Effects of truncations in the N- and C-terminal domains of filensin on filament formation with phakinin in cell-free conditions and cultured cells.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification.

De novo filament formation by human hair keratins K85 and K35 follows a filament development pattern distinct from cytokeratin filament networks.

In human hair follicles, the hair-forming cells express 16 hair keratin genes depending on the differentiation stages. K85 and K35 are the first hair keratins expressed in cortical cells at the early stage of the differentiation. Two types of mutations in the gene encoding K85 are associated with ectodermal dysplasia of hair and nail type. Here, we transfected cultured SW-13 cells with human K85 and K35 genes and characterized filament formation. The K85-K35 pair formed short filaments in the cytoplasm, which gradually elongated and became thicker and entangled around the nucleus, indicating that K85-K35 promotes lateral association of short intermediate filaments (IFs) into bundles but cannot form IF networks in the cytoplasm. Of the K85 mutations related to ectodermal dysplasia of hair and nail type, a two-nucleotide (C1448 T1449 ) deletion (delCT) in the protein tail domain of K85 interfered with the K85-K35 filament formation and gave only aggregates, whereas a missense mutation (233A>G) that replaces Arg78 with His (R78H) in the head domain of K85 did not interfere with the filament formation. Transfection of cultured MCF-7 cells with all the hair keratin gene combinations, K85-K35, K85(R78H)-K35 and K85(delCT)-K35, as well as the individual hair keratin genes, formed well-developed cytoplasmic IF networks, probably by incorporating into the endogenous cytokeratin IF networks. Thus, the unique de novo assembly properties of the K85-K35 pair might play a key role in the early stage of hair formation.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica.

BACKGROUND: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. OBJECTIVE: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. METHODS: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. RESULTS: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. CONCLUSION: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

High-frequency affine mechanics and nonaffine relaxation in a model cytoskeleton.

The cytoskeleton is a network of crosslinked, semiflexible filaments, and it has been suggested that it has properties of a glassy state. Here we employ optical-trap-based microrheology to apply forces to a model cytoskeleton and measure the high-bandwidth response at an anterior point. Simulating the highly nonlinear and anisotropic stress-strain propagation assuming affinity, we found that theoretical predictions for the quasistatic response of semiflexible polymers are only realized at high frequencies inaccessible to conventional rheometers. We give a theoretical basis for determining the frequency when both affinity and quasistaticity are valid, and we discuss with experimental evidence that the relaxations at lower frequencies can be characterized by the experimentally obtained nonaffinity parameter.

In vitro assembly properties of human type I and II hair keratins.

Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations.

The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 A resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (alpha-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg(2+) ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism.

Vimentin intermediate filaments as a template for silica nanotube preparation.

Organic compounds are used as templates to regulate the morphology of inorganic nanostructures. In the present study, we used intermediate filaments (IFs), the major cytoskeleton component of most eukaryotic cells, as a template for hollow silica nanotube preparation. Sol-gel polymerization of tetraethoxysilane proceeded preferentially on the surface of IFs assembled from vimentin protein in vitro, resulting in silica-coated fibres. After removing IFs by calcination, electron microscopy revealed hollow silica nanotubes several micrometers long, with outer diameters of 35-55 nm and an average inner diameter of 10 nm (comparable to that of IFs). Furthermore, the silica nanotubes exhibited a gnarled surface structure with an 18-26 nm repeating pattern (comparable to the 21-nm beading pattern along IFs). Thus, the characteristic morphology of IFs were well replicated into hollow silica nanotubes, suggesting that IFs maybe useful as an organic template.

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro.

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.

The last twenty residues in the head domain of mouse lamin A contain important structural elements for formation of head-to-tail polymers in vitro.

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a alpha-helical rod domain flanked by non-alpha-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.

A case of extragingival peripheral ameloblastoma in the buccal mucosa.

Peripheral ameloblastomas (PAs) of the extragingival areas are extremely rare. To the best of our knowledge, only five cases of extragingival PA have been reported. We present here a sixth case of extragingival PA of the buccal mucosa in an 80-year-old male. The tumor was surgically removed by blunt dissection and there is no evidence of recurrence for 7 months. We also discuss here the clinical characteristics, the origin, and the management of the tumor by reference to the relevant literature.

Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy.

Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of approximately 28 nm, a height of approximately 4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be approximately 25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of approximately 10 nm. Vimentin IFs in air clearly demonstrated approximately 21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of approximately 78 nm (evaluated length approximately 69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of approximately 40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.