Ralf Zimbelmann

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 α‐helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4–2.3 Å resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic α‐helix, which is compatible with a coiled‐coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer–dimer interactions during IF assembly. The 2B segment reveals a double‐stranded coiled coil, which unwinds near residue Phe351 to accommodate a ‘stutter’. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.

Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA.

Synaptophysin is a major glycoprotein of Mr approximately 38,000 (in deglycosylated form: Mr approximately 34,000) characteristic of a certain class of small (30‐80 nm diameter) neurosecretory vesicles, including presynaptic vesicles, but also vesicles of various neuroendocrine cells of both neuronal and epithelial phenotype. Using synaptophysin‐specific antibodies we have isolated cDNA clones from rat nervous tissue libraries, which identify an approximately 2.5‐kb mRNA in rat and human cells, including neuroendocrine tumours, that contains a reading frame for a polypeptide of 307 amino acids with a total mol. wt of 33 312. The deduced amino acid sequence, which was partly confirmed by comparison with sequences of two tryptic peptides obtained from purified synaptophysin, revealed four hydrophobic regions of 24 amino acids each, which are characterized, according to conformation prediction analyses, by marked alpha‐helicity. The sequence shows a single potential N‐glycosylation site, which is assigned to the vesicle interior, and a carboxy‐terminal tail of 89 amino acids which contains glycine‐rich tetrapeptide repeats, the epitope of monoclonal antibody SY38, and a number of collagenase‐sensitive sites accessible on the surface of the intact vesicles. These features suggest that the polypeptide spans the vesicle membrane four times, with both N and C termini located on the outer, i.e. cytoplasmic, surface of the vesicles.

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

Abstract. 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. This reflects the splicing of the 21 aa-encoding exon 7 of the human plakophilin-1 gene and that each mRNA splice form can occur in two polyadenylation forms of 2.7 kb and 5.3 kb. Antibodies recognizing either isoform and/or others that are specific for the exon-encoded sequence of form 1b have allowed, in combination with immunolocalization protocols minimizing losses of diffusible proteins, the detection of both isoforms in the nucleoplasm of diverse kinds of cultured cells and tissues, including desmosome-forming cells as well as cells that never form desmosomes. The protein has also been identified in manually isolated nuclei (germinal vesicles) of Xenopus laevis oocytes. Plakophilin 1a accumulates in nuclei as shown by suitable immunolocalization protocols and upon overexpression following transfection with cDNAs, but is also located in desmosomes of stratified and complex epithelia. By contrast, isoform 1b has been found exclusively in nuclei, even in cells connected by desmosomes immunostained with plakophilin 1a-reactive antibodies. We conclude that plakophilins 1a and 1b are constitutive nuclear proteins encoded by the same gene, which is not expressed in relation to epithelial differentiation pathways, whereas the additional appearance of plakophilin 1a in desmosomal plaques of stratified and complex epithelia is regulated by an as yet unknown mechanism of differentiation-dependent topogenic recruitment. Possible functions of plakophilins are discussed in relation to recent reports of the involvement of other members of the armadillo/plakoglobin multigene family of proteins in cell surface-gene regulation signalling pathways.

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. Adipophilin and TIP47 are expressed in LDs of vitamin A‐storing hepatic stellate cells and additionally in LDs of steatotic hepatocytes. Perilipin, which was thought to be characteristic for LDs of adipocytes and steroidogenic cells, becomes de novo expressed in hepatocytes of human steatotic liver. Perilipin splice variant A was found in human steatotic hepatocytes by biochemical, molecular biological, and immunohistochemical methods. Its association with LDs is different from TIP47 and adipophilin, and depends on size and localization of the LDs, suggesting that the different PAT proteins play specific roles during maturation of LDs. (HEPATOLOGY 2008.)

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. We have comprehensively investigated PAT-expression in neoplastic steatogenesis as well as in respective normal tissues with immunohistology and electron microscopy as well as protein biochemical and molecular biological methods. By staining for PAT-proteins, we found lipid droplet accumulation to be a frequent phenomenon of carcinoma cells. Although adipophilin and TIP47 stained almost ubiquitously the rim of lipid droplets in various tumor types, especially those with clear cell phenotype, perilipin was restricted to lipid droplets of hepatocellular adenoma and carcinoma, sebaceous adenoma and carcinoma, and lipomatous tumors. In hepatocellular carcinoma, perilipin, adipophilin, and TIP47 were coexpressed, and showed regional heterogeneity with a predominantly mutually exclusive localization pattern. In step-wise carcinogenesis, adipophilin expression correlated with the proliferation rate and was upregulated during early tumorigenesis, whereas perilipin was often lost during hepatocarcinogenesis. In conclusion, expression analysis of PAT-proteins showed that by far more carcinomas contain (PAT-positive) lipid droplets than expected by conventional light microscopy. PAT-proteins, such as perilipin, are differentially expressed in different tumor types and thus may support diagnostic considerations. Because inhibition of lipogenesis has been shown to exert antineoplastic effects, PAT-proteins may represent targets for interventive strategies.

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.

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.

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. In biochemical analyses, rigorous immunoprecipitation experiments have revealed N‐cadherin, desmoplakin, desmoglein‐2, plakophilin‐2, plakoglobin and plectin as very stably bound complex partners. We conclude that myozap is a general component of cell–cell junctions not only in the myocardium but also in diverse endothelia of the blood and lymph vascular systems of adult mammals, suggesting that this protein not only serves a specific role in the heart but also a broader set of functions in the vessel systems. We also propose to use myozap as an endothelial cell type marker in diagnoses.

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). 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.

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. In all these cells, LUMA has not been detected in the nuclear envelope. Surprisingly, under certain conditions, similar CJ localizations have also been seen with some antibodies commercially available for some time. The identification of LUMA as a plaque component of myocardiac CJs leads to reconsiderations of the molecular composition and architecture, the development, the functions, and the pathogenic states of CJs and IDs. These findings now also allow the general conclusion that LUMA has to be added to the list of mutations of cardiomyocyte junction proteins that may be involved in cardiomyopathies.