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). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E–N-cadherin heterodimers and with the plaque proteins α- and β-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the “multimodulator” scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs.German-Israeli Foundation for Scientific Research and Development (GIF grant I-1098-43.11/2010

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

Contradicting the “cadherin switch” model, mixed E-cadherin–N-cadherin heterodimeric adherens junctions are prevalent in a variety of endodermal cells and endoderm-derived tumors

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., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or “desmosome-like” junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and “desmosome-like” junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the “areae adhaerentes”, i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5–7 nm internal diameter (“cribelliform junctions”). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.German-Israeli Foundation for Scientific Research and Development (GIF grant I-1098-43.11/2010

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. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker

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. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation

Lipid Droplets, Perilipins and Cytokeratins – Unravelled Liaisons in Epithelium-Derived Cells

<div><p>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 <i>Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5)</i>. Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron– in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.</p></div

Summary scheme and brief description of stimulation methods used for adipose conversion and for the generation of different, distinct types of lipid droplets.

<p>Major treatments and involved PLIN proteins are shown. Preadipocytes containing many small LDs are <u>not</u> differentiated for several weeks with AIM containing media as conventionally described (top row; cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386.s001" target="_blank">Fig. S1</a>), but <u>only</u> very briefly (1–3 days), giving rise to “Adipocytes” (boxed area). Additional, short OA-treatment leads to “OA-Adipocytes” (boxed area, right side). Treatment with OA only - without AIM stimulation - leads to “OA-Preadipocytes” (bottom). Note the huge heterogeneity of sizes and colors of LDs seen within “OA-Adipocytes”. LDs are endogenously generated at the endoplasmic reticulum and stained positively for perilipin (“Endogenous-LDs”, red). Other LDs are obtained from the exogenous uptake of OA and stained positively for adipophilin, TIP47 and S3-12 (“Exogenous-LDs”, green). Merged LDs by fusion and mixed-type expression are seen by yellow color. The backway arrows indicate possible routes of LDs during lipolysis.</p

<i>Immunoelectron microscopic localization of perilipin in briefly AIM-stimulated human preadipocytes.</i>

<p>(<b>a</b>) Shown are groups of LDs positive for perilipin by nanogold-label and silver enhancement. Localization was with mab Peri112.17. The LDs are seen closely associated and anchored with IF bundles. (<b>b</b>) Enlarged perilipin labeling with two small LDs (approximately 300–400 nm in diameter) approaching a big LD (approximately 2.0–2.5 µm in diameter) for combining and coalescence. Bundles of intermediate-sized filaments are associated to grains of the immunolabeled perilipin (arrows). Bars: 0.50 µm.</p

Immunoelectron microscopic localization of adipophilin in briefly AIM-stimulated and OA-treated human preadipocytes.

<p>(a) Cells reveal besides perilipin-positive also adipophilin-positive LDs. Localization was performed with mab AP125. A few positive, mostly smaller LDs are seen as well as plenty of non-labeled, larger LDs. Almost all LDs are closely associated and anchored with IF bundles. (b) A small, strongly immunolabeled LD is seen approaching a big, scarcely labeled LD. These LDs are obviously at the rim of combining. (c,d) Enlargements with details of filament attachment and immunolabeling sites which were highlighted by arrows (vimentin IFs) and arrowheads (adipophilin immunoreaction). Bars: a,b: 1 µm; c,d: 0.50 µm.</p