The exact sites, structures, and molecular mechanisms of interaction between junction organizing zona occludence protein 1 (ZO-1) and the tight junction protein occludin or the adherens junction protein ␣-catenin are unknown. Binding studies by surface plasmon resonance spectroscopy and peptide mapping combined with comparative modeling utilizing crystal structures led for the first time to a molecular model revealing the binding of both occludin and ␣-catenin to the same binding site in ZO-1. Our data support a concept that ZO-1 successively associates with ␣-catenin at the adherens junction and occludin at the tight junction. Strong spatial evidence indicates that the occludin C-terminal coiled-coil domain dimerizes and interacts finally as a four-helix bundle with the identified structural motifs in ZO-1. The helix bundle of occludin 406 -521 and ␣-catenin Different junctional complexes such as adherens junctions and, in specialized tissues, tight junctions, gap junctions, and desmosomes connect cells in multicellular organisms. TJ 1 seal the most apical-lateral parts of cells and constitute a diffusion barrier for the paracellular flow of molecules and serve as a fence between the apical and basolateral membrane compartment (1). In contrast, AJ play important roles in cell adhesion, migration, morphogenesis, and proliferation. AJ represent Ca 2ϩ -dependent cell-cell contacts localized basolateral of TJ, where transmembrane proteins of the cadherin family mediate adhesion. Assembly of AJ is a prerequisite for the formation of TJ and desmosomes (2). ␤-and ␣-catenin bind to the cadherin cytoplasmic domain and link the cadherin-catenin complex to the F-actin cytoskeleton. An important scaffolding protein in cell-cell contacts is the zona occludens protein 1. ZO-1 is a membrane associated guanylate kinase homologue protein composed of the following domains: three PDZ (PSD95/Dlg/ZO-1), a SH3, a GuK (3), an actin binding region (4), and a ZU5 (ZO-1 and Unc5-like netrin receptor domain) according SMART (Simple Modular Architecture Research Tool (smart.embl-heidelberg.de) data base (5)). In non-epithelial cells ZO-1 is a major component of AJ, whereas in epithelial cells it is localized at TJ by directly binding to claudins (6). Occludin, one of the transmembrane proteins of TJ, is a multiphosphoprotein involved in regulation of TJ (7). It has four transmembrane domains with two extracellular loops and a cytosolic N and C terminus. A sequence of 244 amino acids in human ZO-1 containing the GuK domain and an acidic region C terminus to GuK binds to the complete intracellular C-terminal tail of chicken occludin (8).ZO-1 also binds to the AJ protein ␣-catenin (9) and to connexins in gap junctions (10), indicating a general scaffolding function of ZO-1 in junctional complexes. ␣-Catenin consists of several four-helix-bundle domains (vinculin homology domains, VH1-3) and binds ␤-catenin via an intermolecular helix bundle mechanisms within the E-cadherin-catenin complex at the intracellular side of AJ, where one helix of ...

OPA1, a nuclear encoded mitochondrial protein causing autosomal dominant optic atrophy, is a key player in mitochondrial fusion and cristae morphology regulation. In the present study, we have compared the OPA1 transcription and translation products of different mouse tissues. Unlike in humans, we found only two exons (4b and 5b) to be involved in alternative splicing. The relative abundance of the resulting four different splice variants is tissue-dependent. Proteolytic cleavage by mitochondrial processing peptidase generates two long forms, isoforms 1 and 7, which lead to three short forms representing the end products after further proteolytic processing. In contrast, isoforms 5 and 8 are directly processed into their corresponding short forms. Short form 1 molecules form 184 kDa dimers, whereas all other isoforms contribute to 285 kDa complexes. Coiled-coil domains of the OPA1 protein specifically homo-associate and may be involved in the formation of these complexes. Furthermore, the region encoded by exon 5b inhibits the self-association of coiled-coil domain-I. Finally, our data pinpoint isoform 1 as the, by far, most abundant isoform in the nervous tissue. We postulate that manipulation of isoform 1 protein levels in relation to the other isoforms induces changes in the mitochondrial network in the cell and therefore, mutations affecting the level of functional isoform 1 could lead to devastating effects on retinal ganglion cells.

Two is better than one: Coiled‐coil sequence motifs are versatile protein–protein interaction modules. Single and double substitutions of the GCN4 coiled coil (1, 2, and 3) showed that the core positions a and d in the third heptad act as a switch for the transition from the native homodimeric to homotrimeric structures. This creates a synthetic three‐membered association network with monomeric, homotrimeric, and heterodimeric coiled‐coil states.

The standard methods currently used to read out microarrays are fluorescent and chemiluminescent imaging techniques. These methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected. A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.

No abstract