News | IMPRS for Molecules of Life

Publication of IMPRS-LS student Milena Dürrbaum

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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.

Publication of IMPRS-LS students Jonas Mücksch and Philipp Blumhardt

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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.

Publication of IMPRS-LS student Sigrun Schmähling

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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.

Publication of IMPRS-LS student Sandra Lemke

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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.

Publication of IMPRS-LS Student Mehrshad Pakdel

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Pakdel, M., and von Blume, J.
Mol Biol Cell, 2018 29, 235-240.
doi: 10.1091/mbc.E17-02-0117

Exploring new routes for secretory protein export from the trans-Golgi network

Sorting of soluble proteins for transport to intracellular compartments and for secretion from cells is essential for cell and tissue homeostasis. The trans-Golgi network (TGN) is a major sorting station that sorts secretory proteins into specific carriers to transport them to their final destinations. The sorting of lysosomal hydrolases at the TGN by the mannose 6-phosphate receptor is well understood. The recent discovery of a Ca2+-based sorting of secretory cargo at the TGN is beginning to uncover the mechanism by which cells sort secretory cargoes from Golgi residents and cargoes destined to the other cellular compartments. This Ca2+-based sorting involves the cytoplasmic actin cytoskeleton, which through membrane anchored Ca2+ ATPase SPCA1 and the luminal Ca2+ binding protein Cab45 sorts of a subset of secretory proteins at the TGN. We present this discovery and highlight important challenges that remain unaddressed in the overall pathway of cargo sorting at the TGN.

Co-Author Publication of IMPRS-LS Student Carina Lehmer

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Guo, Q., Lehmer, C., Martinez-Sanchez, A., Rudack, T., Beck, F., Hartmann, H., Perez-Berlanga, M., Frottin, F., Hipp, M.S., Hartl, F.U., Edbauer, D., Baumeister, W., and Fernandez-Busnadiego, R
Cell 2018, [Epub ahead of print].
doi: 10.1016/j.cell.2017.12.030

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

Publication of IMPRS-LS student Roman Prytuliak

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Prytuliak, R., Pfeiffer, F., and Habermann, B.H.
BMC Bioinformatics 2018, 19, 24.
doi: 10.1186/s12859-018-2020-x

SLALOM, a flexible method for the identification and statistical analysis of overlapping continuous sequence elements in sequence- and time-series data.

BACKGROUND:
Protein or nucleic acid sequences contain a multitude of associated annotations representing continuous sequence elements (CSEs). Comparing these CSEs is needed, whenever we want to match identical annotations or integrate distinctive ones. Currently, there is no ready-to-use software available that provides comprehensive statistical readout for comparing two annotations of the same type with each other, which can be adapted to the application logic of the scientific question.
RESULTS:
We have developed a method, SLALOM (for StatisticaL Analysis of Locus Overlap Method), to perform comparative analysis of sequence annotations in a highly flexible way. SLALOM implements six major operation modes and a number of additional options that can answer a variety of statistical questions about a pair of input annotations of a given sequence collection. We demonstrate the results of SLALOM on three different examples from biology and economics and compare our method to already existing software. We discuss the importance of carefully choosing the application logic to address specific scientific questions.
CONCLUSION:
SLALOM is a highly versatile, command-line based method for comparing annotations in a collection of sequences, with a statistical read-out for performance evaluation and benchmarking of predictors and gene annotation pipelines. Abstraction from sequence content even allows SLALOM to compare other kinds of positional data including, for example, data coming from time series.

Publication of IMPRS-LS student Yan Xiao

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Yan X, Shi Q, Bracher A, Miličić G, Singh AK, Hartl FU, Hayer-Hartl M. (IMPRS-LS students are in bold)
Cell 2017, [Epub ahead of print].
doi: 10.1016/j.cell.2017.12.010

GroEL Ring Separation and Exchange in the Chaperonin Reaction

The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism.

Publication of IMPRS-LS student Mathias Rosam

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Rosam, M., Krader, D., Nickels, C., Hochmair, J., Back, K.C., Agam, G., Barth, A., Zeymer, C., Hendrix, J., Schneider, M., Antes, I., Reinstein, J., Lamb, D.C., and Buchner, J.
Nat Struct Mol Biol 2018, 25, 90-100.
doi: 10.1038/s41594-017-0012-6

Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate

BiP is the endoplasmic member of the Hsp70 family. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). Bap is a two-domain protein. The interaction of the Bap C-terminal domain with the BiP ATPase domain is sufficient for its weak NEF activity. However, stimulation of the BiP ATPase activity requires full-length Bap, suggesting a complex interplay of these two factors. Here, single-molecule FRET experiments with mammalian proteins reveal that Bap affects the conformation of both BiP domains, including the lid subdomain, which is important for substrate binding. The largely unstructured Bap N-terminal domain promotes the substrate release from BiP. Thus, Bap is a conformational regulator affecting both nucleotide and substrate interactions. The preferential interaction with BiP in its ADP state places Bap at a late stage of the chaperone cycle, in which it coordinates release of substrate and ADP, thereby resetting BiP for ATP and substrate binding.

Publication of IMPRS-LS student Dongik Park

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Park, D.I., Stambuk, J., Razdorov, G., Pucic-Bakovic, M., Martins-de-Souza, D., Lauc, G., and Turck, C.W.
Sci Rep 2018, 8, 179.
doi: 10.1038/s41598-017-17500-0

Blood plasma/IgG N-glycome biosignatures associated with major depressive disorder symptom severity and the antidepressant response

While N-linked glycosylation has been extensively studied in the context of inflammatory and metabolic disorders, its relationship with major depressive disorder (MDD) and antidepressant treatment response has not been investigated. In our exploratory study, we analysed N-glycan profiles in blood plasma samples collected from MDD patients (n = 18) and found gender-dependent correlations with severity of depressive symptoms prior to initiating antidepressant treatment. In addition, several N-glycosylation traits showed gender-dependent associations with clinical antidepressant response. Follow up proteomics analysis in peripheral blood mononuclear cells (PBMCs) collected from MDD patients (n = 20) identified baseline and post-antidepressant treatment pathway differences between responder and non-responder patients. Reactome data analysis further delineated potential biological reaction differences between responder and non-responder patients. Our preliminary results suggest that specific glycosylation traits are associated with depressive symptom severity and antidepressant response and may be of use as biomarkers.