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graduationCongratulations on your PhD!

Michael Bartoschek


Deciphering context-dependent amber suppression efficiency in mammalian cells with an expanded genetic code

RG: Heinrich Leonhardt

 


 

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Reifschneider, A., Robinson, S., van Lengerich, B., Gnörich, J., Logan, T., Heindl, S., Vogt, M.A., Weidinger, E., Riedl, L., Wind, K., Zatcepin, A., Pesämaa, I., Haberl, S., Nuscher, B., Kleinberger, G., Klimmt, J., Götzl, J.K., Liesz, A., Bürger, K., Brendel, M., Levin, J., Diehl-Schmid, J., Suh, J., Di Paolo, G., Lewcock, J.W., Monroe, K.M., Paquet, D., Capell, A., and Haass, C.
(IMPRS-LS students are in bold)
EMBO J, 2022, e109108,  online ahead of print.
DOI: 10.15252/embj.2021109108

Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.

 


 

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Klumpe, S., Fung, H.K., Goetz, S.K., Zagoriy, I., Hampoelz, B., Zhang, X., Erdmann, P.S., Baumbach, J., Müller, C.W., Beck, M., Plitzko, J.M., and Mahamid, J.
Elife, 2021, 10.
DOI: 10.7554/eLife.70506

A modular platform for automated cryo-FIB workflows

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga(+) ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

 


 

graduationCongratulations on your PhD!

Friederike Pennemann


Antiviral properties of species conserved nucleic acid-binding proteins

RG: Andreas Pichlmair

 


 

graduationCongratulations on your PhD!

Laura Lindenthal


Interaction of macrophages and diseased cells

RG: Peter Murray

 


 

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Pennemann, F.L., Mussabekova, A., Urban, C., Stukalov, A., Andersen, L.L., Grass, V., Lavacca, T.M., Holze, C., Oubraham, L., Benamrouche, Y., Girardi, E., Boulos, R.E., Hartmann, R., Superti-Furga, G., Habjan, M., Imler, J.L., Meignin, C., and Pichlmair, A.
(IMPRS-LS students are in  bold)
Nat Commun, 2021, 12, 7009.
DOI: 10.1038/s41467-021-27192-w

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, -2 and -3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.

 


 

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Bauernfried, S., and Hornung, V.
J Exp Med, 2022, 219, online ahead of print.
DOI: 10.1084/jem.20211405

Human NLRP1: From the shadows to center stage

In response to infection or cell damage, inflammasomes form intracellular multimeric protein complexes that play an essential role in host defense. Activation results in the maturation and subsequent secretion of pro-inflammatory cytokines of the IL-1 family and a specific cell death coined pyroptosis. Human NLRP1 was the first inflammasome-forming sensor identified at the beginning of the millennium. However, its functional relevance and its mechanism of activation have remained obscure for many years. Recent discoveries in the NLRP1 field have propelled our understanding of the functional relevance and molecular mode of action of this unique inflammasome sensor, which we will discuss in this perspective.

 


 

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Laudenbach, B.T., Krey, K., Emslander, Q., Andersen, L.L., Reim, A., Scaturro, P., Mundigl, S., Dächert, C., Manske, K., Moser, M., Ludwig, J., Wohlleber, D., Kröger, A., Binder, M., and Pichlmair, A.
(IMPRS-LS students are in bold)
Nat Commun, 2021, 12, 6918
DOI: 10.1038/s41467-021-27239-y

NUDT2 initiates viral RNA degradation by removal of 5'-phosphates

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5'-3' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.

 


 

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Qutbuddin, Y., Krohn, J.H., Brüggenthies, G.A., Stein, J., Gavrilovic, S., Stehr, F., and Schwille, P.
(IMPRS-LS students are in bold)
J Phys Chem B, 2021, 125, 13181-13191
DOI: 10.1021/acs.jpcb.1c07694

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.

 


 

graduationCongratulations on your PhD!

Rohit Agarwal


Ultra-high Throughput Studies of Rare Events and Multi-molecular Complexes using Flow Magnetic Tweezers

RG: Karl Duderstadt