Functional Molecular Microscopy

Objectives

Genetically modified or engineered tissue must be properly characterized before their clinical application. Functional molecular microscopy represents one important method for such characterization. Current microscopic approaches are often restricted to the qualitative interpretation of cellular events. The central focus of our group is thus the development and optimization of novel optical detection paradigms (quantitative detection) in combination with global multi-parametric data analysis (quantitative analysis) to characterize functional properties of individual cells (e.g. induced pluripotent stem cells) as well as of cellular networks and tissues (e.g. bioartificial cardiac tissue) with subcellular resolution. By combined application of molecular microscopy, FRET-based biosensors, optogenetic methods, and multi-parametric data analysis, we will develop a standardized, microscopy-based protocol for quality control of engineered cells and tissues. We will also provide the REBIRTH members with personalized assistance in developing and implementation of project-based imaging techniques.

Research Focus

Quantitative analysis of intracellular processes in living cell

One of our central focus is the combined application of multiple fluorescent biosensors in order to characterize functional properties of individual cells as well as of cellular networks and tissues. Förster resonance energy transfer (FRET) has become an essential tool for the analysis of different functional aspects of proteins in their native environment. FRET analysis was also successfully applied to study spatiotemporal regulation of cellular processes using genetically encoded biosensors. To study molecular details of protein-protein interaction and intracellular signalling in living cells with a high spatio- temporal resolution, we combine confocal time-lapse microscopy with FRET techniques. For the quantitative analysis of FRET signals, we developed novel evaluation algorithms. Moreover, we have established a set of FRET-based biosensors for detection of a broad ensemble of physiological parameters at the single-cell level including ion composition (Ca2+), concentration of second messengers (cAMP), activity of the small GTPases of the Rho family, autophagy, and cytoskeleton dynamics. Our analytic platform thus allows for the investigation of complex cellular processes and is of particular importance for the functional evaluation of engineered cells and tissues.

Role of serotonin receptors in remodeling of the extracellular matrix

Fig. 1 Image of hippocampal neurons showing an increase in gelatinase activity at elongating dendritic spines after stimulation of the serotonin 5-HT7 receptor

Understanding brain function is one of the primary concerns of our modern society – the reason for this being social changes such as stress or sleep problems, ageing of the population and the associated greater prevalence of neurological and psychiatric illness. It is therefore extremely important to improve our awareness of the origin and mechanisms of the neurological and psychiatric disorders that are a major financial burden on our society. Many of these conditions, such as depression, addictions, autism, schizophrenia, stroke, epilepsy, Alzheimer’s and Parkinson’s disease arise from impairment of communication between individual nerve cells – interaction that takes place at highly specialized intercellular contacts, the synapses.We recently investigated the interaction between serotonin receptors and the extracellular matrix (ECM), which coats the neurons like a mesh and has a protective and stabilizing effect. Using high-resolution imaging in conjunction with electrophysiological and biophysical methods, we were able to elucidate the role of serotonin receptor 5-HT7 in controlling ECM-mediated processes in the brain. We were able to show that activation of the 5-HT7 receptor initiates local remodelling of the ECM (Fig. 1). This results in long-term impairment of neuronal processes that control learning and memory in relation to the physiological and pathophysiological status of the brain. On this basis, a new therapeutic strategy for treating various forms of dementia could be developed.

The mechanisms we have discovered by which the ECM is modulated may also be highly relevant for other cell types such as myocardial cells. The ECM of the myocardium plays a key role in maintaining the integrity and function of the heart. This is why it is essential to precisely unravel the pathomechanisms involved in regulating the complex process by which the ECM is remodelled in patients with heart failure – so that new therapeutic strategies can potentially be devised to prevent the progression of heart failure.

Functional analysis of bioartificial cardiac tissue (BCT)

This subproject is centred on functional characterization of bioartificial cardiac tissue (BCT) on a subcellular level. For that we established a fast acquisition protocol which allows us to image the BCT in 3-D with high temporal resolution (up to 3 z-stacks per second). Further we developed stimulation protocol of global BCT contraction using channel-rhodopsin paralleled by Ca2+ imaging. Such experiments allow for detailed functional characterization of BCT at the cellular level by visualizing the intercellular signalling. During these recordings huge amount of information (more than 10GB per experiment) were generated. In order to handle and analyse such big datasets with appropriate speed, we developed automated image processing and data evaluation algorithms. In addition, we can now provide a compact internet visualization of experimental results on the basis of html scripts, which enables the collaboration partner to study each desired step of data evaluation using standard computer hardware.

Virtual Research Center Light Microscopy

Our group is member of the MHH Virtual Research Center Light Microscopy to facilitate access to research infrastructure and resources and provide knowledge and the possibility for easy collaborations for associated and non-associated departments. Within this Center we provide the access to three high-end microscopes (i.e. Till Photonics iMIC for TIRF/FRET, Zeiss LSM 780 and Andor Spinning Disk System CSU-X1) and high resolution spectroscope Fluorolog-322 for all REBIRTH’s members. In January 2018 funding for purchasing of the new multi-photon FLIM-FRET microscopy system combined with electrophysiological set-up was approved within the framework of large-scale equipment proposal (Großgerätantrag) by the Ministry for Science and Culture of Lower Saxony and DFG. From the autumn 2018 this system will be available to all REBIRTH members.

Collaborations

  • AG Kraft, Unit 9.1B and AG Zweigerdt, Unit 10.2
    Characterization of local calcium signalling in human stem cell derived cardiomyocytes
  • AG Thum, Unit 5.5.
    Identification of GATA2-regulated microRNAs for therapy of vascular disease. Telomere length and integrity analysis.
  • AG Chichkov, Unit 7.2.
    3D in vitro platform produced by two-photon polymerization for the analysis of cellular network formation and function.
  • AG. Gruh, Unit 5.7
    Structural and functional analysis of bioartificial cardiac tissue.
  • AG Hilfiker-Kleiner
    Protective role of serotonin receptor by heart failures
  • S. Sigrist, FU Berlin, Super-resolution microscopy of synaptic structures.
  • A. Dityatev, DZNE, Magdeburg, Role of the extracellular matrix in regulation of cellular functions.
  • D. Rusakov, UCL, London, UK, Calcium imaging.
  • M. Niv, Hebrew University of Jerusalem, Computational analysis of protein-protein interaction.

Further Research Projects

  • DFG: “Cooperative interaction between serotonergic signalling and extracellular matrix in regulation of synaptic plasticity under physiological and pathological conditions”.
  • DFG: “Dynamic regulation of small Rho GTPases via serotonin receptors in neurons: Effects on the cytoskeleton, neuronal morphology and functions”.
  • DFG: “Small GTPase activity and astrocyte morphology as determinants of astrocyte Ca2+ signaling”
  • DFG: “Role of palmitoylation of the serotonin 5-HT1A receptor in regulation of physiological and pathological receptor functions”.
  • DFG: “Interplay between serotonergic and L1-mediated signaling in regulation of neuronal morphology and functions under physio-logical and pathological conditions”
  • BMBF: “Neurobiological basis of polyamine protection from age-induced memory decline (SMARTAGE)”

Publications

2013 - ongoing

2017

Bijata M, Labus J, Guseva D, Stawarski M, Butzlaff M, Dzwonek J, Schneeberg J, Bohm K, Michaluk P, Rusakov DA, Dityatev A, Wilczynski G, Wlodarczyk J, Ponimaskin E. Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix. Cell Rep. 2017/06/01 ed2017. p. 1767-82.

Zeug A, Muller FE, Anders S, Herde MK, Minge D, Ponimaskin E, Henneberger C. Control of astrocyte morphology by Rho GTPases. Brain Res Bull. 2017/05/16 ed2017.

Speranza L, Labus J, Volpicelli F, Guseva D, Lacivita E, Leopoldo M, Bellenchi GC, di Porzio U, Bijata M, Perrone-Capano C, Ponimaskin E. Serotonin 5-HT7 receptor increases the density of dendritic spines and facilitates synaptogenesis in forebrain neurons. J Neurochem. 2017/01/26 ed2017. p. 647-61.

Wehr MC, Hinrichs W, Brzozka MM, Unterbarnscheidt T, Herholt A, Wintgens JP, Papiol S, Soto-Bernardini MC, Kravchenko M, Zhang MY, Nave KA, Wichert SP, Falkai P, Zhang WQ, Schwab MH, Rossner MJ. Spironolactone is an antagonist of NRG1-ERBB4 signaling and schizophrenia-relevant endophenotypes in mice. EMBO Molecular Medicine2017. p. 1448-62.

2016

Moutin E, Nikonenko I, Stefanelli T, Wirth A, Ponimaskin E, De Roo M, Muller D. Palmitoylation of cdc42 Promotes Spine Stabilization and Rescues Spine Density Deficit in a Mouse Model of 22q11.2 Deletion Syndrome. Cereb Cortex. 2016/07/02 ed2017. p. 3618-29.

Wirth A, Holst K, Ponimaskin E. How serotonin receptors regulate morphogenic signalling in neurons. Prog Neurobiol. 2016/03/26 ed2017. p. 35-56.

Timashev PS, Vedunova MV, Guseva D, Ponimaskin E, Deiwick A, Mishchenko TA, Mitroshina EV, Koroleva AV, Pimashkin AS, Mukhina IV, Panchenko VY, Chichkov BN, Bagratashvili VN. 3D in vitro platform produced by two-photon polymerization for the analysis of neural network formation and function. Biomedical Physics & Engineering Express. 2016;2(3):035001.

Lievens PM, Kuznetsova T, Kochlamazashvili G, Cesca F, Gorinski N, Galil DA, Cherkas V, Ronkina N, Lafera J, Gaestel M, Ponimaskin E, Dityatev A. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation. Mol Cell Biol. 2016;36(17):2208-25.

Hartmann D, Fiedler J, Sonnenschein K, Just A, Pfanne A, Zimmer K, Remke J, Foinquinos A, Butzlaff M, Schimmel K, Maegdefessel L, Hilfiker-Kleiner D, Lachmann N, Schober A, Froese N, Heineke J, Bauersachs J, Batkai S, Thum T. MicroRNA-Based Therapy of GATA2-Deficient Vascular Disease. Circulation. 2016;134(24):1973-90.

2015

Steinke KV, Gorinski N, Wojciechowski D, Todorov V, Guseva D, Ponimaskin E, Fahlke C, Fischer M. Human Clc-K Channels Require Palmitoylation of Their Accessory Subunit Barttin to Be Functional. J Biol Chem. 2015;290(28):17390-400. Epub 2015/05/28.

Holst K, Guseva D, Schindler S, Sixt M, Braun A, Chopra H, Pabst O, Ponimaskin E. The Serotonin Receptor 5-Ht(7)R Regulates the Morphology and Migratory Properties of Dendritic Cells. J Cell Sci. 2015;128(15):2866-80. Epub 2015/06/21.

Butzlaff M, Weigel A, Ponimaskin E, Zeug A. Esip: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration. PLoS One. 2015;10(8):e0134980.

Butzlaff M, Hannan SB, Karsten P, Lenz S, Ng J, Vossfeldt H, Prussing K, Pflanz R, Schulz JB, Rasse T, Voigt A. Impaired Retrograde Transport by the Dynein/Dynactin Complex Contributes to Tau-Induced Toxicity. Hum Mol Genet. 2015;24(13):3623-37.

Bazovkina DV, Kondaurova EM, Naumenko VS, Ponimaskin E. Genotype-Dependent Difference in 5-Ht2c Receptor-Induced Hypolocomotion: Comparison with 5-Ht2a Receptor Functional Activity. Neural Plast. 2015;2015:846589.

2014

Zeug A, Stawarski M, Bieganska K, Korotchenko S, Wlodarczyk J, Dityatev A, Ponimaskin E. Current Microscopic Methods for the Neural Ecm Analysis. Prog Brain Res. 2014;214:287-312.

Stawarski M, Rutkowska-Wlodarczyk I, Zeug A, Bijata M, Madej H, Kaczmarek L, Wlodarczyk J. Genetically Encoded Fret-Based Biosensor for Imaging Mmp-9 Activity. Biomaterials. 2014;35(5):1402-10.

Skupien A, Konopka A, Trzaskoma P, Labus J, Gorlewicz A, Swiech L, Babraj M, Dolezyczek H, Figiel I, Ponimaskin E, Wlodarczyk J, Jaworski J, Wilczynski GM, Dzwonek J. Cd44 Regulates Dendrite Morphogenesis through Src Tyrosine Kinase-Dependent Positioning of the Golgi. J Cell Sci. 2014;127(23):5038-51.

Naumenko VS, Popova NK, Lacivita E, Leopoldo M, Ponimaskin EG. Interplay between Serotonin 5-Ht1a and 5-Ht7 Receptors in Depressive Disorders. CNS Neurosci Ther. 2014;20(7):582-90.

Guseva D, Wirth A, Ponimaskin E. Cellular Mechanisms of the 5-Ht7 Receptor-Mediated Signaling. Front Behav Neurosci. 2014;8:306.

Guseva D, Holst K, Kaune B, Meier M, Keubler L, Glage S, Buettner M, Bleich A, Pabst O, Bachmann O, Ponimaskin EG. Serotonin 5-Ht7 Receptor Is Critically Involved in Acute and Chronic Inflammation of the Gastrointestinal Tract. Inflamm Bowel Dis. 2014;20(9):1516-29.

Glebov K, Voronezhskaya EE, Khabarova MY, Ivashkin E, Nezlin LP, Ponimaskin EG. Mechanisms Underlying Dual Effects of Serotonin During Development of Helisoma Trivolvis (Mollusca). BMC Dev Biol. 2014;14:14.

Berod L, Friedrich C, Nandan A, Freitag J, Hagemann S, Harmrolfs K, Sandouk A, Hesse C, Castro CN, Bahre H, Tschirner SK, Gorinski N, Gohmert M, Mayer CT, Huehn J, Ponimaskin E, Abraham WR, Muller R, Lochner M, Sparwasser T. De Novo Fatty Acid Synthesis Controls the Fate between Regulatory T and T Helper 17 Cells. Nat Med. 2014;20(11):1327-33.

Bang C, Batkai S, Dangwal S, Gupta SK, Foinquinos A, Holzmann A, Just A, Remke J, Zimmer K, Zeug A, Ponimaskin E, Schmiedl A, Yin X, Mayr M, Halder R, Fischer A, Engelhardt S, Wei Y, Schober A, Fiedler J, Thum T. Cardiac Fibroblast-Derived Microrna Passenger Strand-Enriched Exosomes Mediate Cardiomyocyte Hypertrophy. J Clin Invest. 2014;124(5):2136-46.

2013

Wirth A, Chen-Wacker C, Wu YW, Gorinski N, Filippov MA, Pandey G, Ponimaskin E. Dual Lipidation of the Brain-Specific Cdc42 Isoform Regulates Its Functional Properties. Biochem J. 2013;456(3):311-22.

Prasad S, Zeug A, Ponimaskin E. Analysis of Receptor-Receptor Interaction by Combined Application of Fret and Microscopy. Methods Cell Biol. 2013;117:243-65.

Gorinski N, Ponimaskin E. Palmitoylation of Serotonin Receptors. Biochem Soc Trans. 2013;41(1):89-94.

2006 - 2012

2012

Zeug A, Woehler A, Neher E, Ponimaskin EG. Quantitative Intensity-Based Fret Approaches--a Comparative Snapshot. Biophys J. 2012;103(9):1821-7.

Renner U, Zeug A, Woehler A, Niebert M, Dityatev A, Dityateva G, Gorinski N, Guseva D, Abdel-Galil D, Frohlich M, Doring F, Wischmeyer E, Richter DW, Neher E, Ponimaskin EG. Heterodimerization of Serotonin Receptors 5-Ht1a and 5-Ht7 Differentially Regulates Receptor Signalling and Trafficking. J Cell Sci. 2012;125(Pt 10):2486-99.

Kobe F, Guseva D, Jensen TP, Wirth A, Renner U, Hess D, Muller M, Medrihan L, Zhang W, Zhang M, Braun K, Westerholz S, Herzog A, Radyushkin K, El-Kordi A, Ehrenreich H, Richter DW, Rusakov DA, Ponimaskin E. 5-Ht7r/G12 Signaling Regulates Neuronal Morphology and Function in an Age-Dependent Manner. J Neurosci. 2012;32(9):2915-30.

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