some cases with the morphological defect remain molecularly undefined, particularly those studied before molecular studies were available. We have studied three families with congenital onset, each with a dominantly inherited mutation in a C-terminal exon of the ryanodine receptor 1. They illustrate the spectrum of pathology that can be observed in patients with the myopathic features of central core disease. We show that extensive fibrosis and fat may be present, type 1 fibre uniformity may occur in the absence of cores; cores may be central or peripheral, single or multiple; and that an appearance of multiple focal minicores might cause a diagnostic pathological dilemma. In addition, we show the value of immunocytochemistry in identifying cores, in particular the use of antibodies to desmin and gamma-filamin.

Adult↗
PubMed2003

Filling gaps in signaling to actin cytoskeletal remodeling.

A recent publication in the April 4 issue of Cell advances our understanding of stimulus response coupling leading to actin remodeling. It describes the identification of a novel membrane component Mig-2 that engages filamin A through a new intermediary, migfilin, to stimulate actin assembly and cell spreading on a substrate of extracellular matrix.

Actins↗
PubMed1999

Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod.

Gelation factor (ABP120) is one of the principal actin-cross-linking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue repeats that have an Ig fold. The ability to dimerize is crucial to the actin cross-linking function of gelation factor and is mediated by the rod in which the two chains are arranged in an antiparallel fashion. We report the 2.2 A resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, and domains 1-5 do not pair with domains from the other chain. This information allows construction of a model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact.

Amino Acid Sequence↗