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Fabritius, A., Ng, D., Kist, A.M., Erdogan, M., Portugues, R., and Griesbeck, O.
Cell Chem Biol, 2018, [Epub ahead of print].
(IMPRS-LS students are in bold)
doi: 10.1016/j.chembiol.2018.08.008

Imaging-Based Screening Platform Assists Protein Engineering.

Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies. It allows tracking each individual colony in a large population and collecting quantitative information on library composition during the protein evolution process. We demonstrate the power of the screening platform by optimizing a dim far-red-emitting fluorescent protein whose brightness increased several fold using iterative cycles of mutagenesis and platform-based screening. The resulting protein variant mCarmine is useful for imaging cells and structures within live tissue as well as for molecular tagging. Overall, the platform presented provides powerful, flexible, and low-cost instrumentation to accelerate many fluorescence-based protein optimization projects.


 

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Kabacaoglu, D., Ciecielski, K.J., Ruess, D.A., and Algul, H.
Front Immunol, 2018, 9, 1878.
doi: 10.3389/fimmu.2018.01878

Immune Checkpoint Inhibition for Pancreatic Ductal Adenocarcinoma: Current Limitations and Future Options.

Pancreatic ductal adenocarcinoma (PDAC), as the most frequent form of pancreatic malignancy, still is associated with a dismal prognosis. Due to its late detection, most patients are ineligible for surgery, and chemotherapeutic options are limited. Tumor heterogeneity and a characteristic structure with crosstalk between the cancer/malignant cells and an abundant tumor microenvironment (TME) make PDAC a very challenging puzzle to solve. Thus far, targeted therapies have failed to substantially improve the overall survival of PDAC patients. Immune checkpoint inhibition, as an emerging therapeutic option in cancer treatment, shows promising results in different solid tumor types and hematological malignancies. However, PDAC does not respond well to immune checkpoint inhibitors anti-programmed cell death protein 1 (PD-1) or anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) alone or in combination. PDAC with its immune-privileged nature, starting from the early pre-neoplastic state, appears to escape from the antitumor immune response unlike other neoplastic entities. Different mechanisms how cancer cells achieve immune-privileged status have been hypothesized. Among them are decreased antigenicity and impaired immunogenicity via both cancer cell-intrinsic mechanisms and an augmented immunosuppressive TME. Here, we seek to shed light on the recent advances in both bench and bedside investigation of immunotherapeutic options for PDAC. Furthermore, we aim to compile recent data about how PDAC adopts immune escape mechanisms, and how these mechanisms might be exploited therapeutically in combination with immune checkpoint inhibitors, such as PD-1 or CTLA-4 antibodies.


 

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Kozlowski, M., Corujo, D., Hothorn, M., Guberovic, I., Mandemaker, I.K., Blessing, C., Sporn, J., Gutierrez-Triana, A., Smith, R., Portmann, T., Treier, M., Scheffzek, K., Huet, S., Timinszky, G., Buschbeck, M., and Ladurner, A.G.
(IMPRS-LS students are in bold)
EMBO Rep, 2018, [Epub ahead of print].
doi: 10.15252/embr.201744445

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms.

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.


 

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Spadaro, M., Winklmeier, S., Beltran, E., Macrini, C., Hoftberger, R., Schuh, E., Thaler, F.S., Gerdes, L.A., Laurent, S., Gerhards, R., Brandle, S., Dornmair, K., Breithaupt, C., Krumbholz, M., Moser, M., Kirshnamoorthy, G., Kamp, F., Jenne, D., Hohlfeld, R., Kumpfel, T., Lassmann, H., Kawakami, N., and Meinl, E.
Ann Neurol., 2018, [Epub ahead of print].
doi: 10.1002/ana.25291

Pathogenicity of human antibodies against myelin oligodendrocyte glycoprotein

OBJECTIVE:
Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the CNS. We analyzed their pathogenic activity by affinity-purifying these Abs from patients and transferring them to experimental animals.
METHODS:
Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and determined the recognized MOG-epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in two models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically.
RESULTS:
We identified 17 patients with MOG Abs from our outpatient clinic and selected two with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the two patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients these Abs persisted during our observation period of 2-3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in two different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T cell infiltration; together with MBP-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology.
INTERPRETATION:
MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon co-transfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients.


 

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Keplinger, S., Beiderbeck, B., Michalakis, S., Biel, M., Grothe, B., and Kunz, L.
Front Cell Neurosci, 2018, 12, 111.
doi: 10.3389/fncel.2018.00111

Optogenetic Control of Neural Circuits in the Mongolian Gerbil

The Mongolian gerbil (Meriones unguiculatus) is widely used as a model organism for the human auditory system. Its hearing range is very similar to ours and it uses the same mechanisms for sound localization. The auditory circuits underlying these functions have been characterized. However, important mechanistic details are still under debate. To elucidate these issues, precise and reversible optogenetic manipulation of neuronal activity in this complex circuitry is required. However, genetic and genomic resources for the Mongolian gerbil are poorly developed. Here, we demonstrate a reliable gene delivery system using an AAV8(Y337F)-pseudotyped recombinant adeno-associated virus (AAV) 2-based vector in which the pan-neural human synapsin (hSyn) promoter drives neuron-specific expression of CatCH (Ca2+-permeable channelrhodopsin) or NpHR3.0 (Natronomonas pharaonis halorhodopsin). After stereotactic injection into the gerbil's auditory brainstem (medial nucleus of the trapezoid body, dorsal nucleus of the lateral lemniscus) and midbrain [inferior colliculus (IC)], we characterized CatCH- and/or NpHR3.0-transduced neurons in acute brain slices by means of whole-cell patch-clamp recordings. As the response properties of optogenetic tools strongly depend on neuronal biophysics, this parameterization is crucial for their in vivo application. In a proof-of-principle experiment in anesthetized gerbils, we observed strong suppression of sound-evoked neural responses in the dorsal nucleus of the lateral lemniscus (DNLL) and IC upon light activation of NpHR3.0. The successful validation of gene delivery and optogenetic tools in the Mongolian gerbil paves the way for future studies of the auditory circuits in this model system.


 

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Strauss, M.T., Schueder, F., Haas, D., Nickels, P.C., and Jungmann, R.
Nat Commun 2018, 9, 1600.
doi: 10.1038/s41467-018-04031-z

Quantifying absolute addressability in DNA origami with molecular resolution

Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.


 

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Durrbaum, M., Kruse, C., Nieken, K.J., Habermann, B., and Storchova, Z.
BMC Genomics, 2018,  19, 197.
doi: 10.1186/s12864-018-4556-6

The deregulated microRNAome contributes to the cellular response to aneuploidy

BACKGROUND:
Aneuploidy, or abnormal chromosome numbers, severely alters cell physiology and is widespread in cancers and other pathologies. Using model cell lines engineered to carry one or more extra chromosomes, it has been demonstrated that aneuploidy per se impairs proliferation, leads to proteotoxic as well as replication stress and triggers conserved transcriptome and proteome changes.
RESULTS:
In this study, we analysed for the first time miRNAs and demonstrate that their expression is altered in response to chromosome gain. The miRNA deregulation is independent of the identity of the extra chromosome and specific to individual cell lines. By cross-omics analysis we demonstrate that although the deregulated miRNAs differ among individual aneuploid cell lines, their known targets are predominantly associated with cell development, growth and proliferation, pathways known to be inhibited in response to chromosome gain. Indeed, we show that up to 72% of these targets are downregulated and the associated miRNAs are overexpressed in aneuploid cells, suggesting that the miRNA changes contribute to the global transcription changes triggered by aneuploidy. We identified hsa-miR-10a-5p to be overexpressed in majority of aneuploid cells. Hsa-miR-10a-5p enhances translation of a subset of mRNAs that contain so called 5'TOP motif and we show that its upregulation in aneuploids provides resistance to starvation-induced shut down of ribosomal protein translation.
CONCLUSIONS:
Our work suggests that the changes of the microRNAome contribute on one hand to the adverse effects of aneuploidy on cell physiology, and on the other hand to the adaptation to aneuploidy by supporting translation under adverse conditions.


 

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Mucksch, J.*, Blumhardt, P.*, Strauss, M.T., Petrov, E.P., Jungmann, R., and Schwille, P.
* equal contribution
(IMPRS-LS students are in bold)
Nano Lett, 2018, [Epub ahead of print].
doi: 10.1021/acs.nanolett.8b00875

Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.


 

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Schmahling, S., Meiler, A., Lee, Y., Mohammed, A., Finkl, K., Tauscher, K., Israel, L., Borath, M., Philippou-Massier, J., Blum, H., Habermann, B., Imhof, A., Song, J.J., and Muller, J.
Development, 2018, [Epub ahead of print].
doi: 10.1242/dev.163808

Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15 null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the critical physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.


 

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Lemke, S., and Schnorrer, F.
J Vis Exp, 2018, 132.
doi: 10.3791/57312

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events - occurring on the time scale of hours and days - to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.