News

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.

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.

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.

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.