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.     

Purification and characterization of GP-55, a protein associated with actin-based cytoplasmic gels derived from brain tissue

Incubation of rat brain cytoplasmic extracts results in the formation of three-dimensional gels that can be collapsed by low-speed centrifugation. Electrophoresis in polyacrylamide gels indicate that these cytoplasmic gels are composed of actin and several associated proteins. Among the latter we have identified a component with an apparent molecular mass of 55,000 daltons. The results of two-dimensional electrophoresis, peptide mapping, and in vivo labeling experiments with [35S]methionine, indicate that the 55-kDa protein is composed of two different gene products we have named alpha and beta; a third polypeptide, named beta', probably derives from the beta polypeptide as a result of some posttranslational modification(s). These three polypeptides, which have the same molecular weight but different isoelectric points, are present in a rather pure preparation of the 55-kDa protein, obtained from cytoplasmic gels by ion-exchange chromatography. In vitro, purified 55-kDa protein interacts in a specific manner with F-actin, as shown by viscosimetry and electron microscopy, leading to the formation of a complex network of cross-linked microfilament bundles. The results of immunofluorescence experiments indicate that the 55-kDa protein is located intracellularly in structures belonging to the Golgi apparatus, and that it is not only present in every type of rat cell tested but also in primary cultures of chick embryo neurons. Based on its intracellular location, we have named the 55-kDa protein as Golgi Protein-55 (GP-55).     

Integrin-type fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties

The spreading of freshly isolated rat arterial smooth muscle cells (SMCs) on a substrate of fibronectin (FN) is associated with marked changes in fine structure and function of the cells, collectively referred to as a modulation from a contractile to a synthetic phenotype. Recent studies have indicated that this process is mediated via an interaction between the minimal cell-attachment sequence of FN (RGDS) and cell surface receptors. Here, we report the isolation of such receptors by sequential chromatography on affinity columns of wheat germ agglutinin (WGA) and a 105-kDa cell-binding fragment of FN (105-kDa fragment). The receptor was composed of two proteins with electrophoretic mobilities in SDS-polyacrylamide gels of 160 and 115 kDa under nonreducing conditions and 150 and 130 kDa under reducing conditions. Immunoprecipitation of surface-labeled cells with a rabbit antiserum against the beta chain of the rat hepatocyte FN receptor similarly yielded two proteins of 160 and 115 kDa. In metabolically labeled cells an additional component of 105 kDa was precipitated, presumably representing a precursor of the 115-kDa protein. Immunocytochemical studies demonstrated that SMCs grown on laminin formed FN fibrils and actin filament bundles in close alignment with cell surface receptors after a few days of culture. In cells seeded on the 105-kDa fragment, the receptors were already arranged in parallel with actin filaments on the first day of culture. Later on, the cells secreted FN and laid down FN fibrils along the receptors on the cell surface and the actin filament bundles in the cytoplasm. Taken together, the findings indicate that arterial SMCs are equipped with FN receptors that belong to the integrin family of proteins and consists of alpha (160-kDa) and beta (115-kDa) subunits. The receptor complexes apparently play an important role in determining the differentiated characteristics of the cells, possibly by mediating a linkage between the extracellular matrix and the cytoskeleton.     

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.     

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.     

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.     

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.     

Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.     

Mutual effects of α-actinin,calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (α-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin α-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of α-actinin and calponin to actin bundles. Higher ability of calponin to depress α-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin–α-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with α-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that α-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Phosphorylation of filamin (ABP-280) regulates the binding to the lipid membrane, integrin, and actin

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm, where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, the hypothesis that it serves as a substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins is considered. The results suggest conformationally-induced regulation of filamin (ABP-280).     

Mutual effects of alpha-actinin, calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Filamin A protein interacts with human immunodeficiency virus type 1 Gag protein and contributes to productive particle assembly

HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection.     

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.     

A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract

The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.     

MARCKS-Related Protein Binds to Actin without Significantly Affecting Actin Polymerization or Network Structure

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Cytochalasin B and the structure of actin gels

We analyzed the structure of gels formed when macrophage actin-binding protein crosslinks skeletal muscle actin polymers and the effect of the fungal metabolite cytochalasin B on this structure. Measurement of the actin filament length distribution permitted calculation of the critical concentration of crosslinker theoretically required for gelation of actin polymer networks. The experimentally determined critical concentration of actin-binding protein agreed sufficiently with the theoretical to conclude that F-actin-actin-binding protein gels are networks composed of isotropically oriented filaments crosslinked at intervals. The effects of cytochalasin B on these actin networks fits this model. Cytochalasin B (1) bound to F-actin (but not to actin-binding protein), (2) decreased the length of actin filaments without increasing the quantity of monomeric actin, (3) decreased the rigidity of actin networks both in the presence and absence of crosslinking proteins and (4) increased the critical concentration of actin-binding protein required for incipient gelation by a magnitude predicted from network theory if filaments were divided and shortened by the extents observed. The effects of cytochalasin B on gelation were highly dependent on actin concentration and were inhibited by the actin-stabilizing agent phalloidin. Therefore, cytochalasin B diminishes actin gel structure by severing actin filaments at limited sites. The demonstration of gel-sol transformations in actin networks caused by limited actin filament cleavage suggests a new mechanism for the control of cytoplasmic structure.     

MARCKS-related protein binds to actin without significantly affecting actin polymerization or network structure. Myristoylated alanine-rich C kinase substrate

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Mitogen-induced preferential synthesis of proteins during the G0 to S phase transition in human lymphocytes

We have followed the induction of protein synthesis in mitogen-activated human peripheral blood mononuclear cells during the transition from quiescence, or G0, through the prereplicative phase and into first S phase. Doses of mitogens optimal for proliferative response preferentially enhance the synthesis of a subset of intracellular proteins during the approximately 24-h lag interval. The mitogenic lectin phytohemagglutinin (PHA) and OKT3, a mitogenic monoclonal antibody to the CD3 component of the T cell antigen receptor, preferentially enhance bands of the same molecular weight in one-dimensional SDS-PAGE. The proteins are low detergent soluble (0.1% Triton X-100) "cytoplasmic" cellular components and some have been identified as single spots on two-dimensional gels. Bands of 51 and 66 kDa are induced early in lag phase (4 h after stimulation) but are transiently synthesized, decreasing later in lag phase. The majority of the mitogen-induced proteins, 39, 51, 55, 60, 73, and 95 kDa are enhanced by mid lag phase (12 h after stimulation). With the exception of the 55-kDa band, five of these proteins are clearly enhanced in T cells purified after mitogen stimulation. The same five bands show sustained synthesis in actively cycling cells 42-48 h after stimulation and are major synthesized proteins, and corresponding bands are synthesized in a transformed T cell line, MOLT-4. Two of the proteins in this group that are most prominently synthesized during the lag interval have been previously identified as the heat shock proteins, HSP 90 (95-kDa band) and HSC 70 (73-kDa band). We speculate that this group of five proteins, including HSP 90 and HSC 70, may be coordinately expressed in actively replicating T cells and may have some common structural or functional role in sustaining the replicative state.