Formation and degradation of lipid droplets in human adipocytes and the expression of aldehyde oxidase (AOX).

Lipid droplet (LD) binding proteins in mammary glands and in adipocytes were previously compared and striking similar sets of these specific proteins demonstrated. Xanthine oxidoreductase (XOR) together with perilipins and the lactating mammary gland protein butyrophilin play an important role in the secretion process of LDs into milk ducts. In contrast, in adipose tissue and in adipocytes, mainly perilipins have been described.

The cell-cell junctions of mammalian testes. III. Absence of an endothelial cell layer covering the peritubular wall of the seminiferous tubules-an immunocytochemical correction of a 50-year-old error in the literature.

In the molecular biological and ultrastructural studies of the peritubular wall cells encasing the seminiferous tubules of mammalian testes, we found it necessary to characterize the outermost cell layer bordering on the interstitial space in detail. For half a century, the extremely thin cells of this monolayer have in the literature been regarded as part of a lymphatic endothelium, in particular in rodents. However, our double-label immunofluorescence microscopical results have shown that in all six mammalian species examined, including three rodent ones (rat, mouse, guinea pig), this classification is not correct: the very attenuated cells of this monolayer are not of lymphatic endothelial nature as they do not contain established endothelial marker molecules.

Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells.

Proteins of the striatin family (striatins 1-4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs).

The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures.

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the "blood-testis barrier", formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e.

Protein LUMA is a cytoplasmic plaque constituent of various epithelial adherens junctions and composite junctions of myocardial intercalated disks: a unifying finding for cell biology and cardiology.

In a series of recent reports, mutations in the gene encoding a protein called LUMA (or TMEM43), widely speculated to be a tetraspan transmembrane protein of the nuclear envelope, have been associated with a specific subtype of cardiomyopathy (arrhythmogenic cardiomyopathies) and cases of sudden death. However, using antibodies of high specificity in immunolocalization experiments, we have discovered that, in mammals, LUMA is a component of zonula adhaerens and punctum adhaerens plaques of diverse epithelia and epithelial cell cultures and is also located in (or in some species associated with) the plaques of composite junctions (CJs) in myocardiac intercalated disks (IDs). In CJs, LUMA often colocalizes with several other CJ marker proteins.

On the formation of lipid droplets in human adipocytes: the organization of the perilipin-vimentin cortex.

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12.

Lipid droplets, perilipins and cytokeratins--unravelled liaisons in epithelium-derived cells.

Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5).

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells.

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts.

Protein myozap--a late addition to the molecular ensembles of various kinds of adherens junctions.

The protein myozap, a polypeptide of 54 kDa, has recently been identified as a component of the cytoplasmic plaques of the composite junctions (areae compositae) in the myocardiac intercalated disks and of the adherens junctions (AJs) in vascular endothelia. Now we report that using very sensitive new antibodies and drastic localization methods, we have also identified this protein as a component of the AJ plaques in simple and complex epithelia, in the adluminal cell layer of the transitional epithelium of the urinary tract and in certain cell layers of diverse stratified epithelia, including gingiva, tongue, pharynx and esophagus, cervix, vagina and epidermis. Myozap has not been identified in desmosomal and tight junction plaques.

E-N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells.

Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this "cadherin switch" hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them.

The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems.

Recently the protein myozap, a 54-kD polypeptide which is not a member of any of the known cytoskeletal and junctional protein multigene families, has been identified as a constituent of the plaques of the composite junctions in the intercalated disks connecting the cardiomyocytes of mammalian hearts. Using a set of novel, highly sensitive and specific antibodies we now report that myozap is also a major constituent of the cytoplasmic plaques of the adherens junctions (AJs) connecting the endothelial cells of the mammalian blood and lymph vascular systems, including the desmoplakin-containing complexus adhaerentes of the virgultar cells of lymph node sinus. In light and electron microscopic immunolocalization experiments we show that myozap colocalizes with several proteins of desmosomal plaques as well as with AJ-specific transmembrane molecules, including VE-cadherin.

Lipid droplet-associated PAT-proteins show frequent and differential expression in neoplastic steatogenesis.

In many human cancers, lipogenic pathways are activated; in some tumors, such as hepatocellular carcinoma, this is reflected by the presence of visible lipid droplets. Yet, the biology of steatogenesis in malignant tumors is largely unknown. We have recently shown that lipid droplet-associated proteins of the PAT-family, named after their constituents perilipin (perilipin 1), adipophilin (perilipin 2), and TIP47 (perilipin 3) are differentially expressed in hepatic steatogenesis.

Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis.

Fatty change (steatosis) is the most frequent liver pathology in western countries and is caused by a broad range of disorders such as alcohol abuse and metabolic syndrome. The surface layer of lipid droplets (LDs) contains members of a protein family that share homologous sequences and domains, the so-called PAT proteins, named after their constituents, perilipin, adipophilin, and TIP47. We characterized the LD-associated proteins in normal and diseased liver connected with LD accumulation.

Novel actin-related proteins Arp-T1 and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head.

The calyx is a large cytoskeletal component of the perinuclear theca of the mammalian sperm head, displaying remarkable morphological interspecies differences, which is biochemically characterized by resistance to high ionic strength and detergents and by a special protein composition, including the basic proteins calicin, cylicin I and II, and two major actin-capping proteins. In our calyx preparations from bull spermatozoa we have noted two major acidic components which upon partial amino acid sequencing have been identified as novel members of the subfamily of actin-related proteins (Arps). Antibodies raised against the corresponding human proteins, termed Arp-T1 and Arp-T2, have been used to detect the proteins by immunoblotting and immunofluorescence microscopy, demonstrating their specific synthesis in the testis, late in spermatid differentiation, and their localization in the calyx.

Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly.

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive alpha-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly.

Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments.

Intermediate filaments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded alpha-helical coiled coil. Filament formation proceeds via a specific multi-step association of the dimers into the unit-length filaments, which subsequently anneal longitudinally and finally radially compact into mature filaments.

Plakophilins 1a and 1b: widespread nuclear proteins recruited in specific epithelial cells as desmosomal plaque components.

The cytokeratin-binding, basic 80.5 kDa polypeptide plakophilin 1 ("band 6 protein" of bovine muzzle desmosome fractions) has originally been described as a single molecular species, localized to desmosomal plaques of certain cell types, mostly stratified squamous epithelia and complex epithelia. We now report that this protein exists in at least two different isoforms: 726 amino acids (aa), plakophilin 1a; and 747 aa, plakophilin 1b.

CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head.

In the mammalian sperm head, the nucleus is tightly associated with the calyx, a cell type-specific cytoskeletal structure. Previously, we have identified and characterized some basic proteins such as calicin and cylicins I and II as major calyx components of bovine and human spermatids and spermatozoa. Surprisingly we have now discovered another calyx constituent which by amino acid sequencing and cDNA cloning was recognized as a novel isoform of the widespread beta subunit of the heterodimeric actin-binding "capping protein" (CP).

The protein complexity of the cytoskeleton of bovine and human sperm heads: the identification and characterization of cylicin II.

The cytoskeletal calyx of mammalian sperm heads surrounding the basolateral part of the nucleus contains two kinds of basic proteins: Calicin, a polypeptide of approximate M(r) 60,000 as estimated from SDS-PAGE, and the group of the very basic cylicins (pI > 10.0), formerly designated as "multiple-band polypeptides." Recently, bovine cylicin I has been cDNA cloned and identified as a new type of a cytoskeletal protein, which contains numerous Lys-Lys-Asp tripeptides accumulated in nine central repetitive units predicted to form alpha-helices.

Cell type-specific desmosomal plaque proteins of the plakoglobin family: plakophilin 1 (band 6 protein).

Desmosomes represent a special type of the plaque-bearing adhering junctions, characteristic of certain pathways of cell differentiation, which compositionally are not identical in the various kinds of desmosome-forming cells. While all desmosomes contain the cytoplasmic plaque proteins desmoplakin I and plakoglobin, they can vary in their specific complement of desmosomal cadherins and by the presence of additional plaque proteins. We have raised monoclonal antibodies recognizing one such 'accessory' plaque protein, the cytokeratin-binding, basic protein plakophilin 1, originally introduced as 'band 6 protein' or 'polypeptide D6', which is an abundant desmosomal component in certain epithelia.

Desmosomes and cytoskeletal architecture in epithelial differentiation: cell type-specific plaque components and intermediate filament anchorage.

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.

The human gene encoding cytokeratin 20 and its expression during fetal development and in gastrointestinal carcinomas.

The differentiation of the predominant cell types of the mucosal epithelium of the mammalian gastrointestinal tract is characterized by increasing amounts of an intermediate-sized filament (IF) protein designated cytokeratin (CK) 20 which is a major cellular protein of mature enterocytes and goblet cells. Here we report the isolation of the human gene encoding CK 20, its complete nucleotide sequence and the amino acid sequence deduced therefrom that identifies this polypeptide (mol. wt.

Identification of plakoglobin in oocytes and early embryos of Xenopus laevis: maternal expression of a gene encoding a junctional plaque protein.

We have isolated a cDNA encoding the junctional plaque protein plakoglobin of Xenopus laevis and determined its amino acid sequence. Comparisons with sequences of related proteins of the same and other species revealed that in Xenopus plakoglobin and beta-catenin are two different proteins, encoded by separate genes, that both genes are expressed in embryogenesis, and that the amphibian plakoglobin is more closely related to the human plakoglobin than to beta-catenin of the same species. Using this cDNA as a probe, we also show that plakoglobin mRNA is produced and stored in Xenopus oocytes and eggs.

Complexity and expression patterns of the desmosomal cadherins.

Desmosomes are intercellular junctions that contain two major kinds of transmembrane glycoproteins, desmoglein and desmocollins I and II, involved in cell-cell adhesion. Recent sequence analyses have shown that both desmosomal glycoproteins belong to the larger cadherin family of cell adhesion molecules, in which they represent two different subgroups characterized by their specific sequence and topogenesis. In analyses of cDNA sequences and Northern blot experiments we have now found that both desmoglein and desmocollins are not unique gene products but occur in different subtypes produced from different genes.

Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification of a second type of desmoglein gene.

The amino acid sequence of the precursor to desmoglein, a major desmosomal cadherin, has been determined from a cDNA clone from bovine muzzle epithelium, and the transcription start site, i.e., the beginning of the approximately 7.