Multikonfokales Laser-Fluoreszenz-Mikroskop
Final Report Abstract
The Zeiss confocal microscope was extensively used at its location at the Institute for Cell Biology in Münster for collaborative work involving groups within and out of Münster. The main users of the equipment were the groups of E. Raz and J. Schwamborn. The Schwamborn lab used the microscope for visualizing and documenting immunofluorescence stainings of neural stem cells and neurons in the adult mammalian brain. A particular focus of the group is the asymmetric cell division of neural stem cells. There the microscope is used to image the subcellular distribution of cell fate determinants in mitotic neural stem cells. Furthermore, the fate of neural stem cells is manipulated in vivo by the stereotactic injection of viruses that encode for the overexpression or knockdown of those cell fate determinants. Additionally, those viruses contain an expression cassette for GFP. Thereby, transduced cell are identifiable easily. At various time points after viral transduction the brains are fixed and the position and fate of the transduced cells is investigated via confocal microscopy. Additionally, the Schwamborn lab investigates the possibility to use endogenous adult neural stem cells for the induction of regeneration in mouse models for Parkinson’s disease. The main model that is used is a 6-OHDa injection model where dopaminergic neurons in the substantia nigra degenerate because of the injection of this chemical. The confocal microscope is used to image this degeneration, the resulting activation of immune cells in the brain (microglia) and the consequences that this degeneration has on the activity of adult neural stem cells. The Raz lab used the equipment for documenting results relevant for germ-cell migration in zebrafish as well as for experimental manipulations of germ cells for investigating the cellular mechanisms responsible for protrusionformation in these cells. More specifically, the microscope was used for determining the subcellular localization of specific protein fusions in the germ cells as part of functional analysis of proteins relevant for germ-cell development and as part of work aimed at generating and optimizing fluorescence resonance energy transfer (FRET) – based probes. At the level of manipulations of cellular function, we used a 2-photon laser to ablate the cortex of primordial germ cells thereby inducing the formation of belbs at the ablation site. This functionality of the microscope is used the in two directions. First, we investigate the effect of the induction of bleb on other events known to occur at the cell front (e.g. elevation of calcium, elevation of Rac1 activation etc), with the aim of determining the hierarchy among events contributing to the definition of the leading edge of the cell. Second, related to the first goal we determined if other cellular processes in addition to cortex breakage are required for bleb formation. Here, we manipulate the level of certain proteins and evaluate the efficiency of bleb-induction by laser light. This analysis demonstrated that in addition to cortex damage, the function of the RNA-binding protein Dead end and proteins it regulates are essential for bleb formation.
Publications
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(2010). A role for Rho GTPases and cell–cell adhesion in single-cell motility in vivo. Nature Cell Biology 12, 47-53
Kardash, E., Reichman-Fried, M., Maître, JL., Boldajipour, B., Papusheva, E., Messerschmidt, EM., Heisenberg, CP., Raz, E.
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(2010). JAM-A is a novel surface marker for NG2-Glia in the adult mouse brain. BMC Neurosci. 11:27
Stelzer S., Ebnet K., Schwamborn J.C.
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(2011). Cellular organization of adult neurogenesis in the Common Marmoset. Aging Cell 10, 28-38
Bunk E.C., Stelzer S., Hermann S., Schäfers M., Schlatt S., Schwamborn J.C.
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(2011). Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor. Development 138, 2909-14
Boldajipour B, Doitsidou M, Tarbashevich K, Laguri C, Yu SR, Ries J, Dumstrei K, Thelen S, Dörries J, Messerschmidt EM, Thelen M, Schwille P, Brand M, Lortat-Jacob H, Raz E
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(2011). Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nature Protocols 6 1835–1846
Kardash, E., Bandemer, J., Raz. E.
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(2011). Neural progenitor cells maintain their stemness through protein kinase C z mediated inhibition of TRIM32. Stem Cells 9, 1437-47
Hillje A.L., Worlitzer M.M.A., Palm T., Schwamborn J.C.
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(2011). Regulation of hub mRNA stability and translation by miR430 and the dead end protein promotes preferential expression in zebrafishprimordial germ cells. Developmental Dynamics 240, 695-703
Mickoleit M, Banisch TU, Raz E
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(2011). The E3-ubiquitin Ligase TRIM2 regulates Neuronal Polarization. J. Neurochem. 117, 29-37
Khazaei M.R., Bunk E.C., Hillje A.L., Jahn H.M., Riegler E.M., Knoblich J.A., Young P., Schwamborn J.C.
