ABSTRACT
ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR-actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments.
Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/genetics , Actins/metabolism , Promoter Regions, Genetic/genetics , Actin Depolymerizing Factors/genetics , Actin Depolymerizing Factors/ultrastructure , Actins/ultrastructure , Adenosine Diphosphate/metabolism , Animals , Cysteine/genetics , Cysteine/metabolism , Humans , Microscopy, Electron , Models, Molecular , Mutation/genetics , Phalloidine , Protein Binding , Protein Structure, Quaternary , Rabbits , Rhodamines , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Threonine/genetics , Threonine/metabolism , Time FactorsABSTRACT
Cross-links between protomers in F-actin can be used as a very sensitive probe of both the dynamics and structure of F-actin. We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction. The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments. This finding is in conflict with refined models but in agreement with the original Holmes et al. model for F-actin. Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin.
Subject(s)
Actins/chemistry , Actins/genetics , Actins/metabolism , Algorithms , Animals , Deoxyribonuclease I/metabolism , Disulfides/chemistry , Image Processing, Computer-Assisted , Microscopy, Electron , Models, Molecular , Models, Statistical , Muscle, Skeletal/chemistry , Mutation , Protein Conformation , Yeasts/chemistryABSTRACT
Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.
Subject(s)
Actins/metabolism , Microfilament Proteins/metabolism , Actin Depolymerizing Factors , Actins/chemistry , Animals , Destrin , Humans , Microfilament Proteins/chemistry , Models, Molecular , RabbitsABSTRACT
20S-proteasome was purified from rat liver cells by ultracentrifugation, gel-chromatography and ultrafiltration. The ability of 20S-proteasome to interact with fibrillar actin (F-actin) was examined by rapid cosidementation of these dissociated particles with F-actin in isokinetic sucrose gradient. Proteasomes, which were dissociated with Zn2+ ions, can be assembled on the fibrillar actin once again (with the exception of protein 27 kDa) at deleting ions Zn2+ from the solution with the help of EDTA.
Subject(s)
Actins/metabolism , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Animals , Cytosol/metabolism , Liver/metabolism , Male , Proteasome Endopeptidase Complex , Protein Binding , RatsABSTRACT
Nuclear factor-kappa B (NF-kappaB) is a universal transcription factor that participates in induction of a wide variety of cellular genes. In nonstimulated cells, NF-kappaB is sequestered in the cytoplasm. However, little is known about where NF-kappaB is located. We have studied the effect of inducing a reorganization of the actin filament system on NF-kappaB distribution, using normal and E1A+cHa-ras-transformed rat fibroblasts. This paper demonstrates that the p65/RelA subunit of NF-kappaB interacts with actin-containing structures. Immunofluorescence reveals that p65 is concentrated in focal contacts and along stress fibers in normal fibroblasts. Restoration of actin stress fibers in transformants spread on fibronectin is followed by reallocation of p65 to focal contacts and stress fibers, as in normal cells. The p65 is accumulated at the edge of leading lamellae in transformants spread on laminin and is redistributed to cell-to-cell contacts after a prolonged cultivation. Treatment of cells with Cytochalasin D leads to redistribution of p65 into the actin-containing aggregates. Affinity chromatography on matrix-bound F-actin confirms that p65 can bind to filamentous actin. Taken together, these data indicate that distribution of p65 in the cytoplasm depends on the state of the actin cytoskeleton and suggest an additional, yet unknown, function of the NF-kappaB in the cytoplasm.
Subject(s)
Actins/metabolism , NF-kappa B/metabolism , Animals , Cell Adhesion , Cell Line, Transformed , Cells, Cultured , Chromatography, Affinity , Cytochalasin D/pharmacology , Cytoplasm/metabolism , Cytoskeleton/ultrastructure , Electrophoresis, Polyacrylamide Gel , Fibroblasts/metabolism , Fibroblasts/physiology , Fibroblasts/ultrastructure , Fibronectins/physiology , Humans , Immunoblotting , Laminin/physiology , Microscopy, Fluorescence , Rats , Transcription Factor RelASubject(s)
Chromatin/metabolism , DNA/metabolism , Endoribonucleases/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Animals , Antigens/metabolism , Cysteine Endopeptidases/immunology , Humans , Hydrolysis , K562 Cells , Male , Multienzyme Complexes/immunology , Proteasome Endopeptidase Complex , RNA/metabolism , Rats , Rats, WistarABSTRACT
26S-complex was purified from rat liver cells by centrifugation in sucrose gradients and ion exchange chromatography. The ability of 26S-proteasome to interact with fibrillar actin (F-actin) was examined by rapid cosidementation of these particles with F-actin in isokinetic sucrose gradients. It was shown that direct interaction occurred only at a low ATP concentration.
