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PubMed1981

Reconstitution and regulation of actin gel-sol transformation with purified filamin and villin.

Gel-sol transformation of actin filaments, a process essential for cell motility, can be reconstituted in vitro and regulated in a predictable fashion by the combined action of villin and filamin. Measurements made in a low shear falling ball viscometer show that mixtures of actin, villin, and filamin exist either as a gel (yield point greater than or equal to 140 dynes/cm2) or as a low viscosity liquid depending on the relative ration of villin:actin. Filamin induces gelation of F-actin by forming stable cross-links between actin filaments. Villin inhibits filamin-induced F-actin gelation, but the effect can be overcome by increasing the amount of filamin. Sedimentation assays show that villin does not inhibit gelation of actin by preventing filamin from binding to F-actin. Results from viscosity measurements and filament length determinations show that villin increases actin filament number by reducing the average filament length without altering the total amount of polymer. Because the gel point of a fixed amount of polymer is sharply dependent on the ratio of cross-links to number of polymers, the solation effect of villin might be explained by its effect on filament number. Based on the network theory of gel formation, calculations of the amount of additional cross-linker required to overcome the effect of a known increase in the number of actin filaments agree reasonably well with experimental findings. These results document the existence of cellular proteins which could regulate gel-sol transformation in vivo by their effect on actin polymer length and, therefore, on actin filament number.

Actins↗
PubMed1994

Parietal cells contain most of the myosin, filamin and actin present in rat gastric glands.

The association of myosin and a filamin-like protein to the F-actin cytoskeleton of parietal cells was studied in the rat gastric mucosa. Myosin and the filamin-like protein were localized by indirect immunofluorescence microscopy while the distribution of actin was established by using FITC-phalloidin. These cytoskeletal proteins, concentrated in the parietal cells, changed their distribution in correlation with the hydrochloric acid (HCl) secretory state of the cells and the appearance of a developed intracellular canaliculus. Thus, in resting parietal cells, actin showed a patchy distribution, delimiting the poorly developed secretory canaliculi, while myosin and the filamin-like protein distributed diffusely over the cytoplasm. In secreting cells, F-actin was concentrated in the cytoplasmic projections filling the canalicular lumen, while myosin and the filamin-like protein were excluded from this region, concentrating in the adjoining cytoplasm. The present results show that myosin and the filamin-like protein change their association with the secretory membranes in relation to the development of the secretory canaliculus of parietal cells. In resting cells, both proteins associate with the endocellular secretory membranes. In secreting cells, the microvillar projections of the canalicular surface formed by these membranes bind F-actin, but exclude myosin and the filamin-like protein.

Actins↗
PubMed1994

Characterization of the actin binding site on smooth muscle filamin.

We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.

Actinin↗
PubMed1978

Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle.

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.

Actins↗
PubMed1990

Effect of filamin and controlled linear shear on the microheterogeneity of F-actin/gelsolin gels.

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.

Actin Cytoskeleton↗
PubMed1991

Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.

Actinin↗
PubMed1981

Structure of filamin and the F-actin-heavy merofilamin complex.

Rotary-shadowed filamin molecules appear as long, highly flexible rods curved into a variety of configurations. The particles observed were 2.7 nm wide but had contour lengths of either 98 nm or 193 nm. The longer particles are probably end-to-end dimers of the shorter but it is not clear how many polypeptide chains these particles contain. Heavy merofilamin, obtained by digestion of filamin with a calcium-activated protease from muscle, has been used to investigate where filamin binds on the actin filaments. Negatively stained filaments of actin plus heavy merofilamin resemble those of pure actin; occasionally rod-shaped material sticking out from the filament is observed suggesting that the elongated shape of filamin is maintained after digestion. Optical diffraction patterns of electron micrographs of paracrystals of actin plus heavy merofilamin indicate that the helical symmetry of the actin filament is unchanged, but the observed interfilament spacing is larger than in F-actin paracrystals. Increased intensity of the second layer-line reflection is observed, suggesting that additional material is lying along the grooves of the actin helix. The elongated shape of filamin and its ability to bind to F-actin in a way similar to tropomyosin suggest a possible role for this protein in regulating the organization and aggregation of actin filaments.

Actins↗
PubMed1985

Filamin inhibits actomyosin ATPase activity in platelet.

Filamin, an actin cross-linker protein, has been shown to exist in platelet. The role of this protein in the platelet has remained unclear. In this report, we show that filamin inhibits the actin-activated Mg2+ -ATPase activity of platelet myosin. The activation caused by platelet actin is inhibited by 50% at the molar ratio of filamin to actin of 1/50. Platelet tropomyosin, which we showed to enhance the ATPase activity, does not abolish the effect of filamin. The results support the view that filamin stabilizes the actin network in the resting platelet.

Actins↗
PubMed1987

Calpain abolishes the effect of filamin on the actomyosin system in platelets.

Platelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of filamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-actin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for contraction of the activated platelets.

Actins↗
PubMed2006

Structural determinants of LL5beta subcellular localisation and association with filamin C.

PI3K signalling pathways link cell surface receptors to the control of several intracellular functions including cell growth, survival and movement. Filamins are important regulators of cortical actin structure and function. LL5beta is a filamin binding protein that is an effector of the PI3K signalling pathway. We define an N-terminal region of LL5beta that is responsible for binding to the C-terminus of filamins. Under conditions of very low PI3K activity, we show that this region, together with an additional domain of the protein, is responsible for localising the complex to punctate structures that are also decorated by L-FILIP (a protein previously characterised to bind filamin and accelerate its destruction). Under conditions of significant PI3K activity, PtdIns(3,4,5)P(3) binding to the C-terminal PH domain in LL5beta prevents localisation to these structures. These observations start to define the basis for PI3K regulation of filamin through LL5beta.

Actins↗
PubMed2005

Structural basis for vertebrate filamin dimerization.

Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.

Actins↗
PubMed2006

Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP.

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.

Alternative Splicing↗
PubMed2002

The actin-binding protein Filamin-A interacts with the metabotropic glutamate receptor type 7.

A yeast two-hybrid screen identified Filamin-A as a binding partner of the metabotropic glutamate receptor type 7b (mGluR7b) splice variant. In addition, Filamin-A interacted with mGluR4a, mGluR5a, mGluR5b, mGluR7a and mGluR8a. Domain mapping revealed that alternative splicing of mGluR4, mGluR7 and mGluR8 C-termini regulated the interaction. A conserved tyrosine within mGluR C-termini was identified to mediate the binding to Filamin-A. Protein interactions were verified in biochemical assays using recombinant and native proteins. Finally, co-expression of Filamin-A and mGluR7 splice variants was shown in brain regions. These findings suggest that Filamin-A may physically link metabotropic glutamate receptors to the actin cytoskeleton.

Alternative Splicing↗
PubMed2002

Leucine-rich repeat region of decorin binds to filamin-A.

Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane.

Amino Acid Motifs↗
PubMed2004

Presence of filamin in the astrocytic inclusions of Aicardi syndrome.

Aicardi syndrome affects only females and has been hypothesized to be an X-linked dominant male-lethal disorder. Because no familial cases can be studied for genetic linkage analysis, the mutated gene has remained elusive. With the goal of selecting genes for mutation analysis by a functional candidate approach, a detailed pathologic analysis of two brains from deceased Aicardi syndrome patients was performed. The presence of micrencephaly, absent or hypoplastic corpus callosum, polymicrogyria, heterotopia, ventriculomegaly, intracerebral cyst, and intracytoplasmic eosinophilic inclusions was confirmed in glial fibrillary acidic protein-positive astrocytes in the cortex and heterotopias, but not in white matter. The inclusions demonstrated strong immunolabeling with antibodies to filamin and vimentin but weak labeling with antibodies to proteins S100 and microtubule-associated protein 1. These findings suggested that an underlying defect in the cytoskeleton, which involves filamin, may cause this condition. Because the filamin A gene in Xq28 is mutated in another disorder with heterotopia, familial bilateral periventricular heterotopia, mutation analysis of filamin A in Aicardi syndrome patients was pursued. No mutations were found, and the full-length protein was expressed in both brain samples. Future studies will focus on investigation of X-linked genes that may affect function of filamin or other cytoskeletal proteins.

Abnormalities, Multiple↗
PubMed1994

Insertion of filamin into lipid membranes examined by calorimetry, the film balance technique, and lipid photolabeling.

The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studied in reconstituted lipid monolayers and bilayers. Protein-lipid interactions were investigated by differential scanning calorimetry, the film balance technique, and hydrophobic photoradiolabeling. For calorimetric assays, multilamellar vesicles (MLVs) and large unilamellar vesicles produced by the extrusion technique (LUVETs) were used. With MLVs, filamin induced a pronounced drop in phase transition cooperativity. Mixed DMPC/DMPG LUVETs showed a linear decrease of the main phase transition enthalpy and a significant shift in temperature for the solidus and liquidus lines with increasing mole fractions of reconstituted filamin. The insertion of native filamin into uncharged and negatively charged lipid monolayers was measured in time/area diagrams with the film balance technique. Finally, we have newly synthesized a highly sensitive lipid analogue, [125]TID-PC/16, which selectively labels membrane-embedded hydrophobic domains of proteins, and which proved to label filamin, supporting evidence that this protein partially inserts into the hydrophobic domain of liposomes.

Calorimetry, Differential Scanning↗
PubMed1988

Actin-binding and dimerization domains of HeLa cell filamin.

HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.

Actins↗
PubMed2002

FAP52 regulates actin organization via binding to filamin.

FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.

Actins↗