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At least 397 records · Page 22Linked to original sources
PubMed1997

Emerin deletion reveals a common X-chromosome inversion mediated by inverted repeats.

Emery-Dreifuss muscular dystrophy (EMD) is an X-linked disorder characterized by contractures, progressive muscle weakness and cardiomyopathy. The emerin gene, located in human Xq28, is approximately 2 kb in length, is composed of 6 exons and falls within a 219-kb region that has been completely sequenced. Immediately centromeric to emerin is the 26-kb filamin gene (FLN1), composed of 48 exons and encoding the actin-binding protein 280 (refs 7,8). Flanking this 48-kb FLN1/emerin region are two large inverted repeats, each 11.3 kb, that exhibit > 99% sequence identity. The high level of genomic detail in this region allowed us to characterize the first complete emerin gene deletion mutation that also involved a partial duplication of the nearby FLN1 gene. This rearrangement could be explained by mispairing of the large inverted repeats, followed by double recombination among one set of mispaired repeats and internal sequences. Furthermore, our characterization of this rare DNA rearrangement revealed a more common result of the mispairing of these large inverted repeats--recombination contained within the inverted repeats leading to the maintenance of repeat sequence homogeneity and inversion of the 48-kb FLN1/emerin region. The presence of this frequent inversion, found in the heterozygous state in 33% of females, helps to explain the discrepancies observed between the genetic and physical map distances in this region of the X chromosome. It also illustrates the biological insights which can be gleaned by sequencing the human genome.

Adult↗
PubMed2003

A mechanical unfolding intermediate in an actin-crosslinking protein.

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.

Actins↗
PubMed2006

Otopalatodigital syndrome spectrum disorders: otopalatodigital syndrome types 1 and 2, frontometaphyseal dysplasia and Melnick-Needles syndrome.

The term otopalatodigital syndrome spectrum disorders is an umbrella category that includes four phenotypically related conditions, otopalatodigital syndrome types 1 and 2, frontometaphyseal dysplasia and Melnick - Needles syndrome. The phenotype of these conditions in the male ranges from a severe perinatally lethal multiple malformation syndrome to a mild skeletal dysplasia. Most, but not all, instances of these conditions are associated by mutations in the X-linked gene encoding the cytoskeletal protein filamin A. Mutations in this gene are clustered, exhibit a strong genotype-phenotype correlation and are presumed to exert their effect by a gain-of-function mechanism.

Abnormalities, Multiple↗
PubMed2005

The folding pathway of a fast-folding immunoglobulin domain revealed by single-molecule mechanical experiments.

The F-actin crosslinker filamin from Dictyostelium discoideum (ddFLN) has a rod domain consisting of six structurally similar immunoglobulin domains. When subjected to a stretching force, domain 4 unfolds at a lower force than all the other domains in the chain. Moreover, this domain shows a stable intermediate along its mechanical unfolding pathway. We have developed a mechanical single-molecule analogue to a double-jump stopped-flow experiment to investigate the folding kinetics and pathway of this domain. We show that an obligatory and productive intermediate also occurs on the folding pathway of the domain. Identical mechanical properties suggest that the unfolding and refolding intermediates are closely related. The folding process can be divided into two consecutive steps: in the first step 60 C-terminal amino acids form an intermediate at the rate of 55 s(-1); and in the second step the remaining 40 amino acids are packed on this core at the rate of 179 s(-1). This division increases the overall folding rate of this domain by a factor of ten compared with all other homologous domains of ddFLN that lack the folding intermediate.

Animals↗
PubMed1988

Studies on the interaction between actin and cofilin purified by a new method.

Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.

Actin Depolymerizing Factors↗
PubMed1999

Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix.

Most familial early-onset Alzheimer's disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer's disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexes in vivo with actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell-matrix interaction.

Algorithms↗
PubMed2004

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs. The tCaR extracellular domain includes a characteristic hydrophobic segment, conserved cysteine residues that are implicated in receptor dimerization (Cys(129) and Cys(131)) and in coupling to the transmembrane domain (nine conserved cysteine residues), and conserved serine residues (Ser(147) and Ser(169-171)) that are linked to receptor binding of Ca(2+) and L-amino acid-mediated potentiation of function. mRNA expression of tCaR was strong in kidney, brain, and gill. Weaker expression was observed in pituitary, stomach, intestine, urinary bladder, and heart. This distribution is consistent with possible physiological roles in endocrine cells, excitable tissues, and ion-transporting barrier epithelia. Expression of tCaR mRNA in kidney and intestine was salinity-dependent, suggesting a role for the receptor in iono-/osmoregulation in this euryhaline teleost species. Human embryonic kidney-293 cells transiently transfected with tCaR cDNA demonstrated dose-dependent phospholipase C activation in response to elevations in the extracellular Ca(2+) concentration ([Ca(2+)](o)). Functional activation of the mitogen-activated protein kinase cascade by high [Ca(2+)](o) was also confirmed in these cells despite the naturally occurring truncation of the receptor's intracellular tail, which removes segments variably linked in mammalian CaRs to filamin-coupled activation of mitogen-activated protein kinase cascades. Sensitivity of phospholipase C activation to [Ca(2+)](o) was dependent on the ionic strength of the bathing medium, supporting a role in salinity sensing.

Amino Acid Sequence↗
PubMed2006

Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components.

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.

Actin Cytoskeleton↗
PubMed2002

A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate.

Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways.

Amino Acid Sequence↗
PubMed1988

Microheterogeneity of actin gels formed under controlled linear shear.

The diffusion coefficients and fluorescence polarization properties of actin subjected to a known shear have been determined both during and after polymerization, using a modification of a cone-plate Wells-Brookfield rheometer that allows monitoring of samples with an epifluorescence microscope. Fluorescence polarization and fluorescence photobleaching recovery experiments using rhodamine-labeled actin as a tracer showed that under conditions of low shear (shear rates of 0.05 s-1), a spatial heterogeneity of polymerized actin was observed with respect to fluorescence intensity and the diffusion coefficients with actin mobility becoming quite variable in different regions of the sample. In addition, complex changes in fluorescence polarization were noted after stopping the shear. Actin filaments of controlled length were obtained using plasma gelsolin (gelsolin/actin molar ratios of 1:50 to 1:300). At ratios of 1:50, neither spatial heterogeneity nor changes in polarization were observed on subjecting the polymerized actin to shear. At ratios of approximately 1:100, a decrease on the intensity of fluorescence polarization occurs on stopping the shear. Longer filaments exhibit spatial micro-heterogeneity and complex changes in fluorescence polarization. In addition, at ratios of 1:100 or 1:300, the diffusion coefficient decreases as the total applied shear increased. This behavior is interpreted as bundling of filaments aligned under shear. We also find that the F-actin translational diffusion coefficients decrease as the total applied shear increases (shear rates between 0.05 and 12.66 s-1), as expected for a cumulative process. When chicken gizzard filamin was added to gelsolin-actin filaments (at filamin/actin molar ratios of 1:300 to 1:10), a similar decrease in the diffusion coefficients was observed for unsheared samples. Spatial microheterogeneity might be related to the effects of the shear field in the alignment of filaments, and the balance between a three-dimensional network and a microheterogeneous system (containing bundles or anisotropic phases) appears related to both shear and the presence of actin-binding proteins.

Actin Cytoskeleton↗
PubMed1991

Modulation of contraction by gelation/solation in a reconstituted motile model.

The actin-based cytoskeleton is a dynamic component of living cells with major structural and contractile properties involved in fundamental cellular processes. The action of actin-binding proteins can decrease or increase the gel structure. Changes in the actin-based cytoskeleton have long been thought to modulate the myosin II-based contractions involved in these cellular processes, but there has been some debate concerning whether maximal gelation increases or decreases contractile activity. To address this question, we have examined how contractile activity is modulated by the extent of actin gelation. The model system consists of physiologically relevant concentrations and molar ratios of actin filaments (whose lengths are controlled by gelsolin), the actin-cross-linking protein filamin, and smooth muscle myosin II. This system has been studied at the macroscopic and light microscopic levels to relate the gel structure to the rate of contraction. We present results which show that while a minimal amount of structure is necessary to transmit the contractile force, increasing the gel structure inhibits the rate of contraction, despite an increase in the actin-activated Mg(2+)-ATPase activity of myosin. Decreasing the total myosin concentration also inhibits the rate of contraction. Application of cytochalasin D to one side of the contractile network increases the rate of contraction and also induces movement comparable to flare streaming observed in isolated amoeba cytoplasm. These results are interpreted relative to current models of the relationship between the state of gelation and contraction and to the potential effects of such a relationship in the living cell.

Actin Cytoskeleton↗
PubMed1992

The invasin protein of Yersinia enterocolitica: internalization of invasin-bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton.

Yersinia enterocolitica, a facultative intracellular pathogen of mammals, readily enters (i.e., invades) cultured eukaryotic cells, a process that can be conferred by the cloned inv locus of the species. We have studied the mechanism by which the product of inv, a microbial outer membrane protein termed "invasin," mediates the internalization of bacteria by HEp-2 cells and chicken embryo fibroblasts. Invasin-bearing bacteria initially bound the filopodia and the leading edges of cultured cells. Multiple points of contact between the bacterial surface and the surface of the cell ensued and led to the internalization of the bacterium within an endocytic vacuole; the same multi-step process could be induced by an inert particle coated with invasin-containing membranes. Both adherence and internalization were blocked by an antisera directed against the beta 1 integrin cell-adherence molecule. Ultrastructural studies of detergent-insoluble cytoskeletons from infected cells and immunofluorescence microscopy of phalloidin-labeled cells showed alterations in the structure of the cytoskeleton during the internalization process including the accumulation of polymerized actin around entering bacteria. Bacterial entry was prevented by cytochalasin D indicating that the internalization process requires actin microfilament function. Possible linkages between beta 1 containing integrins and the cytoskeleton were examined during the internalization process through the use of protein-specific antibodies and immunofluorescence microscopy. Like actin, the actin-associated proteins filamin, talin and the beta 1 integrin subunit were also found to accumulate around entering bacteria. These findings suggest that the invasin-mediated internalization process is associated with cytoskeletal reorganization.

Adhesins, Bacterial↗
PubMed1993

Structures linking microfilament bundles to the membrane at focal contacts.

We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.

Actin Cytoskeleton↗
PubMed1997

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway.

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.

Amino Acid Sequence↗
PubMed1999

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

14-3-3 Proteins↗
PubMed2005

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.

Animals↗
PubMed2004

A dual phenotype of periventricular nodular heterotopia and frontometaphyseal dysplasia in one patient caused by a single FLNA mutation leading to two functionally different aberrant transcripts.

Two disorders, periventricular nodular heterotopia (PVNH) and a group of skeletal dysplasias belonging to the oto-palato-digital (OPD) spectrum, are caused by FLNA mutations. They are considered mutually exclusive because of the different presumed effects of the respective FLNA gene mutations, leading to loss of function (PVNH) and gain of function (OPD), respectively. We describe here the first patient manifesting PVNH in combination with frontometaphyseal dysplasia, a skeletal dysplasia of the OPD-spectrum. A novel de novo mutation, 7315C-->A in exon 45 of the FLNA gene, was identified. It leads to two aberrant transcripts, one full-length transcript with the point mutation causing a substitution of a highly conserved leucine residue (L2439M) and a second shortened transcript lacking 21 bp due to the creation of an ectopic splice donor site in exon 45. We propose that the dual phenotype is caused by two functionally different, aberrant filamin A proteins and therefore represents an exceptional model case of allelic gain-of-function and loss-of-function phenotypes due to a single mutational event.

Alternative Splicing↗
PubMed1986

A comparative study of high molecular weight proteins in various types of muscle across the animal kingdom.

A wide range of phyla have been surveyed by SDS-PAGE for the new large proteins of the myofibril. Connectin (or titin) appears to be widely distributed. It is seen as a band of constant intensity and mobility in vertebrate striated muscle, but is absent from smooth muscle. It appears in more variable amounts, in a form of constant but greater mobility in many invertebrates: worms, molluscs (adductor but not gastropod feet), insects, a myriapod, and even in human blood platelets. Nebulin shares the same distribution in vertebrate muscles except for its notable absence in all heart muscle examined. It too is found in many invertebrates, not always with titin. It has been found in a worm, molluscs (adductor and gastropod feet), insects, crustaceans and an echinoderm. The mobility of nebulin varies within the vertebrates and more so between invertebrates (where, as with titin, it is greater). The isoforms of filamin in skeletal, cardiac, and smooth muscles of vertebrates are recorded. C-protein in rabbit muscles has four isoforms: white, alpha-red (X-protein), beta-red, and cardiac.

Animals↗