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Schwach, J., Kolobynina, K., Brandstetter, K., Gerlach, M., Ochtrop, P., Helma, J., Hackenberger, C.P.R., Harz, H., Cardoso, M.C., Leonhardt, H., and Stengl, A.
(IMPRS-LS students are in bold)
Chembiochem, 2020, [Epub ahead of print].
doi: 10.1002/cbic.202000727

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences, which was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or PNAs, allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag Ⓡ technology in combination with Cu-catalyzed azide-alkyne-cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.

 


 

The German Society for Cell Biology (DGZ) and ZEISS honor Petra Schwille, Director at the Max Planck Institute of Biochemistry, with the Carl Zeiss Lecture. With this award, the DGZ internationally acknowledges her major contributions to Cell Biology, in particular the introduction of fluorescence cross correlation spectroscopy for understanding fundamental aspects of life.

What is the minimum equipment required by the cell as the smallest living unit in an organism? Petra Schwille is looking into this question with her department "Cellular and Molecular Biophysics". Together they aim to (re)construct cellular processes and ultimately minimal living cells from dramatically simplified functional subsystems such as proteins and protein assemblies. The microscope is not sufficient to observe the interactions between the single, tiny molecules in the cell and the processes underlying them. The Biophysicist has therefore developed the fluorescence cross correlation spectroscopy, a method which visualizes processes in and around the cell. This method allows to analyze the dynamics and interactions of fluorescence-labeled molecules with highest resolution down to the level of single molecules.

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Baek, K., Scott, D.C., and Schulman, B.A.
Curr Opin Struct Biol, 2020, 67, 101-109
doi: 10.1016/j.sbi.2020.10.007

NEDD8 and ubiquitin ligation by cullin-RING E3 ligases

RING E3s comprise the largest family of ubiquitin (UB) and ubiquitin-like protein (UBL) ligases. RING E3s typically promote UB or UBL transfer from the active site of an associated E2 enzyme to a distally-recruited substrate. Many RING E3s - including the cullin-RING ligase family - are multifunctional, interacting with various E2s (or other E3s) to target distinct proteins, transfer different UBLs, or to initially modify substrates with UB or subsequently elongate UB chains. Here we consider recent structures of cullin-RING ligases, and their partner E2 enzymes, representing ligation reactions. The studies collectively reveal multimodal mechanisms - interactions between ancillary E2 or E3 domains, post-translational modifications, or auxiliary binding partners - directing cullin-RING E3-E2 enzyme active sites to modify their specific targets.

 


 

Communication processes between plant cells - Wolfgang Baumeister receives ERC Synergy Grant together with colleagues from Düsseldorf and Hohenheim

The professors Wolfgang Baumeister from the Max Planck Institute of Biochemistry (MPIB) in Martinsried, Wolf B. Frommer and Rüdiger Simon from the Heinrich Heine University Düsseldorf (HHU) and Professor Waltraud Schulze from the University of Hohenheim are jointly funded by the European Research Council (ERC). The "SymPore" project is now being implemented with over 10 million euros. The mission of this Synergy project is to elucidate the structure and function of the puzzling cell-cell connections of plants, the so-called plasmodesmata.

An organism consists of many differentiated cells, each of which has to perform different tasks. To do this, cells have to communicate with each other and exchange information. Only in this way they can coordinate their tasks for the benefit of the whole organism. In animals, this is partly done via protein complexes that form cell-connecting selective channels, so-called "gap junctions". In contrast, plasmodesmata in plants are much more complex: "Plasmodesmata are probably the most important and least studied organelles of a plant cell with immense significance for all aspects of the plant," says Wolf B. Frommer, head of the ERC project. "Plasmodesmata control both the exchange of nutrients and messenger nutrients, as well as the exchange of genetic information and even of proteins". Among other things, they prevent viral proteins or RNA from passing from one cell to the next.

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graduationCongratulations on your PhD!


Carina Trummer
Mechanistic and functional insights into the recognition and regulation of DNA modifications by UHRF1, DNMT1 and TET proteins
RG: Heinrich Leonhardt

 


 

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Pinci, F., Gaidt, M.M., Jung, C., Kuut, G., Jackson, M.A., Bauernfried, S., and Hornung, V.
(IMPRS-LS students are in bold)
J Biol Chem, 2020, [online ahead of print].
DOI: 10.1074/jbc.RA120.015248

C-tag TNF: a reporter system to study TNF shedding

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C-terminus of the transmembrane portion of pro-TNF upon cleavage. In both denatured and non-denatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.


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Alankus, B., Ecker, V., Vahl, N., Braun, M., Weichert, W., Macher-Göppinger, S., Gehring, T., Neumayer, T., Zenz, T., Buchner, M., and Ruland, J.
J Exp Med, 2021, 218, [Epub ahead of print].
DOI: 10.1084/jem.20200517

Pathological RANK signaling in B cells drives autoimmunity and chronic lymphocytic leukemia

Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.


Brenda Schulman, Ulrich Hartl and Wolfgang Baumeister receive a research grant to study Mechanisms of Parkinsons’s disease.

Brenda Schulman, Ulrich Hartl and Wolfgang Baumeister, all directors at the Max Planck Institute of Biochemistry in Martinsried, Germany have received a research grant to study the mechanisms of Parkinson’s disease from the Aligning Science Across Parkinson’s Initiative, the implementing partner of the Michael J. Fox Foundation for Parkinson’s Research is ASAP’s implementation partner. The research project will be led by Wade Harper, head of the Department of Cell Biology in the Blavatnik Institute at Havard Medical School, USA. Harper and co-investigators from the Max-Panck-Institute of Biochemistry as well as Ruben Fernandez-Busnadiego from the University of Göttingen and Judith Frydman at Stanford University, USA aim to elucidate the molecular aberrations in nerve cells that give rise to the Parkinson’s disease.

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Yunmin Wu is interested in how we perceive motion. Inspired by a cat video, she came up with the elegant idea to elicit the waterfall illusion in tiny zebrafish larvae. Thereby, the PhD graduate in Fumi Kubo’s1 group from the department of Herwig Baier gained surprising insights into the neuronal mechanism of seeing motion.

Can you elaborate on your topic of research and why you work with the model organism zebrafish?

Inside the brain, there are many neurons that process motion and its direction. As these neurons are quite abundant, I am interested to find out if all of them are required to recognize motion. For this, larval zebrafish is an amazing animal model – it is small, transparent, and demonstrates so many complex behaviors that we humans also display.

How did you come up with the idea of an optical illusion as a tool to study motion processing?

I was inspired by a video, in which a cat was trying to capture an illusory moving snake. I asked myself if an illusion that affects us humans might also apply to zebrafish. If that is the case, I can then look into the brain and see which neurons are involved. With the help of my supervisor Fumi, I chose the motion aftereffect eventually among many other cool illusions.

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graduationCongratulations on your PhD!


Kira Bartnik
Single-Molecule FRET Studies of Protein Function and Conformational Dynamics -
From DNA Nanotechnology to Viral and Bacterial Infections
RG: Don Lamb