We have examined the role of cell-cell and cell-extracellular matrix (ECM) interactions during mesoderm differentiation and migration at the primitive streak of the mouse embryo with the use of function-perturbing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured on a fibronectin substratum in serum-free, chemically defined medium. After 16-24 hours in culture, cells in explants of epiblast exhibited the typical close-packed morphology of epithelia, and the tissue remained as a coherent patch of cells that were shown to express transcripts of the cytokeratin Endo B by in situ analysis. In contrast, cells in explants of primitive streak mesoderm exhibited a greatly flattened, fibroblastic morphology, did not express Endo B transcripts, and migrated away from the center of the explant. As epiblast cells in vivo undergo the epithelial-mesenchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecule E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadherin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mesenchymal morphology; they were also induced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluorescence studies, anti-E-cadherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in vivo, and they also ceased to express E-cadherin itself. In contrast, these cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoderm at this stage of development. As epiblast cells differentiate into mesoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive interactions between primitive streak tissues and extracellular matrix (ECM) components. Epiblast explants adhered well to fibronectin, more poorly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furthermore, epiblast, but not mesoderm, displayed an anchorage-dependent viability in culture. After anti-E-cadherin treatment, epiblast cells that had assumed the mesenchymal morphology did attach to vitronectin, another characteristic shared with primitive streak mesoderm.(ABSTRACT TRUNCATED AT 400 WORDS)

A genome wide scan in a consanguineous family of Indian origin with autosomal recessive developmental cataracts was performed by two-point linkage analysis with 382 microsatellite markers. It showed linkage to markers on chromosome 20q, between D20S852 and D20S912, with a maximum lod score of 5.4 obtained with D20S860. This region encompasses the beaded filament structural protein 1 (BFSP1) gene. Direct sequencing revealed a 3343 bp deletion including exon 6 (c.736-1384_c.957-66 del) predicted to result in a shift of the open reading frame. This mutation was absent in 50 control individuals from south India. This is the first report of a mutation in the BFSP1 gene associated with human inherited cataracts. This further increases the genetic heterogeneity of inherited cataracts and provides clues as to the importance of BFSP1 in the cell biology of intermediate filaments and their role in the eye lens.

The skin is essential for survival and protects our body against biological attacks, physical stress, chemical injury, water loss, ultraviolet radiation and immunological impairment. The epidermal barrier constitutes the primordial frontline of this defense established during terminal differentiation. During this complex process proliferating basal keratinocytes become suprabasally mitotically inactive and move through four epidermal layers (basal, spinous, granular and layer, stratum corneum) constantly adapting to the needs of the respective cell layer. As a result, squamous keratinocytes contain polymerized keratin intermediate filament bundles and a water-retaining matrix surrounded by the cross-linked cornified cell envelope (CE) with ceramide lipids attached on the outer surface. These cells are concomitantly insulated by intercellular lipid lamellae and hold together by corneodesmosmes. Many proteins essential for epidermal differentiation are encoded by genes clustered on chromosomal human region 1q21. These genes constitute the 'epidermal differentiation complex' (EDC), which is divided on the basis of common gene and protein structures, in three gene families: (i) CE precursors, (ii) S100A and (iii) S100 fused genes. EDC protein expression is regulated in a gene and tissue-specific manner by a pool of transcription factors. Among them, Klf4, Grhl3 and Arnt are essential, and their deletion in mice is lethal. The importance of the EDC is further reflected by human diseases: FLG mutations are the strongest risk factor for atopic dermatitis (AD) and for AD-associated asthma, and faulty CE formation caused by TG1 deficiency causes life-threatening lamellar ichthyosis. Here, we review the EDC genes and the progress in this field.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Although parietal podocytes along the Bowman's capsule have been described by electron microscopy in the normal human kidney, their molecular composition remains unknown. Ten human normal kidneys that were removed for cancer were assessed for the presence and the extent of parietal podocytes along the Bowman's capsule. The expression of podocyte-specific proteins (podocalyxin, glomerular epithelial protein-1, podocin, nephrin, synaptopodin, and alpha-actinin-4), podocyte synthesized proteins (vascular endothelial growth factor and novH), transcription factors (WT1 and PAX2), cyclin-dependent kinase inhibitor p57, and intermediate filaments (cytokeratins and vimentin) was tested. In addition, six normal fetal kidneys were studied to track the ontogeny of parietal podocytes. The podocyte protein labeling detected parietal podocytes in all of the kidneys, was found in 76.6% on average of Bowman's capsule sections, and was prominent at the vascular pole. WT1 and p57 were expressed in some parietal cells, whereas PAX2 was present in all or most of them, so some parietal cells coexpressed WT1 and PAX2. Furthermore, parietal podocytes coexpressed WT1 and podocyte proteins. Cytokeratin-positive cells covered a variable part of the capsule and did not express podocyte proteins. Tuft-capsular podocyte bridges were present in 15.5 +/- 3.7% of the glomerular sections. Parietal podocytes often covered the juxtaglomerular arterioles and were present within the extraglomerular mesangium. Parietal podocytes were present in fetal kidneys. Parietal podocytes that express the same epitopes as visceral podocytes do exist along Bowman's capsule in the normal adult kidney. They are a constitutive cell type of the Bowman's capsule. Therefore, their role in physiology and pathology should be investigated.

The LMNA gene encodes lamins A and C, two intermediate filament-type proteins that are important determinants of interphase nuclear architecture. Mutations in LMNA lead to a wide spectrum of human diseases including autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), which affects skeletal and cardiac muscle. The cellular mechanisms by which mutations in LMNA cause disease have been elusive. Here, we demonstrate that defects in neuromuscular junctions (NMJs) are part of the disease mechanism in AD-EDMD. Two AD-EDMD mouse models show innervation defects including misexpression of electrical activity-dependent genes and altered epigenetic chromatin modifications. Synaptic nuclei are not properly recruited to the NMJ because of mislocalization of nuclear envelope components. AD-EDMD patients with LMNA mutations show the same cellular defects as the AD-EDMD mouse models. These results suggest that lamin A/C-mediated NMJ defects contribute to the AD-EDMD disease phenotype and provide insights into the cellular and molecular mechanisms for the muscle-specific phenotype of AD-EDMD.

There is an accumulation of evidence in the literature demonstrating the integral role of vimentin intermediate filaments (IFs) in the progression of lung cancers. Vimentin IF proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT), and the metastatic spread of cancer. Specifically, vimentin IFs have been recognized as an essential component regulating EMT, major signal transduction pathways involved in EMT and tumor progression, cell migration and invasion, the positioning and anchorage of organelles, such as mitochondria, and cell-cell and cell-substrate adhesion. In tumorgenesis, vimentin forms a complex with 14-3-3 and beclin 1 to inhibit autophagy via an AKT-dependent mechanism. Vimentin is a canonical marker of EMT, and recent evidence has shown it to be an important regulator of cellular motility. Transcriptional regulation of vimentin through hypoxia-inducible factor-1 may be a potential driver of EMT. Finally, vimentin regulates 14-3-3 complexes and controls various intracellular signaling and cell cycle control pathways by depleting the availability of free 14-3-3. There are many exciting advances in our understanding of the complex role of vimentin IFs in cancer, pointing to the key role vimentin IFs may play in tumor progression.

OBJECTIVE

The goal of this study was to analyze the genetic disorder of a family with cardiomyopathy, skin disorder, and woolly hair.

BACKGROUND

Arrhythmogenic right ventricular dysplasia (ARVD) is a heart muscle disorder causing arrhythmia and sudden cardiac death. We report a patient with familial autosomal recessive ARVD, woolly hair, and a pemphigous-like skin disorder with a new mutation in the desmoplakin gene.

METHODS

Genomic deoxyribonucleic acid was extracted from the patient's blood and 12 first- and second-degree family members, and was amplified by polymerase chain reaction. Linkage analysis with polymorphic microsatellites was performed for 11 genes that code for structural desmosomal proteins. The genetic locus of the disease in this family was mapped to the chromosomal region 6p24 that contains the desmoplakin gene. Exons of the desmoplakin gene were analyzed by single-strand conformational polymorphism and direct sequencing. Confirmation of the mutation was carried out by restriction enzyme analysis.

RESULTS

We identified in the patient a homozygous missense mutation in exon 24 of the desmoplakin gene, leading to a Gly2375Arg substitution in the C-terminal of the protein where the binding site to intermediate filaments is located. Eight of 12 family members without hair or skin abnormalities were heterozygous for this mutation. The remaining 4, as well as 90 unrelated healthy control individuals of the same ethnic origin, were homozygous for the normal allele.

CONCLUSIONS

We have described a new mutation in the desmoplakin gene that causes familial ARVD. These findings suggest that desmosomal proteins play an important role in the integrity and function of the myocardium. Dysfunction of these proteins can lead to the development of cardiomyopathies and arrhythmias.

During spermatogenesis, the movement of germ cells across the seminiferous epithelium is associated with extensive junction restructuring. Yet the underlying mechanism (or mechanisms) that regulates these events is largely unknown. If the molecular architecture of the cell-cell actin-based adherens junction (AJ), such as ectoplasmic specialization (ES) and tubulobulbar complex- two testis-specific AJ types, is known, many functional mechanistic studies can be designed. We thus undertook an investigation to study 3 adaptors in the seminiferous epithelium: zyxin, axin, and Wiskott-Aldrich syndrome protein (WASP). All 3 adaptors were shown to be products of Sertoli and germ cells. Zyxin was shown to be a stage-specific protein that was most prominent during stages V-VII and restricted mostly to pachytene spermatocytes, but it could also be detected at the site of basal and apical ectoplasmic specialization (ES). Zyxin, axin, and WASP were shown to be structurally linked to the N-cadherin/beta-catenin/alpha-actinin/actin complex but not to the nectin-3/afadin or the beta 1-integrin-mediated protein complexes. Interestingly, zyxin, axin, and WASP are also structurally linked to vimentin (an intermediate filament protein) and alpha-tubulin (the subunit of a microtubule), which suggests that they have a role (or roles) in the regulation of the dynamics of the desmosome-like junction and microtubule. These results illustrate that zyxin, axin, and WASP are adaptors in both AJs and intermediate filament-based desmosome-like junctions. This raises the possibility that classic cadherins are also associated with vimentin-based intermediate filaments via these adaptors in the testis. While virtually no N-cadherin was found to associate with vimentin in the seminiferous tubules, it did associate with vimentin when testis lysates were used. Interestingly, about 5% of the E-cadherin associated with vimentin in isolated seminiferous tubules, and about 50% of the E-cadherin in the testis used vimentin as its attachment site. These data suggest that cadherins in the testis, unlike those in other epithelia, use different attachment sites to anchor the cadherin/catenin complex to the cytoskeleton. The levels of zyxin, axin, and WASP were also assessed during AF-2364-mediated AJ disruption of the testis, which illustrated a time-dependent protein reduction that was similar to the trends observed in nectin-3 and afadin but was the opposite of those observed for N-cadherin and beta-catenin, which were induced. Collectively, these results illustrate that while these adaptors are structurally associated with the cadherin/catenin complex in the testis, they are regulated differently.

This literature review reflects current knowledge on the intermediate filament protein nestin, which most authors regard as a marker of "neural stem/progenitor cells." The structural-functional characteristics of nestin and its presence in various central nervous system cells at different stages of ontogenesis in normal and pathological conditions are discussed.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Efforts to understand nuclear organization in plant cells have received little assistance from the better-studied animal nuclei, because plant proteomes do not contain recognizable counterparts to the key animal proteins involved in nuclear organization, such as lamin nuclear intermediate filament proteins. Previous studies identified a plant-specific insoluble nuclear protein in carrot (Daucus carota), called Nuclear Matrix Constituent Protein1 (NMCP1), which contains extensive coiled-coil domains and localizes to the nuclear periphery. Here, we describe a genetic characterization of two NMCP1-related nuclear proteins in Arabidopsis thaliana, LITTLE NUCLEI1 (LINC1) and LINC2. Disruption of either gene caused a reduction in nuclear size and altered nuclear morphology. Moreover, combining linc1 and linc2 mutations had an additive effect on nuclear size and morphology but a synergistic effect on chromocenter number (reduction) and whole-plant morphology (dwarfing). The reduction in nuclear size in the linc1 linc2 double mutant was not accompanied by a corresponding change in endopolyploidy. Rather, the density of DNA packaging at all endopolyploid levels in the linc1 linc2 mutants was increased significantly. Our results indicate that the LINC coiled-coil proteins are important determinants of plant nuclear structure.

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.

Lamins A and C are intermediate filament proteins which polymerize into the nucleus to form the nuclear lamina network. The lamina is apposed to the inner nuclear membrane and functions in tethering chromatin to the nuclear envelope and in maintaining nuclear shape. We have recently characterized a globular domain that adopts an immunoglobulin fold in the carboxyl-terminal tail common to lamins A and C. Using an electrophoretic mobility shift assay (EMSA), we show that a peptide containing this domain interacts in vitro with DNA after dimerization through a disulfide bond, but does not interact with the core particle or the dinucleosome. The covalent dimer binds a 30-40 bp DNA fragment with a micromolar affinity and no sequence specificity. Using nuclear magnetic resonance (NMR) and an EMSA, we observed that two peptide regions participate in the DNA binding: the unstructured amino-terminal part containing the nuclear localization signal and a large positively charged region centered around amino acid R482 at the surface of the immunoglobulin-like domain. Mutations R482Q and -W, which are responsible for Dunnigan-type partial lipodystrophy, lower the affinity of the peptide for DNA. We conclude that the carboxyl-terminal end of lamins A and C binds DNA and suggest that alterations in lamin-DNA interactions may play a role in the pathophysiology of some lamin-linked diseases.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Hemidesmosomes are important adhesion devices found in epithelial cells. They connect the intermediate filament cytoskeleton network with components of the basement membrane zone. 804G cells are an unusual epithelial cell line, since they form bona fide hemidesmosomes when plated on glass or plastic. In this study we tested an hypothesis: that this ability is a consequence of an extracellular component produced by the 804G cells. As probes for our study we generated a rabbit antiserum (J18) and monoclonal antibodies against components of urea-solubilized 804G matrix. Antibodies in the J18 serum recognize major lectin-binding polypeptides of 150, 140 and 135 kDa in the 804G matrix. A monoclonal antibody (5C5) that shows reactivity with the 150 and 135 kDa polypeptides in western immunoblots immunoprecipitates all three molecular mass species, indicating that these polypeptides are part of a matrix complex. Moreover, one, at least, of these matrix elements is immunologically related to laminin, since J18 antibodies selected on fusion protein fragments of a newly characterized laminin variant, laminin B2t (Kallunki et al., J. Cell Biol., 119, 679-694, 1992), react with the 140 kDa polypeptide component of the 804G cell matrix. To undertake functional analyses of 804G matrix, cells of the human epidermal carcinoma line SCC12, which do not assemble bona fide hemidesmosomes in vitro, were cultured on 804G matrix for 24 h and then analysed by confocal immunofluorescence and electron microscopy. In SCC12 cells maintained on 804G cell matrix, hemidesmosomal antigens localize in a distinctive leopard spot pattern that mirrors the distribution of 804G matrix elements. Furthermore, ultrastructural analysis reveals that the 804G cell matrix supports the formation of 'mature' hemidesmosomes by SCC12 cells. Thus 804G cell matrix is a remarkable tool for hemidesmosome studies and it will now be of great importance to determine the exact composition of the 804G matrix, especially its structural and antigenic relationship to laminins.

The distribution and time course of postischemic astrocyte hypertrophy and hyperplasia and the relationship to neuronal viability or necrosis was studied in rats subjected to 30 min of carotid and vertebral artery occlusion followed by reperfusion from 3 h to 5 weeks. Intermediate filaments (IFs) were evaluated by electron microscopy, IF proteins by immunohistochemistry, and astrocyte division by [3H]thymidine uptake. Glial fibrillary acidic protein (GFAP) increased in damaged and nondamaged brain regions by 2 days and was associated with cell enlargement, increases in IF, and transformation of GFAP-negative into GFAP-positive glia. Cell hypertrophy and increased GFAP persisted only in regions of neuronal necrosis whereas the number and size of GFAP-positive astrocytes returned to control levels in nondamaged regions by 2 weeks. Astrocyte hyperplasia was not seen until 3 days and was confined to damaged brain regions. Vimentin-positive astrocytes were numerous by 2 days in damaged brain and remained only in those regions at 5 weeks. The data demonstrate that reactive astrocytosis develops in undamaged brain, but is reversible with prolonged survival, whereas reactive astrocytosis that accompanies structural brain damage persists for prolonged periods and is associated with hyperplasia, as well as hypertrophy. In addition, the results show that astrocyte expression of vimentin is more specific than GFAP in identifying regions of permanent ischemic injury during the early postischemic period.

Meiotic recombination between homologous chromosomes ensures their proper segregation at the first division of meiosis and is the main force shaping genetic variation of genomes. The HOP2 and MND1 genes are essential for this recombination: Their disruption results in severe defects in homologous chromosome synapsis and an early-stage failure in meiotic recombination. The mouse Hop2 and Mnd1 proteins form a stable heterodimer (Hop2/Mnd1) that greatly enhances Dmc1-mediated strand invasion. In order to elucidate the mechanism by which Hop2/Mnd1 stimulates Dmc1, we identify several intermediate steps in the homologous pairing reaction promoted by Dmc1. We show that Hop2/Mnd1 greatly stimulates Dmc1 to promote synaptic complex formation on long duplex DNAs, a step previously revealed only for bacterial homologous recombinases. This synaptic alignment is a consequence of the ability of Hop2/Mnd1 to (1) stabilize Dmc1-single-stranded DNA (ssDNA) nucleoprotein complexes, and (2) facilitate the conjoining of DNA molecules through the capture of double-stranded DNA by the Dmc1-ssDNA nucleoprotein filament. To our knowledge, Hop2/Mnd1 is the first homologous recombinase accessory protein that acts on these two separate and critical steps in mammalian meiotic recombination.

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. However, viral tropism and mechanism of spread in the CNS have not been examined. Here we investigate coxsackievirus B3 (CVB3) tropism and pathology in the CNS of neonatal mice, using a recombinant virus expressing the enhanced green fluorescent protein (eGFP). Newborn pups were extremely vulnerable to coxsackievirus CNS infection, and this susceptibility decreased dramatically by 7 days of age. Twenty-four hours after intracranial infection of newborn mice, viral genomic RNA and viral protein expression were detected in the choroid plexus, the olfactory bulb, and in cells bordering the cerebral ventricles. Many of the infected cells bore the anatomical characteristics of type B stem cells, which can give rise to neurons and astrocytes, and expressed the intermediate filament protein nestin, a marker for progenitor cells. As the infection progressed, viral protein was identified in the brain parenchyma, first in cells expressing neuron-specific class III beta-tubulin, an early marker of neuronal differentiation, and subsequently in cells expressing NeuN, a marker of mature neurons. At later time points, viral protein expression was restricted to neurons in specific regions of the brain, including the hippocampus, the entorhinal and temporal cortex, and the olfactory bulb. Extensive neuronal death was visible, and appeared to result from virus-induced apoptosis. We propose that the increased susceptibility of the neonatal CNS to CVB infection may be explained by the virus' targeting neonatal stem cells; and that CVB is carried into the brain parenchyma by developing neurons, which continue to migrate and differentiate despite the infection. On full maturation, some or all of the infected neurons undergo apoptosis, and the resulting neuronal loss can explain the longer-term clinical picture.

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.

OBJECTIVE

Drusen are variably sized extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. They are commonly found in aged eyes, however, numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration. The purpose of this study was to investigate the impact of drusen on overlying cells of the retina.

METHODS

Tissue containing retina and RPE/choroid was dissected from human donor eyes, embedded in agarose, and sectioned at 100 micro m using a vibratome. Sections were immunostained with a panel of antibodies that labeled glial cells, first-, second-, and third-order retinal neurons and processed for confocal microscopy.

RESULTS

Retinal cells that overlie both soft and hard drusen exhibited numerous structural and molecular abnormalities. Normally detectable only in the outer segments of rod photoreceptors, rod opsin immunolabeling was also observed in the inner segment, cell body, axon, and axon terminal of photoreceptors that overlie drusen. Labeling with this antibody also revealed the deflection and shortening of rod inner and outer segments. Cone photoreceptors displayed similar structural abnormalities, as well as a decrease in cone opsin immunoreactivity. Drusen-associated abnormalities in the synaptic terminals of photoreceptor cells were also observed. In addition, an increase in intermediate filament protein immunoreactivity (vimentin and glial fibrillary acidic protein) was observed within Müller glial cells in areas of retina overlying drusen. Both soft and hard drusen were associated with a similar spectrum of effects in both macular and extramacular regions. Second- and third-order neurons, including bipolar, horizontal, amacrine, and ganglion cells all appeared unaffected. The structural and molecular abnormalities observed in photoreceptors and Müller glial cells were confined to retinal regions directly overlying and immediately adjacent to drusen; more distant retinal regions appeared unperturbed. Remarkably, significant abnormalities were observed over small subclinical drusen.

CONCLUSIONS

Retinal cells overlying both soft and hard drusen exhibit structural and molecular abnormalities indicative of photoreceptor degeneration and Müller glial activation. These abnormalities resemble the degenerative effects common to many forms of retinal degeneration, but are confined to areas directly overlying drusen. This suggests that photoreceptor cell function is compromised as a consequence of drusen formation.