Analysis of the Bin1 SH3 interaction with peptides derived from the hepatitis C virus protein NS5A and c-Myc reveals that NS5A can competitively displace c-Myc in vitro

Severe liver damage like cirrhosis and hepatocellular carcinoma (HCC) can be caused by manifestation of the hepatitis C virus (HCV) infection. Constitutively activated c-Myc oncogene has been shown to contribute to the establishment of HCV-mediated HCC. Interestingly, only one of many isoforms of the tumor suppressor protein Bin1 (bridging integrator 1), Bin1+12A, contains an internal, canonical SH3 binding motif that recognizes its own SH3 domain. This leads to the inability of Bin1+12A to interact with c-Myc. The expression of the Bin1+12A isoform is a main phenotype in malignant melanoma cells. We suggest that also other mechanisms that disturb the interaction of Bin1 and c-Myc might have severe consequences since the latter is tightly regulated in healthy cells. The HCV nonstructural protein 5A (NS5A) plays a key role in virus replication and assembly. NS5A plays an intercepting role in several cellular pathways, which are linked to cell growth, cell cycle control, cell survival, cellular stress response, apoptosis as well as HCC. It is known that NS5A contains a highly conserved canonical, polyproline (PxxP) SH3-binding motif, which is located between its D2 and D3 domains. This PxxP motif was described to interact with the SH3 domain of Bin1. In addition to a biophysical analysis of the canonical binding between Bin1 SH3 and the PxxP motif of NS5A [1], we identified two additional low-affinity binding sites for non-canonical SH3 binding on NS5A [2]. The hypothesis underlying the work presented here is that viral NS5A is able to sequester cellular Bin1 from c-Myc

Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes.

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis

Planning for digitalisation in SMEs using tools of the digital factory

Digitalisation offers ways as well as means for manufacturers to adapt their production systems to handle diversifying and rapidly changing market demands. Yet, small and medium sized enterprises are often overwhelmed by the speed of development of ICT solutions and business schemes. This paper discusses how tools of the Digital Factory enable decision-makers to assess digitalisation measures during the planning process to take full advantage of newly available technologies. Potentials and prerequisites are examined for the application of available tools. They are illustrated considering an in-depth example on the simulation of an automotive supplier’s tool management process

STED super-resolution microscopy of mitochondria in the rectal <i>Muscularis externa</i> demonstrates high structural preservation of the stored paraffin-embedded tissue.

<p>STED recordings were performed on 2 µm thick dewaxed sections cut along the longitudinal axis of the rectum. (A) Left: STED overview image of a region of the inner circular layer of the rectal <i>Muscularis externa</i> decorated with an antiserum against Tom20. Right: Magnifications of the areas in the indicated dashed squares showing the distribution of TOM clusters within the mitochondria. (B–E) STED images of tissue sections decorated with antisera against Tom20 (B), Mic60 (mitofilin) (C), aconitase (D), and cyclophilin D (E). In each panel the confocal (top, left) and the corresponding STED image (top, right) is displayed. Bottom: Magnification of the STED image as indicated by a dashed square. Note the different distributions of the four proteins within the mitochondria. Scale bars: 20 µm (A, left); 1 µm (A, right) and (B–E, top); 200 nm (B–E, bottom).</p

STED super-resolution microscopy of archived human tissue samples stored for up to 17 years in a clinical repository.

<p>Representative images of tumor tissues stored at room temperature for less than 1 year (A), 11 years (B) or 17 years (C), were sectioned, dewaxed, decorated with an antiserum against Tom20 and imaged. Left: Representative confocal images. The same color table was used for the three images in order to visualize the relative staining efficiencies. Middle/Right: Comparison of STED (middle) and confocal (right) microscopy of tissue sections of different age. Here, the color tables were adjusted to the signal intensities obtained. Scale bars: 10 µm (left) and 1 µm (middle, right).</p

Immunofluorescence labeling of HER2 positive paraffin-embedded rectal cancer tissue.

<p>Confocal overview image of a region of a HER2 positive rectal cancer tissue section labeled with DAPI (blue) to highlight the nuclei and decorated with antisera against Tom20 (fire) and HER2 (green). (A) overlay, (B) Tom20, and (C) HER2. Scale bars: 25 µm.</p

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with “Universal Hydrophilizer” or Hydroxyl Groups

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a “universal hydrophilizer”) or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of “blinking” spiroamides derived from <i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetramethyl rhodamine

MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation.

Stephan T, Bruser C, Deckers M, et al. MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation. The EMBO journal. 2020: e104105.Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F1 Fo -ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F1 Fo -ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae. © 2020 The Authors. Published under the terms of the CC BY 4.0 license

STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue.

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.Open-Access-Publikationsfonds 2014peerReviewe