Fluorescent measurement of desmin intermediate filament assembly

Intermediate filaments (IF) are cytoskeletal elements that are believed to play a major role in the specification and maintenance of cell form. Although previously thought to be stable and static because of their relative insolubility in physiological solvents, IF have recently been shown to have dynamic properties not unlike those of other cytoskeletal elements. The methodology for measuring this dynamic behavior, however, has been mostly borrowed from studies of other filament proteins and are poorly suited to IF because of their unusual physicochemical properties. In this report we introduce a fluorescence assay for quantifying in vitro IF assembly. Desmin subunits labeled with iodoacetamidofluorescein (IAF) to approximately 0.4 mol/mol retain the ability to polymerize into filaments indistinguishable from unlabeled IF in the electron microscope. By spectrophotometry, however, up to 90% of the starting fluorescence is quenched upon maximal IF assembly from IAF-desmin subunits. This quench is proportional to the total concentration of desmin subunits and is a sensitive measure of the assembly process. The critical concentration of assembly, measured at 170 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.0, is 0.2 microM. This indicates that a significant level of unpolymerized desmin exists in steady-state equilibrium with polymerized filaments under these conditions and suggests that IF subunit-filament equilibria may play a role in cytoskeletal dynamics.     

Modulation of desmin intermediate filament assembly by a monoclonal antibody

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.     

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。     

Towards a molecular description of intermediate filament structure and assembly

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.     

Characterization of distinct early assembly units of different intermediate filament proteins

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.     

Stepwise characterization of the thermodynamics of trichocyte intermediate filament protein supramolecular assembly

Trichocyte intermediate filament protein (IFP) is a heterodimeric complex that plays a pivotal role in the hair shaft for its mechanical strength, hair shape, and so on. Trichocyte IFP consists of acidic-type IFP and basic-type IFP, and the well-studied supramolecular assembly process of the complex occurs via the following steps: dimer formation, tetramer formation, formation of the lateral 32mer, and the elongation of the 32mer. Among these interactions, only the dimer formation, owing to coiled-coil interaction, has been described in detail; the nature of other interactions remains unspecified. For each assembly step, we report interaction isotherms obtained by means of isothermal titration calorimetry at various urea and NaCl concentrations. Decreasing the urea concentration generally promotes protein refolding, and we therefore expected to observe endothermic interactions owing to the refolding process. However, exothermic interactions were observed at 4 and 2 M urea, along with various characteristic endothermic interactions at the other urea concentrations as well as NaCl titration. The thermal responses described herein enabled us to analyze the protein supramolecular assembly process in a stepwise manner.     

Desmosome and intermediate filament assembly during differentiation and stratification of epithelial cells

In the present investigation the sequential expression and organization of keratin intermediate filament proteins were studied in the developing rat palatal epithelia starting from early gestation period to the adult. The distribution and organization of keratin proteins were correlated with the formation and elaboration of desmosornes during differentiation and stratification of the epithelia.     

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.     

Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly.

The assembly of soluble vimentin subunits into intermediate filaments (IFs) is dependent on information located in the amino-terminal domain. Using site-directed mutagenesis of a Xenopus laevis vimentin cDNA and an Escherichia coli production system to obtain pure mutated protein, we have identified, in the head domain, a nine amino acid motif (SSYRRIFGG), evolutionarily conserved from amphibia to man, which plays an important role in the orderly formation of IFs. Exchanges in the central di-arginine and in the two aromatic residues interfere with IF assembly of vimentin in vitro: on assembly under standard assembly conditions (160 mM-NaCl) most of the protein is included in dense aggregates, with a variable and minor proportion of IFs, whereas at lower ionic concentrations short and incomplete IF-like structures are formed. The deletion of the whole motif results in a protein that under standard assembly conditions (e.g. 160 mM-NaCl) predominantly and rapidly precipitates into large aggregates of non-IF material, whereas at lower ionic strength (e.g. 50 mM-NaCl) both IFs and dense aggregates are formed simultaneously. Our results show that the mutated protein can assume different forms at the same time and under the same conditions. This motif alone is insufficient for the formation of normal IFs as demonstrated by a mutant in which the motif has been brought closer to the alpha-helical rod domain by deletion of 55 internal amino acid residues. Corresponding observations have been made, by immunofluorescence microscopy, upon transfection of cultured epithelial cells lacking vimentin IFs. The importance of the head domain motif for the assembly and higher-order arrangement of IFs is discussed.     

Altered chaperone-like activity of alpha-crystallins promotes cataractogenesis

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.     

Regulation of intermediate filament gene expression.

Members of the intermediate filament protein family exhibit complex patterns of development-specific and tissue-specific expression. Studies exploring the mechanisms of gene regulation are underway and key regulatory factors are currently being described and isolated for certain genes encoding intermediate filament proteins. Selected systems from this diverse group of about 50 genes will be discussed.     

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.     

Interactions of intermediate filament proteins from wool.

Filaments of wool are heteropolymers formed by interaction of type I and type II intermediate filament (IF) proteins. There are four proteins in each of these two classes. Interaction of the reduced wool IF proteins was studied by two-dimensional electrophoresis which showed that complexes between type I and type II proteins were formed in solution at urea concentrations below 6 M. Complex formation between the carboxymethyl derivatives of wool IF proteins was studied using a filter binding assay in which radio-labelled individual components were allowed to react under various conditions with SDS-PAGE separated components after transfer to nitrocellulose. The results suggested that (i) absolute type specificity of interaction was maintained, (ii) fine specificity, i.e. preferential reaction between specific components is observed, (iii) wool IF proteins (hard keratins) also react, with the same type specificity, with soft keratins isolated from cow snout, (iv) the initial step in the polymerization sequence that leads to filament formation yields heterodimers.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

p38 MAPK interacts with actin and modulates filament assembly during skeletal muscle differentiation

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.     

A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive alpha-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4-2.3 A resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic alpha-helix, which is compatible with a coiled-coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer-dimer interactions during IF assembly. The 2B segment reveals a double-stranded coiled coil, which unwinds near residue Phe351 to accommodate a 'stutter'. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.