We have analyzed the expression of the three retinoic acid receptor (RAR) (alpha, beta, gamma) mRNAs and the intermediate filament protein keratin 19 (K19) mRNA in cell lines cultured from oral and epidermal human squamous cell carcinoma (SCC) and from benign, hyperplastic, and hyperkeratotic (leukoplakia) lesions arising in various regions of the oral cavity. Seven of the SCC lines were derived from tumors arising in regions of the oral cavity in which the normal epithelial cells (keratinocytes) express RAR beta transcripts. Seven of the nine SCC lines tested did not exhibit detectable RAR beta mRNA levels, even in response to addition of retinoic acid (RA). The RAR beta gene did not appear to be rearranged or deleted in the five nonexpressing SCC lines examined by Southern analysis. The steady-state RAR gamma mRNA levels were 2- to 4-fold lower in 6 of the 9 SCC lines than in their normal counterparts, whereas the RAR alpha message levels in SCC lines were similar to those of the normal cell strains. The expression of keratin 19 message, which is RA inducible in normal keratinocytes, was also abnormal in many of the SCC cell lines. Some SCC lines, e.g., those derived form tumors of the soft palate epithelium, did not express high levels of K19 message even though normal soft palate keratinocytes expressed high levels of K19 mRNA. Two of the nine SCC lines expressed higher than normal levels of K19 mRNA, and this expression was RA independent. Cells cultured from four oral leukoplakia lesions were also examined and found to express RAR beta mRNA at relatively normal levels, but they expressed RAR gamma message at half the level of epithelial cells cultured from normal tissue. These results show that the correlation between RAR beta gene expression and K19 gene expression that we have observed in the various normal keratinocyte subtypes of the oral cavity (D.L. Crowe et al., manuscript in preparation) is not present in transformed keratinocytes (SCC cells). The lack of apparent RA regulation of the K19 gene in SCC lines may be associated with other aberrations in differentiation which have been identified in SCC cells. Abnormally low expression of the RAR beta receptor may contribute to neoplastic progression in stratified squamous epithelia. It may also determine whether a tumor is responsive to RA as a chemotherapeutic agent.

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (approximately 5 nm wide, 100-200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deep-etch technique, these approximately 5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of approximately 5 nm fibrils that are morphologically distinct from the approximately 8 nm myosin-containing fibrils which were identified by anti-myosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actin-vesicle, actin-intermediate filament and vesicle-plasma membrane linkages.

beta-Catenin and plakoglobin (gamma-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and alpha-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, beta-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increased the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolished beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.

Hemidesmosomes (HDs) mediate cell adhesion to the extracellular matrix and have morphological association with intermediate-sized filaments (IFs) through cytoplasmic plaques. Though several proteins have been located in HDs, most of them have not been well characterized, with the exception of the 230-kD antigen of bullous pemphigoid (BP), an autoimmune skin blistering disease. Only recently we have succeeded in isolating HDs from bovine corneal epithelial cells and in identifying five major components on SDS-PAGE (Owaribe K., Y. Nishizawa, and W. W. Franke. 1991. Exp. Cell Res. 192:622-630). In this study we report on immunological characterization of one of the major components, termed HD1, with an apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HD1 with BP antigen at the basement membrane zone of those tissues that have typical HDs, including skin epidermis, corneal and tracheal epithelia, and myoepithelium. In cultured keratinocytes, HD1 demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions. Immunoelectron microscopy revealed that an epitope of HD1 was located on the cytoplasmic side of HDs. Taking all these results together, we conclude that HD1 is a new hemidesmosomal component. Interestingly, HD1 also exists in endothelial and glial cells, which lack typical HDs.

Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

Fibrocartilage is an avascular tissue that is best documented in menisci, intervertebral discs, tendons, ligaments, and the temporomandibular joint. Several of these sites are of particular interest to those in the emerging field of tissue engineering. Fibrocartilage cells frequently resemble chondrocytes in having prominent rough endoplasmic reticulum, many glycogen granules, and lipid droplets, and intermediate filaments together with and actin stress fibers that help to determine cell organization in the intervertebral disc. Fibrocartilage cells can synthesize a variety of matrix molecules including collagens, proteoglycans, and noncollagenous proteins. All the fibrillar collagens (types I, II, III, V, and XI) have been reported, together with FACIT (types IX and XII) and network-forming collagens (types VI and X). The proteoglycans include large, aggregating types (aggrecan and versican) and small, leucine-rich types (decorin, biglycan, lumican, and fibromodulin). Less attention has been paid to noncollagenous proteins, although tenascin-C expression may be modulated by mechanical strain. As in hyaline cartilage, matrix metalloproteinases are important in matrix turnover and fibrocartilage cells are capable of apoptosis.

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard alpha-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft alpha-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (E(i) = 6.4 MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180 MPa) and toughness (130 MJ/m(3)) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible alpha->>beta conformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.

The inner side of the nuclear envelope (NE) is lined with lamins, a meshwork of intermediate filaments that provides structural support for the nucleus and plays roles in many nuclear processes. Lamins, classified as A- or B-types on the basis of biochemical properties, have a conserved globular head, central rod and C-terminal domain that includes an Ig-fold structural motif. In humans, mutations in A-type lamins give rise to diseases that exhibit tissue-specific defects, such as Emery-Dreifuss muscular dystrophy. Drosophila is being used as a model to determine tissue-specific functions of A-type lamins in development, with implications for understanding human disease mechanisms. The GAL4-UAS system was used to express wild-type and mutant forms of Lamin C (the presumed Drosophila A-type lamin), in an otherwise wild-type background. Larval muscle-specific expression of wild type Drosophila Lamin C caused no overt phenotype. By contrast, larval muscle-specific expression of a truncated form of Lamin C lacking the N-terminal head (Lamin C DeltaN) caused muscle defects and semi-lethality, with adult 'escapers' possessing malformed legs. The leg defects were due to a lack of larval muscle function and alterations in hormone-regulated gene expression. The consequences of Lamin C association at a gene were tested directly by targeting a Lamin C DNA-binding domain fusion protein upstream of a reporter gene. Association of Lamin C correlated with localization of the reporter gene at the nuclear periphery and gene repression. These data demonstrate connections among the Drosophila A-type lamin, hormone-induced gene expression and muscle function.

Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. There have been increasing reports of human cardiomyopathies associated with abnormal accumulation and aggregation of desmin filaments. Recently identified desmin mutations in humans suffering from skeletal muscle myopathy and cardiomyopathy suggest that these diseases might arise as a consequence of impaired function of desmin filaments. Previous generation of desmin null mice in our laboratory demonstrated that the absence of desmin results in myocyte ultrastructural defects and myocyte cell death leading to fibrosis and calcification of the myocardium. However, the effects that these defects have on cardiac function were not addressed. To further our understanding of desmin function in vivo, and in order to address the direct involvement of desmin in cardiomyopathy, we investigated the effect of the absence of desmin on myocardial mass, myocyte size and shape, changes in gene expression and cardiac systolic and diastolic function in mice. Morphometric characterization of isolated cardiomyocytes demonstrated a 24% increase in cell volume in the desmin null mice, solely due to an increase in transverse section area, suggesting for the first time that mice lacking the intermediate filament protein desmin develop concentric cardiomyocyte hypertrophy. This type of hypertrophy was accompanied by induction of embryonic gene expression and later by ventricular dilatation, and compromised systolic function. These results demonstrate that desmin is essential for normal cardiac function, and they suggest that the absence of an intact desmin filament system, rather than accumulation of the protein, may be responsible for the pathology seen in some of the desmin associated cardiomyopathies.

Neuronal intermediate filament (IF) inclusion disease (NIFID) is a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal, and extrapyramidal signs. Pathologically, in affected areas, there is neuronal loss, astrocytosis, and neuronal intracytoplasmic aggregates of abnormal neuronal IFs that contain neither tau nor alpha-synuclein. Thus, to characterize the neuronal IF protein profile of inclusions in NIFID, immunohistochemistry (IHC) was performed on 10 cases of NIFID, four normal aged controls (NL), and two cases of Alzheimer's disease (AD) using a panel of anti-neuronal IF proteins. Immunoelectron microscopy was performed on selected cases and frozen tissue from the frontal lobe of four cases was used for biochemical studies including sequential extractions and Western blotting. Based on these studies, we report here for the first time that alpha-internexin, a neuronal IF protein, is present within the inclusions of NIFID as are all three neurofilament subunits: heavy, medium, and light. Thus, all class IV neuronal IF proteins are present within the pathological inclusions of this disease. Biochemistry revealed that IF aggregates were soluble in sodium dodecyl sulfate (SDS) and no post-translational modification was detected when compared with Alzheimer's disease or aged control brains. Hence, we conclude that NIFID is characterized by the pathological cytoplasmic aggregation of all class IV neuronal IF proteins in brain. The discovery of alpha-internexin in the cytoplasmic inclusions implicates novel mechanisms of pathogenesis in NIFID and other neurological diseases with pathological accumulations of IFs.

The 19-kDa protein encoded within the adenovirus E1B gene is essential for transformation by adenovirus and for proper regulation of viral early gene transcription. In order to investigate the biological function of the 19-kDa E1B protein, vectors were constructed to produce the 19-kDa protein in mammalian cells under the direction of heterologous promoters. Surprisingly, during transient expression, the E1B 19-kDa protein specifically associated with and disrupted the organization of intermediate filaments and the nuclear lamina, without disturbing the organization of other cytoskeletal networks. These results directly demonstrate an effect of a viral transforming protein on the cytoskeleton and suggest a role for intermediate filaments and the nuclear lamina in modulation of viral gene expression and the process of oncogenic transformation.

The association of keratin mutations with genetic skin fragility disorders is now one of the best-established examples of cytoskeleton disorders. It has served as a paradigm for many other diseases and has been highly informative for the study of intermediate filaments and their associated components, in helping to understand the functions of this large family of structural proteins. The keratin diseases have shown unequivocally that, at least in the case of the epidermal keratins, a major function of intermediate filaments is to provide physical resilience for epithelial cells. This review article reflects on the variety of phenotypes arising from mutations in keratins and the reasons for this variation.

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

The intermediate filament glial fibrillary acidic protein (GFAP) is a classic marker for several types of glial cells, including astrocytes and nonmyelinating Schwann cells. The pattern of expression of GFAP in the postnatal murine inner ear, from postnatal day 3 (P3) to P38, was studied by anti-GFAP immunostaining in wild-type mice as well as in two lines of transgenic mice expressing either beta-galactosidase (LacZ) or green fluorescent protein (GFP) under the control of the GFAP promoter. Analysis of protein and promoter activity shows that several classes of supporting cells in the sensory epithelia, as well as Schwann cells and satellite cells express GFAP. Early after birth, all cochlear supporting cells express GFAP, in a gradient decreasing in intensity from base to apex. After P15, GFAP expression in the organ of Corti is mostly restricted to supporting cells of the inner hair cell area (i.e., inner border and inner phalangeal cells) and outer hair cell area (i.e., Deiters' cells). A small population of limbic cells also showed expression in a base-to-apex gradient. In the vestibular organs, high expression was detected in supporting cells in extrastriolar regions of the utricular macula and in the canal ampullae, with weaker staining in the saccular macula. These results suggest that supporting cells of the inner ear have important similarities to glial cells and may play roles similar to those of astrocytes or Schwann cells in supporting the normal development and maintenance of neurons and sensory cells of the inner ear.

Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

The desmosome junction is an important component in the cohesion of epithelial cells, especially epidermal keratinocytes. To gain insight into the structure and function of desmosomes, their morphogenesis has been studied in a primary mouse epidermal (PME) cell culture system. When these cells are grown in approximately 0.1 mM Ca2+, they contain no desmosomes. They are induced to form desmosomes when the Ca2+ level in the culture medium is raised to approximately 1.2 mM Ca2+. PME cells in medium containing low levels of Ca2+, and then processed for indirect immunofluorescence using antibodies directed against desmoplakins (desmosomal plaque proteins), display a pattern of discrete fluorescent spots concentrated mainly in the perinuclear region. Double label immunofluorescence using keratin and desmoplakin antibodies reveals that the desmoplakin-containing spots and the cytoplasmic network of tonofibrils (bundles of intermediate filaments [IFB]) are in the same juxtanuclear region. Within 1 h after the switch to higher levels of Ca2+, the spots move toward the cell surface, primarily to areas of cell-cell contact and not to free cell surfaces. This reorganization occurs at the same time that tonofibrils also move toward cell surfaces in contact with neighboring cells. Once the desmoplakin spots have reached the cell surface, they appear to aggregate to form desmosomes. These immunofluorescence observations have been confirmed by immunogold ultrastructural localization. Preliminary biochemical and immunological studies indicate that desmoplakin appears in whole cell protein extracts and in Triton high salt insoluble residues (i.e., cytoskeletal preparations consisting primarily of IFB) prepared from PME cells maintained in medium containing both low and normal Ca2+ levels. These findings show that certain desmosome components are preformed in the cytoplasm of PME cells. These components undergo a dramatic reorganization, which parallels the changes in IFB redistribution, upon induction of desmosome formation. The reorganization depends upon both the extracellular Ca2+ level and the establishment of cell-to-cell contacts. Furthermore, the data suggests that desmosomes do not act as organizing centers for the elaboration of IFB. Indeed, we postulate that the movement of IFB and preformed desmosomal components to the cell surface is an important initiating event in desmosome morphogenesis.

Since their discovery, the function of intermediate filaments (IFs) has remained obscure. In skin, epidermal cells have extensive cytoskeletal architectures of IFs, composed of type I and type II keratin heterodimers. Clues to possible functions of these proteins have come from recent studies showing that several autosomal-dominant, blistering skin disorders are caused by defects in genes that encode epidermal keratins. These diseases all exhibit cell degeneration and keratin network perturbations in cells that express the particular mutant keratin gene. However, it is not clear from these studies whether cytolysis arises from the presence of large insoluble keratin aggregates that compromise cellular physiology or from the absence of an extensive keratin filament network, which jeopardizes mechanical integrity. We report here the analysis of an extremely rare case of severe recessive epidermolysis bullosa simplex (EBS), where the patient lacks a discernible keratin filament network in basal epidermal cells. Genetic analyses revealed a homozygous point mutation that yielded a premature termination codon in the major basal type I keratin gene and caused complete ablation of K14. The consanguineous parents were normal, each harboring one copy of the null K14 mutation. Analysis of cultured keratinocytes enabled us to document that the loss of K14 is not compensated for by the up-regulation of any other type I keratin. When taken together with the in vivo studies showing the presence of cell fragility generated from the lack of an extensive basal keratin network, these findings provide the first clear demonstration of loss of function associated with the absence of an IF protein in vivo.

We have traced the evolutionary origins of keratin-like sequences to the genomes of lower eukaryotes. The proteins encoded by these genes have evolved to form the intermediate filaments that comprise the backbone of vertebrate skin cells. Two related but distinct types of keratins encoded by two separate multigene subfamilies are expressed in the epidermal keratinocytes of vertebrate species from fish to human. Both at the level of protein and at the level of DNA, these two classes of keratins are coordinately conserved throughout vertebrate evolution, indicating the central role that both types of keratins must play in the assembly and structure of the 8-nm filament.

The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 microM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P< or =0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t1/2) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 microM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.