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PubMed2006

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin.

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5 min following ligand addition at concentrations as low as 5 nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with alpha6 and beta1 integrin subunits at the base of the extensions. alpha5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectin-fibronectin interactions during progression of assembly.

Animals↗

PubMed2004

Molecular mechanisms for the assembly of the T cell receptor-CD3 complex.

The T cell receptor (TCR)-CD3 complex represents on of the most intricate membrane receptor structures since it is built from six distinct chains. This complexity led to a number of different proposals for the arrangement of the receptor subunits, its stoichiometry and the mechanisms responsible for receptor triggering. Early work had demonstrated that basic and acidic transmembrane (TM) residues were involved in the assembly but the molecular arrangement could not be deduced due to the complexity of the receptor. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrated that the complex assembled in the ER contains only a single TCRalphabeta heterodimer and one copy of each of the CD3deltaepsilon, CD3gammaepsilon and zeta-zeta signaling dimers. Surprisingly, assembly of each of the three signaling dimers with TCR was dependent on one of the three basic TCR TM residues as well as both acidic residues located in the TM domains of the interacting signaling dimer. Each assembly step thus results in the formation of a three-helix interface in the membrane that involves one basic and two acidic TM residues, and this arrangement effectively shields these ionizable residues at protein-protein interfaces from the lipid. Since proteins whose TM domains have exposed ionizable residues are not stably integrated into the lipid bilayer, assembly based on shielding of ionizable residues permits full equilibration of the receptor into the lipid bilayer and prevents degradation. Assembly, export of intact receptor complexes and degradation of unassembled components thus rely on the same organizing principle.

Amino Acid Sequence↗

PubMed2006

Coiled coils direct assembly of a cold-activated TRP channel.

Transient receptor potential (TRP) channels mediate numerous sensory transduction processes and are thought to function as tetramers. TRP channel physiology is well studied; however, comparatively little is understood regarding TRP channel assembly. Here, we identify an autonomously folded assembly domain from the cold- and menthol-gated channel TRPM8. We show that the TRPM8 cytoplasmic C-terminal domain contains a coiled coil that is necessary for channel assembly and sufficient for tetramer formation. Cell biological experiments indicate that coiled-coil formation is required for proper channel maturation and trafficking and that the coiled-coil domain alone can act as a dominant-negative inhibitor of functional channel expression. Our data define an authentic TRP modular assembly domain, establish a clear role for coiled coils in ion channel assembly, demonstrate that coiled-coil assembly domains are a general feature of TRPM channels, and delineate a new tool that should be of general use in dissecting TRPM channel function.

Amino Acid Sequence↗

PubMed2005

Assembly and regulation of prothrombinase complex on B16F10 melanoma cells.

A number of studies indicate that coagulation proteases play significant roles in cancer biology. Melanoma is a highly metastatic cancer, and there is evidence that thrombin contributes to this aggressive pattern. However, few studies correlate this type of cancer with formation of the prothrombinase complex, which is responsible for conversion of prothrombin into thrombin in the coagulation system. The aim of this study was to investigate the assembly and regulation of prothrombinase complex on the murine melanoma cell line, B16F10. B16F10 cells were unable to activate prothrombin except when previously incubated with factor Xa. This effect was dependent on factor Xa binding to cell membranes, since no activation was detected with Gla-domainless factor Xa. The thrombin formation by B16F10-bound factor Xa was enhanced approximately 10 fold in the presence of factor Va, indicating the assembly of prothrombinase complex. Differently from platelets, B16F10-assembled prothrombinase complex was inhibited by prothrombin fragment 1 but not by fragment 2. In addition, bothrojaracin, a specific ligand of proexosite I on prothrombin, caused a significant decrease in the zymogen activation. Our data demonstrate that B16F10 melanoma cells generate thrombin by promoting assembly of the prothrombinase complex. This ability might be correlated with the increased metastatic potential of this cell line. Moreover, B16F10-assembled prothrombinase complex seems to be modulated in a different way from that found for the physiological complex assembled on platelets.

Animals↗

PubMed2004

A novel role for non-muscle gamma-actin in skeletal muscle sarcomere assembly.

Existing models describing sarcomere assembly have arisen primarily from studies using cardiac muscle. In contrast to cardiac muscle, skeletal muscle differentiation is characterised by dramatic changes in protein expression, from non-muscle to muscle-specific isoforms before organisation of the sarcomeres. Consequently, little is understood of the potential influence of non-muscle cytoskeletal proteins on skeletal sarcomere assembly. To address this issue, transfectant (gamma33-B1) and control mouse C2 myoblasts were differentiated to form myotubes, and various stages of skeletal sarcomere assembly were studied. Organisation of non-muscle gamma-actin and co-localisation with sarcomeric alpha-actinin, an early marker of sarcomere assembly and a major component of Z lines, was noted. gamma-Actin was also identified in young myotubes with developing sarcomeric myofibrils in regenerating adult mouse muscle. Localisation of gamma-actin in a different area of the myotube to the muscle-specific sarcomeric alpha-actin also indicated a distinct role for gamma-actin. The effects of aberrant gamma-actin expression in other myoblast lines, further suggested a sequestering role for gamma-actin. These observations make the novel suggestion that non-muscle gamma-actin plays a role in skeletal sarcomere assembly both in vitro and in vivo. Consequently, a modified model is proposed which describes the role of gamma-actin in skeletal sarcomere assembly.

Actinin↗

PubMed2001

Identification of functional regions of Cbp3p, an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase in yeast mitochondria.

The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain. To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein. Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p. Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability. Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex. Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c(1) assembly. Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity. Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p. Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover. The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Delta12-23, Delta24-54, Delta56-96 and Delta12-96. All mutants were respiratory competent, indicating that residues 12-96 were not essential for Cbp3p function, stability or mitochondrial import. Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity.

Amino Acid Sequence↗

PubMed1986

Macromolecular assemblies of myosin.

The self-assembly of myosin into filamentous structures is a highly cooperative and rapid process. Nevertheless, the presence of nonequivalent bonding interactions within the filament permits differential stabilization of several macromolecular assemblies of myosin under well-controlled ionic conditions in citrate/Tris buffer at pH 8.0. We have detected and characterized bipolar myosin minifilaments, myosin octamers, and tetramers by using light scattering, analytical ultracentrifugation, and viscosity techniques. These structures have molecular weights of 8.0 X 10(6), 3.9 X 10(6) g/mol, sedimentation coefficients of 32S, 22S, and 18S, and radii of gyration of 990 A, 890 A and 790, A, respectively. The similar radii of gyration indicate similar bipolar geometry for all these particles. The 32S minifilaments in 10 mM citrate/Tris buffer (pH 8.0) are the most stable species. The smaller 18S and 22S assemblies in 2 mM and 5 mM citrate/Tris, pH 8.0, are readily affected by low concentrations of KCl and fuse into the minifilament particles. The instability of the 18S and 22S forms of myosin assembly is also revealed by their titration with ATP. These structures are dissociated at lower ATP concentrations than the minifilaments and do not show the cooperative dissociation transitions characteristic of filaments and minifilaments. Sedimentation velocity analysis of the 18S and 22S species in the presence of ATP reveals the involvement of 10S myosin dimer in the dissociation of assembled myosin. The different forms of assembled myosin are discussed in the context of formation of myosin minifilaments.

Adenosine Triphosphate↗

PubMed1998

Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces.

Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.

Adsorption↗

PubMed1999

Identification of 70 amino acids important for GABA(C) receptor rho1 subunit assembly.

gamma-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the mammalian central nervous system and gates at least three subclasses of receptors, termed GABA(A), GABA(B) and GABA(C). Accumulating evidence indicates that GABA(C) receptors are composed exclusively of rho subunits. The N-terminal half of the rho subunits has been shown to mediate formation of homo- and heterooligomeric GABA(C) receptors. In this study, we searched for specific sequences within the N-terminus of the rho1 subunit involved in the assembly process. Assembly sequences were localized to a 128-amino acid region by deletion of progressively larger regions of a chimeric rho1beta1 subunit previously shown to disrupt rho1 and rho2 assembly. To confirm this observation, a series of GABA(A) receptor beta subunit chimeras containing different regions of the rho1 N-terminus were tested for interference with rho1 and rho2 subunit assembly into functional GABA receptors. Transfer of 70 residues within the 128 amino acid region to the beta1 subunit created a chimera that disrupted rho1, but not rho2, assembly into functional receptors. These observations refine the location of signals involved in rho1 subunit assembly, and suggest that different signals exist for the formation of rho1 homooligomeric and rho1/rho2 heterooligomeric GABA(C) receptors.

Amino Acid Sequence↗

PubMed1998

Zomepirac acyl glucuronide covalently modifies tubulin in vitro and in vivo and inhibits its assembly in an in vitro system.

Drugs possessing a carboxylate functional group usually form acyl glucuronides as major metabolites. These electrophilic metabolites can undergo several spontaneous reactions, including covalent adduct formation with proteins. The present study examined whether covalent adducts were formed with microtubular protein (MTP, 85%, alpha/beta-tubulin) and whether this influenced its ability to assemble into microtubules. Bovine brain microtubular protein (MTP) was purified by assembly-disassembly cycles and incubated with the nonsteroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperature and pH 7.5. Assembly was monitored by change in turbidity (increase in absorbance at 340 nm). Both ZAG and iso-ZAG caused dose-dependent inhibition of assembly (50% inhibition at about 1 mM), while ZP caused modest inhibition (< 50% inhibition at 4 mM). In a slightly different system, incubation of performed microtubules with 4 mM ZAG caused about 35% inhibition of reassembly ability, while modification of MTP under similar conditions resulted in about 85% reduction of assembly ability. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG covalently modified MTP in a dose-dependent manner, while ZP itself caused no modification. Tubulin and many minor proteins comprising MTP were modified. ZP-modified tubulin was shown to be present in the cytosol of livers from rats dosed twice daily for 3 days with ZP at 50 mg/kg, using a sandwich ELISA with ZP and tubulin antisera. Whether any perturbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation.

Animals↗

PubMed2001

Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast.

Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo-COX in the SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D-PAGE.

Electron Transport Complex IV↗

PubMed1983

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. II. Purification of the protein products of genes 10 and 11 and the in vitro formation of the P(10/11) complex.

Two bacteriophage T4 proteins which are precursors to the phage baseplate have been purified to homogeneity. These proteins, P10 and P11, are components of the P(10/11) complex, which is the first intermediate in the assembly of T4 baseplate 1/6th arms. Each protein was isolated from cells infected with a T4 amber mutant defective in the production of the other protein. Thus these purified proteins have never been assembled into the P(10/11) complex in vivo. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the ability of these proteins to block the phage killing activity of specific antisera were used to monitor the purification steps. Sedimentation equilibrium experiments reveal a molecular weight of 188,000 g/mol for P10 and 60,000 g/mol for P11. These data together with the previously determined molecular weights of the gene 10 and gene 11 polypeptide chains (King & Mykolajewycz, 1973) and the in vivo assembled P(10/11) complex (Berget & King, 1978b) are consistent with P10 being a dimer of the product of gene 10, P11 being a dimer of the product of gene 11, and P(10/11) being a tetramer containing one of each of these dimers. Purified P10 and P11 are active in assembly because they complement 10- and 11- defective extracts, respectively, to form viable bacteriophage in vitro. Furthermore, these proteins assemble in vitro to form a protein structure identical to the P(10/11) complex formed in vivo as determined by non-denaturing gel electrophoresis. This P(10/11) complex formed in vitro complements 10-/11- defective extracts to form viable phage. The overall extent of this in vitro assembly reaction is not affected by NaCl to 1.5 M or 2% Triton X-100. The reaction is, however, prevented by the denaturing effects of urea and sodium dodecyl sulfate.

Antibodies↗

PubMed1985

Templeting and self-assembly.

Templeting and self-assembly represent the two extremes of the spectrum of determinate pattern-assembly processes. A templeted pattern can be defined as one that requires a prepattern or templet explicitly specifying the final topology of the pattern. Conversely, a self-assembling pattern can be defined as one for which the inherent constraints of the precursor elements alone are sufficient to specify the final pattern. Both concepts can be directly expressed in matrix notation, and a simple matrix measure, the templeting index, characterizes the relative amount of templeting or of self-assembly in any particular system. With this language, a fundamental principle of pattern-assembly becomes evident: in the determinate realm, some patterns can only be assembled using the same-sized templets--templets that are at least as large as the final pattern.

Mathematics↗

PubMed1996

The effect of deamination and/or blocking arginine residues on the molecular assembly of acid-extracted rat tail tendon collagen.

We describe the effect of deamination of lysine and blocking of arginine residues on the assembly of collagen into native fibrils and SLS aggregates. Treatment of collagen solutions with one or both of these procedures does not prevent the formation of fibrils or SLS aggregates but reduces their ability to form assemblies with accurate longitudinal registration. These observations provide direct confirmation that hydrophobic interactions are important in collagen assembly. Unbanded fibrils were formed within the first 24 h at 4 degrees C from both acidic and neutralized deaminated and from neutralized control collagen solutions, transversely banded fibrils appearing later. This is compatible with the suggestion that initially, collagen fibrils are assembled by lyotropic liquid crystallization and with other observations which suggest that collagen molecules are initially free to move laterally within the fibril before being locked into place. Fibrils assembled from deaminated collagen solution show two variant longitudinal registration patterns which grade into one another. This suggests that, with a reduction in positively charged side chains, the thermodynamic energy minima responsible for longitudinal registration are less sharp compared with control collagen solutions. Reduction of positive charge by chemical modification helps to explain why the chemical modifications reduce swelling of collagen fibres. It also helps to explain why fibrils form spontaneously at 4 degrees C in both arginine-blocked and deaminated collagen solutions. Thus chemical modifications of rat tail tendon provides new insight into the mechanisms in collagen assembly.

Acetates↗

PubMed1991

The bacterial flagellum and flagellar motor: structure, assembly and function.

The bacterial flagellum is a complex multicomponent structure which serves as the propulsive organelle for many species of bacteria. Rotation of the helical flagellar filament, driven by a proton-powered motor embedded in the cell wall, enables the flagellum to function as a screw propeller. It seems likely that almost all of the genes required for flagellar formation and function have been identified. Continuing analysis of the portions of the genome containing these genes may reveal the existence of a few more. Transcription of the flagellar genes is under the control of the products of a single operon, and so these genes constitute a regulon. Other controls, both transcriptional and post-transcriptional, have been identified. Many of these genes have been sequenced, and the information obtained will aid in the design of experiments to clarify the various regulatory mechanisms of the flagellar regulon. The flagellum is composed of several substructures. The long helical filament is connected via the flexible hook to the complex basal body which is located in the cell wall. The filament is composed of many copies of a single protein, and can adopt a number of distinct helical forms. Structural analyses of the filament are adding to our understanding of this dynamic polymer. The component proteins of the hook and filament have all been identified. Continuing studies on the structure of the basal body have revealed the presence of several hitherto unknown basal-body proteins, whose identities and functions have yet to be elucidated. The proteins essential for energizing the motor, the Mot and switch proteins, are thought to exist as multisubunit complexes peripheral to the basal body. These complexes have yet to be identified biochemically or morphologically. Not surprisingly, flagellar assembly is a complex process, occurring in several stages. Assembly occurs in a proximal-to-distal fashion; the basal body is assembled before the hook, and the hook before the filament. This pattern is also maintained within the filament, with monomers added at the distal end of the polymer; the same is presumably true of the other axial components. An exception to this general pattern is assembly of the Mot proteins into the motor, which appears to be possible at any time during flagellar assembly. With the identification of the genes encoding many of the flagellar proteins, the roles of these proteins in assembly is understood, but the function of a number of gene products in flagellar formation remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗

PubMed2006

Dynamics of assembly and reorganization of extracellular matrix proteins.

This chapter will review advances in our understanding of the dynamics of assembly and reorganization of extracellular matrix (ECM) proteins and will highlight the role of fibronectin as a key orchestrator for the assembly of multiple ECM proteins. The dynamic rather than static nature of the ECM will be emphasized by reviewing time-lapse imaging studies in living cell and embryo systems, with a particular focus on fibronectin and members of the fibrillin superfamily. These studies have provided new insights into the assembly and reorganization of ECM fibrillar networks, suggesting that fibril assembly is a hierarchical process, with increasingly larger fibrillar structures formed by the progressive aggregation of smaller units. These studies have also revealed that motile cells appear to be actively involved in the assembly and reorganization of ECM fibrillar networks by shunting fibrillar material from one location to another, adding fibrillar material to the ends of growing fibrils, and exchanging material between fibrils. A common theme emerging from these studies is that cell- and tissue-generated mechanical forces are critical in the assembly and remodeling of the ECM.

Animals↗

PubMed2001

Major histocompatibility class I folding, assembly, and degradation: a paradigm for two-stage quality control in the endoplasmic reticulum.

Protein folding in living cells is a complex process involving many interdependent factors. The primary site for folding of nascent proteins destined for secretion is the endoplasmic reticulum (ER). Several disease states, including cystic fibrosis, are brought about because of irregularities in protein folding. Under normal cellular conditions, "quality control" mechanisms ensure that only correctly folded proteins are exported from the ER, with incorrectly folded or incompletely assembled proteins being degraded. Quality control mechanisms can be divided into two broad processes: (1) Primary quality control involves general mechanisms that are not specific for individual proteins; these monitor the fidelity of nascent protein folding in the ER and mediate the destruction of incompletely folded proteins. (2) Partially folded or assembled proteins may be subject to secondary quality control mechanisms that are protein- or protein-family-specific. Here we use the folding and assembly of major histocompatibility complex (MHC) class I as an example to illustrate the processes of quality control in the ER. MHC class I, a trimeric complex assembled in the ER of virally infected or malignant cells, presents antigenic peptide to cytotoxic T lymphocytes; this mediates cell killing and thereby prevents the spread of infection or malignancy. The folding and assembly of MHC class I is subjected to both primary and secondary quality control mechanisms that lead either to correct folding, assembly, and secretion or to degradation via a proteasome-associated mechanism.

Animals↗

PubMed1998

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression.

To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a cDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick filaments when assembled at temperatures below 21 degrees C. At 37 degrees C few filaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 degrees C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 degrees C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normal IFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Amino Acid Sequence↗