Focal point: Biochemistry of stem cells

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1 Focal point: Biochemistry of stem cells Stem cells have become a major impact penetrating nearly all disciplines of live sciences dealing with eukaryotic cells or organisms. We predict that in the near future a strong demand on educated scientists in various fields will arise in many sections, such as in basic research, in pharmaceutical industry and in medicine. This demands in turn an advanced interdisciplinary educational approach starting in the field of chemistry of small molecules, bioinformatics of nucleic acids and protein networks, biochemistry of transcription factors, genetics and epigenetics, systems biology and possible applications for treating degenerative diseases of the brain, liver and muscle etc. More recently, it has become possible to reprogram patient derived cells allowing to develop strategies of cell replacement therapy and patient specific drug development. Defined combinations of transcription factors may even allow certain types of trans-differentiation such as from fibroblasts into neurons. The application of nanoparticles may be useful to introduce proteins into cells thus avoiding any permanent genetic manipulation of cells and stressing the importance of gaining knowledge in epigenetic mechanism controlling patterns of gene expression.

2 Research Focus in Biochemistry of Stem Cells Faculty of Chemistry and Biochemistry Faculty of Biology and Biotechnology External Groups 1) The Extracellular matrix influences differentiation and neurite growth of embryonic motoneurons Ig superfamily signal transduction mediated through ECM molecules Prof. S. Wiese 2) Modes Stem Cell Division : Prof. A. Faissner 3) Molecular programming and induction of dopaminergic neurons phenotype in h human ipsc: Dr. K Chakrabarty/ Prof Heumann Protein purification and transduction : Dr. S. Neumann/ Prof R.Heumann 4) Migratory behavior of adult stem cell/ tumor cell hybrids: Prof Th. Dittmar/ University Witten 5) Perturbation and Measurement of Rho GTPase Activity in Living Cells Dr. L. Demehlt/ MPI Molecular Physiology Dortmund

3 Research Focus Prof. Dr. Stefan Wiese Faculty of Biology and Biotechnology AG Molecular Cellbiology NDEF 05/598

4 The Extracellular matrix influences differentiation and neurite growth of embryonic motoneurons cells migrate laterally from the ventricular zone and turn along the dorso-ventral axis commissural (C) and association (A) neurons differentiate in the dorsal part dorsal root ganglion (DRG) neurons differentiate from neural crest progenitors motor neurons (M) and ventral interneurons (V) develop in the ventral half Motor axons emigrate from spinal cord to innervate their muscle target On their way to their targets they traverse a dense jungle of many different cells, expressing a variety of different molecular guidance cues Jessell T Questions: What are the ECM guidance cues that help motoneurons to differentiate from stem cells? What are the intracellular signaling events? (Polleux F. 2007)

5 Ig superfamily signal transduction mediated through ECM molecules Tenascin R Tenascin C Neurocan Hyaloronan L1/NgCAM NCAM F3/ F11/ Contactin TAG-1- Axonin DAG FGF-R PLCg Frs2 Grb2 Sos Raf Ras Sos Grb2 Shc Fyn Ankyrin FAK Actin Cytoskeleton PKC GAP43 MEK CAM initiates two pathways of signaling via FAK + tyrosine Cytoskeletal dynamics Erk kinases Fyn, which is associated with lipid rafts and via FGFR and activation of protein kinase C CAM activation leads to gene transcription and/or changes Gene transcription in cytoskeletal dynamics

6 Research Focus Prof. Andreas Faissner Faculty of Biology and Biotechnology Dept. CellMorphology&MolecularNeurobiology NDEF 05/586

7 When do neuralstem cells divide? What controls thecellfate of thedaughter cells? Howisneurogenesisregulated as opposed to gliogenesis? Whatistherole of the immediate environment theneuralstemcellniche in thesecellularprocesses? in particulartherole of chondroitinsulfates (CS-carbohydrates) How do neuralstem cells integrateenvironmentalinformation? wehaveidentifiedthestar-familyprotein Sam68 and theguaninenucleotideexchangefactor (GEF) Vav3 as potential integrators of bygenetrapping

8 Research Focus Prof. Rolf Heumann Molecular Neurobiochemistry Faculty of Chemistry and Biochemistry NC7/174 A) Molecular programming and induction of dopaminergic neurons phenotype in human ipsc: Dr. Koushik Chakrabarty B) Protein transduction of transcription factors: Dr. Sebastian Neumann

9 ONECUT1 in midbrain DA development Onecut1 Onecut1 Onecut1 Dr. Koushik Chakrabarty

10 Protein transduction Edenhofer, Curr Pharm Des. 14, , 2008 adopted from Peitz 2007 Dr. Sebastian Neumann

11 Proof of principle: Protein transduction of HTNCre on CV1-5B cells Cell permeable Cre recombinase: HTN-Cre Cre reporter cell line: CV1-5B adopted from Peitz et al., PNAS 99(7), , 2002 adopted from Kellendonk et al., Nucleic Acids Research 24(8), , 1996 Fabian Raudzus & Sebastian Neumann Dr. Sebastian Neumann

12 Reprogramming of stem cells to induced dopaminergic neurons (ida) by protein transduction viral transduction our approach: protein transduction adopted from Caiazzo et al., Nature 476, , 2011 Dr. Koushik Chakrabarty and Dr. Sebastian Neumann

13 Stem Cell Research Group Migratory behavior of adult stem cell/ tumor cell hybrids Prof. Dr. Thomas Dittmar Stem Cell Research Group Institute of Immunology Center for Biomedical Education and Research (ZBAF) Witten/ Herdecke University

14 Stem Cell Research Group Cell fusion and cancer Cancer Fusion of tumor cells with normal cells, e.g., adult stem cells, can give rise to hybrid cells exhibiting novel properties, such as: - an enhanced metastatic potential (due to an altered migratory activity) - an increased drug resistance Duelli & Lazebnik Cancer Cell 2003; 3: a higher proliferation rate - an increased resistance to apoptosis

15 Stem Cell Research Group Breast stem/ breast cancer hybrids are sensitive to the lymph node metastasis directing chemokine CCL21 Green: EGFP (breast epithelial cell) Red: BrdU (tumor cell) Blue: Syto60 (nuclear stain) M13HS-8

16 Perturbation and Measurement of Rho GTPase Activity in Living Cells Abram Calderon, Tomas Mazel, Aline Timmermann and Leif Dehmelt Max-Planck-Institute for Molecular Physiology and TU Dortmund

17 Experimental Approach to Study Rho GTPase Crosstalk Cdc42? Rac1 Cdc42? Rac1 Cdc42? Rac1?????? RhoA RhoA RhoA Equilibrium Perturbation Measurement of the system s response

18 Spatio-temporal Perturbation with Light input Rac1 GDP Rac1 GTP output Rac1 GTP LOV Rac1 GTP LOV Rac1 GTP Wu et al., Nature 461:104

19 Measurement of RhoGTPase Activity Based on Effector WASP-GBD binds only to GTP-Cdc42 (not to GDP-Cdc42) and get recruited to the plasma membrane, where GTP-Cdc42 is located. Binding WASP-GBD WASP-GBD mcherry mcherry selective illumination in evanescent wave of TIRF microscope WASP-GBD mcherry WASP-GBD mcherry WASP-GBD mcherry WASP-GBD mcherry ~200nm GDP Cdc42 WASP-GBD GTP Cdc42 mcherry Glass Coverslip / Substrate

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