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Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena
A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.
Sci Rep, 7 Art. No. 23 (2017)
  PubMed Source   

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
@article{Müller6796,
author={Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena},
title={A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.},
journal={Scientific reports},
volume={7},
pages={null--null},
year=2017
}

Karl Hoffmann, Anja Voss-Böhme, Jochen Rink, Lutz Brusch
A dynamically diluted alignment model reveals the impact of cell turnover on the plasticity of tissue polarity patterns.
J R Soc Interface, 14(135) Art. No. doi: 10.1098/rsif.2017.0466 (2017)
PubMed Source   

The polarization of cells and tissues is fundamental for tissue morphogenesis during biological development and regeneration. A deeper understanding of biological polarity pattern formation can be gained from the consideration of pattern reorganization in response to an opposing instructive cue, which we here consider using the example of experimentally inducible body axis inversions in planarian flatworms. We define a dynamically diluted alignment model linking three processes: entrainment of cell polarity by a global signal, local cell-cell coupling aligning polarity among neighbours, and cell turnover replacing polarized cells by initially unpolarized cells. We show that a persistent global orienting signal determines the final mean polarity orientation in this stochastic model. Combining numerical and analytical approaches, we find that neighbour coupling retards polarity pattern reorganization, whereas cell turnover accelerates it. We derive a formula for an effective neighbour coupling strength integrating both effects and find that the time of polarity reorganization depends linearly on this effective parameter and no abrupt transitions are observed. This allows us to determine neighbour coupling strengths from experimental observations. Our model is related to a dynamic 8-Potts model with annealed site-dilution and makes testable predictions regarding the polarization of dynamic systems, such as the planarian epithelium.
@article{Hoffmann6955,
author={Karl Hoffmann, Anja Voss-Böhme, Jochen Rink, Lutz Brusch},
title={A dynamically diluted alignment model reveals the impact of cell turnover on the plasticity of tissue polarity patterns.},
journal={Journal of the Royal Society, Interface},
volume={14},
issue ={135},
pages={null--null},
year=2017
}

Sven Karol, Tobias Nett, Pietro Incardona, Nesrine Khouzami, Jeronimo Castrillon, Ivo F. Sbalzarini
A Language and Development Environment for Parallel Particle Methods
In: V. International Conference on Particle-based Methods - Fundamentals and Applications (2017)(Eds.) Peter Wriggers, Barcelona, International Center for Numerical Methods in Engineering (CIMNE) (2017), 1-10
   

@proceedings{Karol6891,
title = {A Language and Development Environment for Parallel Particle Methods},
year = 2017,
editor = {Sven Karol, Tobias Nett, Pietro Incardona, Nesrine Khouzami, Jeronimo Castrillon, Ivo F. Sbalzarini},
volume = {V. International Conference on Particle-based Methods - Fundamentals and Applications},
series = {},
publisher = {International Center for Numerical Methods in Engineering (CIMNE)}
}

Guruchandar Arulmozhivarman, Martin Kräter, Manja Wobus, Jens Friedrichs, Elham Pishali Bejestani, Katrin Müller, Katrin Lambert, Dimitra Alexopoulou, Andreas Dahl, Martin Stöter, Marc Bickle, Nona Shayegi, Jochen Hampe, Friedrich Stölzel, Michael Brand, Malte von Bonin, Martin Bornhäuser
Zebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells.
Sci Rep, 7(1) Art. No. 12084 (2017)
PubMed Source   

The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34(+) cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34(+) cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.
@article{Arulmozhivarman6953,
author={Guruchandar Arulmozhivarman, Martin Kräter, Manja Wobus, Jens Friedrichs, Elham Pishali Bejestani, Katrin Müller, Katrin Lambert, Dimitra Alexopoulou, Andreas Dahl, Martin Stöter, Marc Bickle, Nona Shayegi, Jochen Hampe, Friedrich Stölzel, Michael Brand, Malte von Bonin, Martin Bornhäuser},
title={Zebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells.},
journal={Scientific reports},
volume={7},
issue ={1},
pages={null--null},
year=2017
}

Gretchen Meinke, Janet Karpinski, Frank Buchholz, Andrew Bohm
Crystal structure of an engineered, HIV-specific recombinase for removal of integrated proviral DNA.
Nucleic Acids Res, 45(16) 9726-9740 (2017)
PubMed Source   

As part of the HIV infection cycle, viral DNA inserts into the genome of host cells such that the integrated DNA encoding the viral proteins is flanked by long terminal repeat (LTR) regions from the retrovirus. In an effort to develop novel genome editing techniques that safely excise HIV provirus from cells, Tre, an engineered version of Cre recombinase, was designed to target a 34-bp sequence within the HIV-1 LTR (loxLTR). The sequence targeted by Tre lacks the symmetry present in loxP, the natural DNA substrate for Cre. We report here the crystal structure of a catalytically inactive (Y324F) mutant of this engineered Tre recombinase in complex with the loxLTR DNA substrate. We also report that 17 of the 19 amino acid changes relative to Cre contribute to the altered specificity, even though many of these residues do not contact the DNA directly. We hypothesize that some mutations increase the flexibility of the Cre tetramer and that this, along with flexibility in the DNA, enable the engineered enzyme and DNA substrate to adopt complementary conformations.
@article{Meinke6954,
author={Gretchen Meinke, Janet Karpinski, Frank Buchholz, Andrew Bohm},
title={Crystal structure of an engineered, HIV-specific recombinase for removal of integrated proviral DNA.},
journal={Nucleic acids research},
volume={45},
issue ={16},
pages={9726--9740},
year=2017
}

Sara Carvalhal, Michelle Stevense, Katrin Koehler, Ronald Naumann, Angela Huebner, Rolf Jessberger, Eric R Griffis
ALADIN is required for the production of fertile mouse oocytes.
Mol Biol Cell, 28(19) 2470-2478 (2017)
PubMed Source   

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.
@article{Carvalhal6921,
author={Sara Carvalhal, Michelle Stevense, Katrin Koehler, Ronald Naumann, Angela Huebner, Rolf Jessberger, Eric R Griffis},
title={ALADIN is required for the production of fertile mouse oocytes.},
journal={Molecular biology of the cell},
volume={28},
issue ={19},
pages={2470--2478},
year=2017
}

Matthias Höllerhage, Claudia Moebius, Johannes Melms, Wei-Hua Chiu, Joachim N Goebel, Tasnim Chakroun, Thomas Koeglsperger, Wolfgang H Oertel, Thomas W Rösler, Marc Bickle, Günter U Höglinger
Protective efficacy of phosphodiesterase-1 inhibition against alpha-synuclein toxicity revealed by compound screening in LUHMES cells.
Sci Rep, 7(1) 11469-11469 (2017)
PubMed Source   

α-synuclein-induced neurotoxicity is a core pathogenic event in neurodegenerative synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. There is currently no disease-modifying therapy available for these diseases. We screened 1,600 FDA-approved drugs for their efficacy to protect LUHMES cells from degeneration induced by wild-type α-synuclein and identified dipyridamole, a non-selective phosphodiesterase inhibitor, as top hit. Systematic analysis of other phosphodiesterase inhibitors identified a specific phosphodiesterase 1 inhibitor as most potent to rescue from α-synuclein toxicity. Protection was mediated by an increase of cGMP and associated with the reduction of a specific α-synuclein oligomeric species. RNA interference experiments confirmed PDE1A and to a smaller extent PDE1C as molecular targets accounting for the protective efficacy. PDE1 inhibition also rescued dopaminergic neurons from wild-type α-synuclein induced degeneration in the substantia nigra of mice. In conclusion, this work identifies inhibition of PDE1A in particular as promising target for neuroprotective treatment of synucleinopathies.
@article{Höllerhage6946,
author={Matthias Höllerhage, Claudia Moebius, Johannes Melms, Wei-Hua Chiu, Joachim N Goebel, Tasnim Chakroun, Thomas Koeglsperger, Wolfgang H Oertel, Thomas W Rösler, Marc Bickle, Günter U Höglinger},
title={Protective efficacy of phosphodiesterase-1 inhibition against alpha-synuclein toxicity revealed by compound screening in LUHMES cells.},
journal={Scientific reports},
volume={7},
issue ={1},
pages={11469--11469},
year=2017
}

Cordula Reuther, Matthaus Mittasch, Sundar Naganathan, Stephan W. Grill, Stefan Diez
Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.
Nano Lett, 17(9) 5699-5705 (2017)
PubMed Source   

Molecular motors, highly efficient biological nanomachines, hold the potential to be employed for a wide range of nanotechnological applications. Toward this end, kinesin, dynein, or myosin motor proteins are commonly surface-immobilized within engineered environments in order to transport cargo attached to cytoskeletal filaments. Being able to flexibly control the direction of filament motion, and in particular on planar, non-topographical surfaces, has, however, remained challenging. Here, we demonstrate the applicability of a UV-laser-based ablation technique to programmably generate highly localized patterns of functional kinesin-1 motors with different shapes and sizes on PLL-g-PEG-coated polystyrene surfaces. Straight and curved motor tracks with widths of less than 500 nm could be generated in a highly reproducible manner and proved to reliably guide gliding microtubules. Though dependent on track curvature, the characteristic travel lengths of the microtubules on the tracks significantly exceeded earlier predictions. Moreover, we experimentally verified the performance of complex kinesin-1 patterns, recently designed by evolutionary algorithms for controlling the global directionality of microtubule motion on large-area substrates.
@article{Reuther6943,
author={Cordula Reuther, Matthaus Mittasch, Sundar Naganathan, Stephan W. Grill, Stefan Diez},
title={Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.},
journal={Nano letters},
volume={17},
issue ={9},
pages={5699--5705},
year=2017
}

Martin Weigert, Loic Royer, Florian Jug, Gene Myers
Isotropic reconstruction of 3D fluorescence microscopy images using convolutional neural networks
In: Medical Image Computing and Computer-Assisted Intervention -- MICCAI 2017 : 20th International Conference, Quebec City, QC, Canada, September 10-14, 2017, Proceedings, Part II (2017)(Eds.) Maxime Descoteaux (Lecture Notes in Computer Science ; 10434), Cham, Springer International Publishing (2017), 126-134
  Source   

Fluorescence microscopy images usually show severe anisotropy in axial versus lateral resolution. This hampers downstream processing, i.e. the automatic extraction of quantitative biological data. While deconvolution methods and other techniques to address this problem exist, they are either time consuming to apply or limited in their ability to remove anisotropy. We propose a method to recover isotropic resolution from readily acquired anisotropic data. We achieve this using a convolutional neural network that is trained end-to-end from the same anisotropic body of data we later apply the network to. The network effectively learns to restore the full isotropic resolution by restoring the image under a trained, sample specific image prior. We apply our method to 3 synthetic and 3 real datasets and show that our results improve on results from deconvolution and state-of-the-art super-resolution techniques. Finally, we demonstrate that a standard 3D segmentation pipeline performs on the output of our network with comparable accuracy as on the full isotropic data. © Springer International Publishing AG 2017.
@proceedings{Weigert6948,
title = {Isotropic reconstruction of 3D fluorescence microscopy images using convolutional neural networks},
year = 2017,
editor = {Martin Weigert, Loic Royer, Florian Jug, Gene Myers},
volume = {Medical Image Computing and Computer-Assisted Intervention -- MICCAI 2017 : 20th International Conference, Quebec City, QC, Canada, September 10-14, 2017, Proceedings, Part II },
series = {(Lecture Notes in Computer Science ; 10434)},
publisher = {Springer International Publishing}
}

Kamran Rizzolo, Jennifer Huen, Ashwani Kumar, Sadhna Phanse, James Vlasblom, Yoshito Kakihara, Hussein A Zeineddine, Zoran Minic, Jamie Snider, Wen Wang, Carles Pons, Thiago V Seraphim, Edgar Boczek, Simon Alberti, Michael Costanzo, Chad L Myers, Igor Stagljar, Charles Boone, Mohan Babu, Walid A Houry
Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.
Cell Rep, 20(11) 2735-2748 (2017)
PubMed Source   

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.
@article{Rizzolo6932,
author={Kamran Rizzolo, Jennifer Huen, Ashwani Kumar, Sadhna Phanse, James Vlasblom, Yoshito Kakihara, Hussein A Zeineddine, Zoran Minic, Jamie Snider, Wen Wang, Carles Pons, Thiago V Seraphim, Edgar Boczek, Simon Alberti, Michael Costanzo, Chad L Myers, Igor Stagljar, Charles Boone, Mohan Babu, Walid A Houry},
title={Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.},
journal={Cell reports},
volume={20},
issue ={11},
pages={2735--2748},
year=2017
}