Factors modulating filament formation by bovine glial fibrillary acidic protein, the intermediate filament component of astroglial cells

Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.     

Intermediate filament structure

In a previous communication (Biosci. Rep. 3, 517–525, 1993) we described quantitative X-ray diffraction studies of -keratin which were shown to be consistent with the presence of finite arrays of repeating units, successive arrays being set down at axial intervals of 470 Å. In addition the axial interval between repeating units in an array was shown to be 197.9 Å. It was suggested that this could most readily be explained by supposing that a surfacelattice was present which contained a dislocation along a helical path with unit heighth = 470 Å and unit twist |t| = 49.1° . The number of repeating units was shown to be in the range 7–9. With 7 repeats the mismatch of the lattice along the dislocation is small and this choice was used to develop a detailed model for the filament. Subsequent studies of molecular interactions have shown however that the coiled-coil rope segments in the rod domain of the molecule are most probably oriented parallel to the dislocation, and so minimization of lattice mismatch may be less important than originally supposed. In the present communication it is shown that the choice of 8, rather than 7, for the number of repeating units yields a model which is more compatible with estimates of the linear density and also provides the basis for a general model for polymorphism in intermediate filament lattices.     

Softness, strength and self-repair in intermediate filament networks

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.     

Filament-guided filament assembly provides structural memory of filament alignment during cytokinesis

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Intermediate filament structure: 2. Molecular interactions in the filament

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.     

Metal ion-dependent,reversible, protein filament formation by designed beta-roll polypeptides

Background  

A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca²⁺. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide.     

Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation

We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation.     

Actin filament nucleation by endosomes, lysosomes and secretory vesicles

Intracellular pathogens such as Listeria monocytogenes and vaccinia virus propel themselves through the cytoplasm of mammalian cells by nucleating actin filaments. Recently, actin assembly has also been shown to power the movement of intracellular vesicles, and this may be a mechanism underlying endomembrane movement in a variety of physiological contexts. Surprisingly, class I myosins have been found to play important roles in both actin nucleation and endomembrane trafficking.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

真核细胞的中间纤维结构、组装和功能的研究

在真核细胞中普遍存在中间纤维(IF),不同类细胞的中间纤维都有相似的“头部 1A L1 1B L1-2 2A L2 2B 尾部”结构,其中：头部有一个β—折叠区,1A上有七个残基的亚结构,1B和2A L2 2B上有规则的轴向排列的氨基酸残基,2B的铲螺旋C末端有一个高度保守的氨基酸残基框,这些结构都有其独特的功能。各种IF组装方式不同,但至少都经历了非纤维性颗粒、波形短纤维和长纤维的形态变化过程。研究发现,IF具有阻止细胞凋亡的功能;在细胞凋亡过程中IF发生磷酸化和降解;细胞质中间纤维(CIF)在染色质一核纤层／CIF结合中发挥DNA选择功能。     

Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein

A set of monoclonal antibodies to desmin has been isolated from a fusion of mouse myeloma cells with spleen cells from mice immunized with purified porcine desmin. Eleven group I antibodies recognized desmin in the immune blot, and using defined desmin fragments the epitope has been tentatively assigned as lying between residues 325 and 372. When cell lines were tested in immunofluorescence only the human line RD and hamster BHK-21 were positive. When tissue sections were used, skeletal, cardiac, visceral and some vascular smooth muscle cells were positive. Thus, the group I antibodies appear specific for desmin and do not recognize other intermediate filament proteins. Group II monoclonals recognized not only desmin in the immune blot but also other polypeptides. The epitope of this class is located between residues 70 and 280. In immunofluorescence on cell lines and tissues, the staining patterns of group II antibodies were more complicated and demonstrate that not only other intermediate filament proteins but also additional antigenic determinants are being recognized. The group I antibodies stain, as expected from their desmin specificity, rat and human rhabdomyosarcomas and thus appear to be useful reagents in pathology.     

Intermediate filament structure.

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.     

Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes

Shortly after Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. 1 h later, actin filaments coat the Listeria and then become rearranged to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. If infected macrophages are treated with cytochalasin D, all the actin filaments associated with the Listeria break down leaving a fine, fibrillar material that does not decorate with subfragment 1 of myosin. This material is associated with either the surface of the Listeria (the cloud stage) or one end (the tail stage). If the cytochalasin-treated infected macrophages are detergent extracted and then incubated in nuclei-free monomeric actin under polymerizing conditions, actin filaments assemble from the fine, fibrillar material, the result being that each Listeria has actin filaments radiating from its surface like the spokes of a wheel (cloud form) or possesses a long tail of actin filaments formed from the fine, fibrillar material located at one end of the Listeria. Evidence that the fine fibrillar material is involved in nucleating actin assembly comes from a Listeria mutant. Although the mutant replicates at a normal rate in macrophages, actin filaments do not form on its surface (cloud stage) or from one end (tail stage), nor does the bacterium spread. Furthermore it does not form the fine fibrillar material. Evidence that the nucleating material is a secretory product of Listeria and not the macrophage comes from experiments using chloramphenicol, which inhibits protein synthesis in Listeria but not in macrophages. If chloramphenicol is applied 1 h after infection, a time before actin filaments are found attached to the Listeria in untreated macrophages, actin filaments never assemble on the Listeria even when fixed 3 h later. Furthermore the fine fibrillar material is absent, although there is a coat of dense granular material.     

Intermediate filament dynamics.

The view of intermediate filaments as static cytoskeletal elements is changing. Studies of exogenous intermediate filament proteins, either microinjected or expressed from transfected genes, have demonstrated that a continuous incorporation of subunits into the polymerized filaments is taking place. This incorporation appears to be required for maintaining normal cytoplasmic networks of intermediate filaments. At the post-translational level, phosphorylation is an important factor in regulating dynamic aspects of intermediate filament organization and structure.