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Kashammer, L., Saathoff, J.H., Lammens, K., Gut, F., Bartho, J., Alt, A., Kessler, B., and Hopfner, K.P.
Mol Cell, 2019, [Epub ahead of print].
doi: 10.1016/j.molcel.2019.07.035

Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.


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Gehrlach, D.A., Dolensek, N., Klein, A.S., Roy Chowdhury, R., Matthys, A., Junghanel, M., Gaitanos, T.N., Podgornik, A., Black, T.D., Reddy Vaka, N., Conzelmann, K.K., and Gogolla, N.
Nat Neurosci, 2019, 22, 1424-1437.
(IMPRS-LS students are in bold)
doi: 10.1038/s41593-019-0469-1

Aversive state processing in the posterior insular cortex

Triggering behavioral adaptation upon the detection of adversity is crucial for survival. The insular cortex has been suggested to process emotions and homeostatic signals, but how the insular cortex detects internal states and mediates behavioral adaptation is poorly understood. By combining data from fiber photometry, optogenetics, awake two-photon calcium imaging and comprehensive whole-brain viral tracings, we here uncover a role for the posterior insula in processing aversive sensory stimuli and emotional and bodily states, as well as in exerting prominent top-down modulation of ongoing behaviors in mice. By employing projection-specific optogenetics, we describe an insula-to-central amygdala pathway to mediate anxiety-related behaviors, while an independent nucleus accumbens-projecting pathway regulates feeding upon changes in bodily state. Together, our data support a model in which the posterior insular cortex can shift behavioral strategies upon the detection of aversive internal states, providing a new entry point to understand how alterations in insula circuitry may contribute to neuropsychiatric conditions.


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La Chioma, A., Bonhoeffer, T., and Hubener, M.
Curr Biol, 2019, 29, 2954-2960 e2955.
doi: 10.1016/j.cub.2019.07.037

Area-Specific Mapping of Binocular Disparity across Mouse Visual Cortex

Depth perception is a fundamental feature of many visual systems across species. It is relevant for crucial behaviors, like spatial orientation, prey capture, and predator detection. Binocular disparity, the difference between left and right eye images, is a powerful cue for depth perception, as it depends on an object's distance from the observer [1,2]. In primates, neurons sensitive to binocular disparity are found throughout most of the visual cortex, with distinct disparity tuning properties across primary and higher visual areas, suggesting specific roles of different higher areas for depth perception [1-3]. Mouse primary visual cortex (V1) has been shown to contain disparity-tuned neurons, similar to those found in other mammals [4,5], but it is unknown how binocular disparity is processed beyond V1 and whether it is differentially represented in higher areas. Beyond V1, higher-order, lateromedial (LM) and rostrolateral (RL) areas contain the largest representation of the binocular visual field [6,7], making them candidate areas for investigating downstream processing of binocular disparity in mouse visual cortex. In turn, comparison of disparity tuning across different mouse visual areas might help delineating their functional specializations, which are not well understood. We find clear differences in neurons' preferred disparities across areas, suggesting that higher visual area RL is specialized for encoding visual stimuli very close to the mouse. Moreover, disparity preference is related to visual field elevation, likely reflecting an adaptation to natural image statistics. Our results reveal ethologically relevant areal specializations for binocular disparity processing across mouse visual cortex.


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Balaji, R., Weichselberger, V., and Classen, A.K.
Development, 2019, 146.
doi: 10.1242/dev.171256

Response of epithelial cell and tissue shape to external forces in vivo

How actomyosin generates forces at epithelial adherens junctions has been extensively studied. However, less is known about how a balance between internal and external forces establishes epithelial cell, tissue and organ shape. We used the Drosophila egg chamber to investigate how contractility at adherens junctions in the follicle epithelium is modulated to accommodate and resist forces arising from the growing germ line. We found that between stages 6 and 9, adherens junction tension in the post-mitotic epithelium decreases, suggesting that the junctional network relaxes to accommodate germline growth. At that time, a prominent medial Myosin II network coupled to corrugating adherens junctions develops. Local enrichment of medial Myosin II in main body follicle cells resists germline-derived forces, thus constraining apical areas and, consequently, cuboidal cell shapes at stage 9. At the tissue and organ level, local reinforcement of medial junction architecture ensures the timely contact of main body cells with the expanding oocyte and imposes circumferential constraints on the germ line guiding egg elongation. Our study provides insight into how adherens junction tension promotes cell and tissue shape transitions while integrating the growth and shape of an internally enclosed structure in vivo.


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Lingaraju, M., Johnsen, D., Schlundt, A., Langer, L.M., Basquin, J., Sattler, M., Heick Jensen, T., Falk, S., and Conti, E.
Nat Commun, 2019, 10, 3393.
doi: 10.1038/s41467-019-11339-x

The MTR4 helicase recruits nuclear adaptors of the human RNA exosome using distinct arch-interacting motifs

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.


graduationCongratulations on your PhD!

Sandra Lemke
Analysis of molecular forces transmitted by Talin during muscle development in vivo
RG: Frank Schnorrer

Philipp Blumhardt
Surface-Integrated Fluorescence Correlation Spectroscopy (SI-FCS) for the quantification of transient membrane and surface binding
RG: Petra Schwille

Mehrshad Pakdel
Activity of the SPCA1 calcium ATPase couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network
RG: Julia von Blume


 

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Konjevic Sabolek, M., Held, K., Beltran, E., Niedl, A.G., Meinl, E., Hohlfeld, R., Lassmann, H., and Dornmair, K.
Ann Clin Transl Neurol, 2019, 6, 1151-1164
doi: 10.1002/acn3.783

Communication of CD8+ T cells with mononuclear phagocytes in multiple sclerosis

OBJECTIVE: CD8+ T cells are the most prevailing lymphocyte population in inflammatory lesions of patients with multiple sclerosis (MS) but it is not even known whether they are merely passive bystanders or actively communicate with other cells in the brain. To identify their potential interaction partners, we analyzed CD8+ T cells that contained vectorially oriented cytotoxic granules and analyzed the areas to which the granules pointed.
METHODS: We stained cryo-sections of active MS lesions of an index patient with antibodies to CD8 and perforin, searched for vectorially oriented perforin granules, and isolated target areas opposing the granules and control areas by laser-microdissection. From both areas, we analyzed cell-type specific transcripts by next-generation sequencing. In parallel, we stained samples from the index-patient and other patients by four-color immunohistochemistry (IHC).
RESULTS: We found transcripts of the mononuclear phagocyte (MP) specific markers CD163 and CD11b only in the microdissected target areas but not in control areas. We validated the finding that MPs are communication partners of CD8+ T cells in MS lesions by classical IHC in samples from the index-patient and other patients with acute and progressive MS and other inflammatory neurological diseases.
INTERPRETATION: Because CD163 and CD11b are specifically expressed in MPs, our findings suggest that CD8+ T cells communicate with local MPs. Although it is still unclear if these interactions lead to killing of the communication partners by CD8+ T cells, our data underline that CD8+ T cells play an active role in the pathogenesis of MS.


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Matscheko, N., Mayrhofer, P., Rao, Y., Beier, V., and Wollert, T.
PLoS Biol, 2019, 17, e3000377
doi: 10.1371/journal.pbio.3000377

Atg11 tethers Atg9 vesicles to initiate selective autophagy

Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.


graduation

Congratulations on your PhD!

Nicole Teichmann
Preclinical evaluation of an oral MEK1/2 inhibitor as a therapeutic strategy for GEMM-based Pancreatic ductal adenocarcinoma (PDAC)
RG: Jens Siveke

Sigrun Schmähling
Biochemical purification and functional characterization of a novel trithorax-group protein complex
RG: Jürg Müller


 

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Cramer, K., Bolender, A.L., Stockmar, I., Jungmann, R., Kasper, R., and Shin, J.Y.
Int J Mol Sci, 2019, 20, [Epub ahead of print].
doi: 10.3390/ijms20143376

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.