Among the diverse kinds of intercellular, plaque-bearing, cadherin-containing junctions, desmosomes (maculae adhaerentes) represent a major type characterized by the presence of specific transmembrane glycoproteins, i.e. desmosomal cadherins of the desmoglein and desmocollin categories, and the cytoplasmic plaque proteins, desmoplakin I and plakoglobin. Recent studies, however, have shown that the composition of desmosomes is not identical in the various normal and tumorous desmosome-forming tissues and cell cultures, including diverse forms of epithelia and carcinomas, meningothelia and meningiomas, myocardium and the lymph node follicle reticulum. Desmosomes can differ in their specific complement of desmogleins, Dsg1-3, and desmocollins, Dsc1a-3b, as well as in the additional presence and in their relative amounts of certain accessory plaque proteins such as desmoplakin II and plakophilin 1, a basic member of the larger plakoglobin family of proteins ("band 6 protein"). Assembly and function of desmosomes are effected by the interaction of the specific complement of desmosomal cadherins with certain cytoplasmic proteins. In particular, the cytoplasmic portions ("tails") of the desmosomal cadherins contain certain domains and amino acid sequence motifs, identified by mutagenesis and transfection assays, that are essential elements in desmosome formation, notably the assembly of plaque proteins, and in the site-specific anchorage of intermediate-sized filaments (IFs) of the cytoskeleton, thereby contributing to the specific intracellular as well as supracellular, i.e. tissue, architecture.

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.

We have determined the structural organization of the human gene (LMNB1) that encodes nuclear lamin B1, an intermediate filament protein of the nuclear envelope. The transcription unit spans more than 45 kb and the transcription start site is 348 nucleotides upstream from the translation initiation codon. Lamin B1 is encoded by 11 exons. Exon 1 codes for the amino-terminal head domain and the first portion of the central rod domain, exons 2 through 6 the central rod domain, and exons 7 through 11 the carboxyl-terminal tail domain of this intermediate filament protein. Intron positions are conserved in other lamin genes from frogs, mice, and humans but different in lamin genes from Drosophila melanogaster and Caenorhabditis elegans. In the region encoding the central rod domain, intron positions are also similar to those in the gene for an invertebrate nonneuronal cytoplasmic intermediate filament protein and the genes for most vertebrate cytoplasmic intermediate filament proteins except neurofilaments and nestin.

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the 'a' form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter 'b' form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

A new protein found at sites of cell-substrate adhesion has been identified by analysis of a nonimmune rabbit serum. By indirect immunofluorescence this serum stains focal contacts (adhesion plaques) and the associated termini of actin filament bundles in cultured chicken cells. Western immunoblot analysis of total chick embryo fibroblast protein demonstrated an 82-kD polypeptide to be the major protein recognized by the unfractionated serum. This 82-kD protein is immunologically distinct from other known adhesion plaque proteins such as vinculin, talin, alpha-actinin, and fimbrin. Antibody affinity-purified against the electrophoretically isolated, nitrocellulose-bound 82-kD protein retained the ability to stain the area of the adhesion plaque, which confirms that the 82-kD protein is indeed a constituent of the focal contact. The 82-kD polypeptide has a basic isoelectric point relative to actin and fibronectin, and it appears to be very low in abundance. The 82-kD protein is ubiquitous in chicken embryo tissues. However, it appears to be more abundant in fibroblasts and smooth muscle than in brain or liver. Intermediate levels of the protein were detected in skeletal and cardiac muscle. The subcellular distribution of the 82-kD protein raises the possibility that this polypeptide is involved in linking actin filaments to the plasma membrane at sites of substrate attachment or regulating these dynamic interactions.

Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the osteocyte cytoskeleton. This study provides an extensive analysis of the osteocyte cytoskeleton, based on the successful isolation of osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in osteocyte processes. The prominence and unique distribution of fimbrin in osteocyte processes provides the possibility of its use as an intracellular marker to distinguish osteocytes from osteoblasts.

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin, whereas a derivative highly tumorigenic cell line transformed by both v-ras-H and T-antigen did express vimentin. Analysis of several other kinds of epithelial carcinoma cell lines showed only rare examples of vimentin expression.

Keratins 8 and 18 (K8 and K18) are heteropolymeric intermediate filament phosphoglycoproteins of simple-type epithelia. Mutations in K8 and K18 predispose the affected individual to liver disease as they protect hepatocytes from apoptosis. K18 undergoes dynamic O-linked N-acetylglucosamine glycosylation at Ser 30, 31 and 49. We investigated the function of K18 glycosylation by generating mice that overexpress human K18 S30/31/49A substitution mutants that cannot be glycosylated (K18-Gly(-)), and compared the susceptibility of these mice to injury with wild-type and other keratin-mutant mice. K18-Gly(-) mice are more susceptible to liver and pancreatic injury and apoptosis induced by streptozotocin or to liver injury by combined N-acetyl-D-glucosaminidase inhibition and Fas administration. The enhanced apoptosis in the livers of mice that express K18-Gly(-) involves the inactivation of Akt1 and protein kinase Ctheta as a result of their site-specific hypophosphorylation. Akt1 binds to K8, which probably contributes to the reciprocal hyperglycosylation and hypophosphorylation of Akt1 that occurs on K18 hypoglycosylation, and leads to decreased Akt1 kinase activity. Therefore, K18 glycosylation provides a unique protective role in epithelial injury by promoting the phosphorylation and activation of cell-survival kinases.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.

A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon-shaped tail-less particles, after which they develop long tails at each pointed end, at temperatures close to that of the natural habitat, 85 degrees C. The extracellularly developed tails constitute tubes, which terminate in an anchor-like structure that is not observed in the tail-less particles. A thin filament is located within the tube, which exhibits a periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable elements as well as genes, which previously have been associated only with archaeal self-transmissable plasmids. In total, it encodes 72 predicted proteins, including 11 structural proteins with molecular masses in the range of 12 to 90 kDa. Several of the larger proteins are rich in coiled coil and/or low complexity sequence domains, which are unusual for archaea. One protein, in particular P800, resembles an intermediate filament protein in its structural properties. It is modified in the two-tailed, but not in the tail-less, virion particles and it may contribute to viral tail development. Exceptionally for a crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and this process can be interrupted by different stress factors.

MRP8 and MRP14 are two Ca(2+)-binding proteins of the S-100 family expressed by myelomonocytic cells. Both proteins assemble to noncovalently associated complexes in a Ca(2+)-dependent manner. Members of the S-100 family are known to play a role in cytoskeletal-membrane interactions; therefore, we investigated the subcellular distribution of MRP8/MRP14 and their complexes in human monocytes. Using differential centrifugation and subsequent Western blot or enzyme-linked immunosorbent assay analysis, we found that MRP8/MRP14 were almost completely translocated from the cytoplasma to membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Using a cross-linking technique, complexed forms of MRP8/MRP14 were found to be associated with the plasma membrane. Analysis of MRP-transfected L132 cells showed that the MRP8 as well as the MRP14 component of the MRP8/MRP14 complex may independently bind to membrane and cytoskeletal structures. Furthermore, immunogold electron microscopy showed a colocalization of MRP8/MRP14 and the intermediate filament type III protein vimentin in A23187-treated monocytes. Our data indicate that, in analogy to other S-100-like proteins, MRP8 and MRP14 play a role in Ca(2+)-dependent cytoskeletal-membrane interactions. Restriction of MRP8/MRP14 expression to distinct stages of myelomonocytic differentiation suggests that these proteins are involved in highly specific pathways of intracellular signaling in phagocytes.

The crucial role of cancer stem cells (CSCs) in the pathology of malignant diseases has been extensively studied during the last decade. Nestin, a class VI intermediate filament protein, was originally detected in neural stem cells during development. Its expression has also been reported in different tissues under various pathological conditions. Specifically, nestin has been shown to be expressed in transformed cells of various human malignancies, and a correlation between its expression and the clinical course of some diseases has been proved. Furthermore, the coexpression of nestin with other stem cell markers was described as a CSC phenotype that was subsequently verified using tumorigenicity assays. The primary aim of this review is to summarize the recent findings regarding nestin expression in CSCs, its possible role in CSC phenotypes, particularly with respect to capacity for self-renewal, and its utility as a putative marker of CSCs.

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.

Desmosomes are highly organized intercellular adhesive junctions that are particularly prominent in epidermis and other tissues experiencing mechanical stress. Desmoplakin, a constitutive component of the desmosomal plaque, is the most abundant protein present in such junctions and plays a critical role in linking the intermediate filament network to the plasma membrane in these tissues. Here we report the first mutation in the gene encoding desmoplakin. The identified mutation, resulting in a null allele and haploinsufficiency, was observed in genomic DNA from a kindred with the dominantly inherited skin disorder, striate palmoplantar keratoderma. Affected individuals had a linear pattern of skin thickening on the fingers and palms and circumscribed areas of skin thickening on the soles. Affected skin demonstrated loosening of intercellular connections, disruption of desmosome-keratin intermediate filament interactions and a proportion of rudimentary desmosomal structures. The disorder mapped to chromosome 6p21 with a maximum lod score of 10.67. The mutation was a heterozygous C->>T transition in exon 4 of the desmoplakin gene and predicted a premature termination codon in the N-terminal region of the peptide. This is the first reported mutation of desmo-plakin and also the first inherited skin disorder in which haploinsufficiency of a structural component has been implicated. It identifies dosage of desmoplakin as critical in maintaining epidermal integrity.

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.

Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of microfilaments, intermediate filaments, and tight junction-specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub-apical region of the dying hair cell. This region becomes cytokeratin-positive. The two supporting cells meet at the mid-line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens-type junction, but desmosomes are absent.

OBJECTIVE

The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies.

METHODS

The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp.

RESULTS

The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity.

CONCLUSIONS

The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.

The Arp2/3 complex, a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, initiates formation of actin filament networks in response to intracellular signals. The molecular mechanism of filament nucleation, however, is not well understood. Arp2 and Arp3 are predicted to bind ATP via a highly conserved nucleotide-binding domain found in all members of the actin superfamily and to form a heterodimer than mimics a conventional actin dimer. We show here that adenosine nucleotides bind with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable ATP is required for actin nucleation activity. Binding of N-WASP WA increases the affinity of both Arp2 and Arp3 for ATP but does not alter the stoichiometry of nucleotides bound in the presence of saturating concentrations of ATP. The Arp2/3 complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments, but addition of the phosphate analogue BeF(3) partially restores activity to the ADP-Arp2/3 complex. Bound nucleotide also regulates the affinity of the Arp2/3 complex for its upstream activators N-WASP and ActA. We propose that the active nucleating form of the Arp2/3 complex is the ADP-P(i) intermediate in the ATPase cycle and that the ATPase activity of the Arp2/3 complex controls both nucleation of new filaments and release of the Arp2/3 complex from membrane-associated activators.