Konfokales Laserscanning-Mikroskop
Final Report Abstract
Vacuole: Vacuoles are multifunctional organelles that are essential for the sessile lifestyle of plants. To study physiology, morphology and biogenesis of the large central vacuole we use the Leica SP5II to image the fluorescent dye BCECF, that accumulates efficiently into the vacuolar lumen. In order to study vacuole biogenesis we performed high precision recording of z-stacks and subsequent surface 3D rendering to create highly plastic images of developing vacuoles in Arabidopsis roots. With this we were able to show that already in young cells, the vacuole is represented by only one continuous compartment that is tubular organised and highly complex. In combination with the analyses of fluorescence protein (FP)-labelled markers for different subcellular compartments and fluorescence recovery after photobleaching (FRAP) we were able to demonstrate that the endoplasmic reticulum is the major membrane source for the biogenesis of the lytic vacuole in Arabidopsis. Z-stacking and subsequent surface 3D rendering was also used to show the importance of vacuolar potassium uptake for proper vacuolar morphology and stomatal movement. TGN/EE: The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrincoated vesicles, although direct proof for their participation is missing. We used the SP5II to perform colocalisation analyses of fluorophores-tagged proteins in order to demonstrate that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. Along this line, we were able to show that the Arabidopsis SAND1 protein is an essential player for the fusion of MVBs with the vacuole, which constitutes a major difference in SAND1 function between plant and non plant organisms, since SAND1 in animals and yeast is required for early-to-late endosomal maturation. Calcium imaging: We use the SP5II routinely in our lab to study Ca2+ dynamics in vivo. The SP5II provides a suitable platform for high precision-imaging, which is a prerequisite to study Ca2+ signalling with high temporal and spatial resolution. To facilitate Ca2+ imaging for plant researchers, we developed and tested a set of vectors that encode different versions of the Förster resonance energy transfer (FRET)-based Ca2+ indicator yellow cameleon 3.6. We were able to establish transgenic Arabidopsis lines in which the Ca2+ sensor is targeted to different subcellular compartments. In an ongoing process we are continuously improving our protocol for live cell imaging in plants. pH imaging: To investigate the role of vacuolar H+-ATPases (V-ATPases) with respect to intracellular acidification we extensively use the SP5II to improve and develop microscope-based methods for quantitative pH measurements in Arabidopsis. The pH-sensitive fluorescent dye BCECF accumulates specifically in vacuoles of roots, which allows precise measurements of steady-state vacuolar pH. Moreover this method is suited to follow pH dynamics over time. Besides vacuolar pH measurements we developed a genetically encoded pH sensor that is capable to measure pH in the trans-Golgi network/early endosome (TGN/EE) (Scholl and Schumacher, unpublished). This sensor will be of great importance in the future to clarify the role of TGN/EE localised V-ATPase for pH regulation in the endomembrane system. Protein-Protein interaction: Due to the installation of the fluorescence lifetime imaging (FLIM) upgrade package for the SP5II, we were able to set up a platform to study in vivo protein-protein interactions via Förster resonance energy transfer (FRET) by measuring the lifetime of GFP-tagged proteins. Using this method we were able to show in vivo interaction of transcription factors controlling the proliferative potential of stem cells in the shoot apical meristem. Moreover we used this technique to establish in vivo interaction of WNK8 kinase with PP2CA phosphatase, both involved in ABA signal transduction.
Publications
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Multivesicular Bodies Mature from the Trans-Golgi Network/Early Endosome in Arabidopsis. Plant Cell 23: 3463-3481. (2011)
Scheuring, D., Viotti, C., Krueger, F., Kuenzl, F., Sturm, S., Bubeck, J., Hillmer, S., Frigerio, L., Robinson, D.G., Pimpl, P., and Schumacher, K.
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FRET-based genetically encoded sensors allow highresolution live cell imaging of Ca2+ dynamics. The Plant journal 69: 181-92. (2012)
Krebs, M., Held, K., Binder, A., Hashimoto, K., Herder, den, G., Parniske, M., Kudla, J., and Schumacher, K.
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Lack of the Golgi phosphate transporter PHT4;6 causes strong developmental defects, constitutively activated disease resistance mechanisms and altered intracellular phosphate compartmentation in Arabidopsis. The Plant journal 72: 732-44. (2012)
Hassler, S., Lemke, L., Jung, B., Möhlmann, T., Krüger, F., Schumacher, K., Espen, L., Martinoia, E., and Neuhaus, H.E..
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Regulation of the V-type ATPase by redox modulation. The Biochemical journal 448: 243-51. (2012)
Seidel, T., Scholl, S., Krebs, M., Rienmüller, F., Marten, I., Hedrich, R., Hanitzsch, M., Janetzki, P., Dietz, K., and Schumacher, K.
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Live cell imaging of cytoplasmic and nuclear Ca2+ dynamics in Arabidopsis roots. Cold Spring Harbor protocols 2013: 776-80. (2013)
Krebs, M. and Schumacher, K.
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PDMP induces rapid changes in vacuole morphology in Arabidopsis root cells. J. Exp. Bot. 64: 529-540. (2013)
Krüger, F., Krebs, M., Viotti, C., Langhans, M., Schumacher, K., and Robinson, D.G.
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The endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis. Plant Cell 25: 3434-49. (2013)
Viotti, C., Krüger, F., Krebs, M., Neubert, C., Fink, F., Lupanga, U., Scheuring, D., Boutté, Y., Frescatada-Rosa, M., Wolfenstetter, S., Sauer, N., Hillmer, S., Grebe, M. and Schumacher, K.
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(2014). Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion. Current biology : CB 24: 1383-9. (2014)
Singh, M.K., Krüger, F., Beckmann, H., Brumm, S., Vermeer, J.E.M., Munnik, T., Mayer, U., Stierhof, Y., Grefen, C., Schumacher, K., and Jürgens, G.
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Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake. Proc. Natl. Acad. Sci. U.S.A. 111: E1806-14. (2014)
Andrés, Z., Pérez-Hormaeche, J., Leidi, E.O., Schlücking, K., Steinhorst, L., McLachlan, D.H., Schumacher, K., Hetherington, A.M., Kudla, J., Cubero, B., and Pardo, J.M.
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Regulatory Framework for Shoot Stem Cell Control Integrating Metabolic, Transcriptional, and Phytohormone Signals. Developmental Cell 28: 438-449. (2014)
Schuster, C., Gaillochet, C., Medzihradszky, A., Busch, W., Daum, G., Krebs, M., Kehle, A., and Lohmann, J. A
