HAV-Peptides Attached to Colloidal Probes Faithfully Detect E-Cadherins Displayed on Living Cells.

Cell adhesion molecules are crucial for a variety of biological processes, including wound healing, barrier formation and tissue homeostasis. One of them is E-cadherin which is generally found at adherent junctions between epithelial cells. To identify this molecule on the surface of cells, E-cadherin mimetic peptides with a critical amino acid sequence of HAV (histidine-alanine-valine) were synthesized and attached to solid-supported membranes covering colloidal probes. Two different functionalization strategies were established, one based on the complexation of DOGS-NTA(Ni) with a polyhistidine-tagged HAV-peptide and the other one relying on the formation of a HAV-lipopeptide using in situ maleimide-thiol coupling. Binding studies were performed to verify the ability of the peptides to attach to the membrane surface. Compared to the non-covalent attachment via the His-tag, we achieved a higher yield by lipopeptide formation. Colloidal probes functionalized with HAV-peptides were employed to measure the presence of E-cadherins on living cells either using video particle tracking or force spectroscopy. Here, human HaCaT cells were examined confirming the specific interaction of the HAV-peptide with the E-cadherin of the cells. Statistical methods were also used to determine the number of single-bond ruptures and the force of a single bond. These findings may be essential for the development of novel biosynthetic materials given their potential to become increasingly relevant in medical applications.

Identification of an aquaculture poriferan "Pest with Potential" and its phylogenetic implications.

Correct identification and classification of sponges is challenging due to ambiguous or misleading morphological features. A particular case is a blue keratose sponge occasionally referred to as the "Blue Photo Sponge" among aquarists, which appears frequently (and in several cases unintended) in private aquaria. This spicule-less species, occasionally specified as Collospongia auris Bergquist, Cambie & Kernan 1990, not only displays a high phenotypic plasticity in growth form and colour, it also proliferates in aquacultures under standard conditions unlike most other sponges. Therefore, this species is regarded as a pest for most aquarists. In turn, the ease of cultivation and propagation in aquacultures qualifies this species as a model organism for a wide array of scientific applications. For these purposes, correct identification and classification are indispensable. We reconstructed ribosomal gene trees and determined this species as Lendenfeldia chondrodes (De Laubenfels, 1954) (Phyllospongiinae), distant to Collospongia auris, and corroborated by skeletal features. Additionally, the resulting phylogeny corroborated major shortcomings of the current Phyllospongiinae classification-its consequences are discussed.

Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification.

In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3'-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI-RNA-ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI-RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI.

Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase-RNA complex.

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI from Thermotoga maritima was cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 102.9, b = 112.8, c = 132.8 Å.