Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP

Chris Hawes1, Federica Brandizzil1, Henri Batoko2, Ian Moore2

1 Oxford Brookes University, Oxford, 2 University of Oxford, Oxford
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.3
DOI:  10.1002/0471143030.cb1303s09
Online Posting Date:  May, 2001
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Abstract

By the addition of plant or mammalian targeting sequences, green fluorescent protein (GFP) can be directed to the endoplasmic reticulum, the Golgi apparatus, or both organelles in plant cells. This unit describes the application of rapid Agrobacterium and virusā€mediated transient expression systems in leaf tissue to permit visualization of ER and Golgi dynamics in vivo by epifluorescence or confocal microscopy.

     
 
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Table of Contents

  • Basic Protocol 1: Transient Expression for Visualization of ER and Golgi Probes in Leaves
  • Alternate Protocol 1: Virus Mediated Expression of GFP Constructs
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transient Expression for Visualization of ER and Golgi Probes in Leaves

  Materials
  • Suitable Agrobacterium vector (e.g., pVKH18En6) with multiple cloning site and appropriate selectable marker
  • Agrobacterium tumefaciens(e.g., GV3101::pMP90)
  • YEB medium containing appropriate selective antibiotic (see recipe)
  • 5% (v/v) sodium hypochlorite or 1% (w/v) Virkon (Amtec Int. Ltd.)
  • Infiltration medium (INM; see recipe)
  • Four‐week‐old greenhouse plants of Nicotiana tabacum, N. clevelandii, and N. benthamiana
  • Shaking incubator, 28°C
  • 1.5‐ml microcentrifuge tubes, sterile
  • Spectrophotometer
  • 1‐ml disposable plastic syringe, without needle
  • 22° to 25°C greenhouse for growing plants
  • Permanent marker pen
  • Fine scissors
  • Slides and coverslips (use thickness 0 for confocal microscopes)
  • Electrical or waterproof tape
  • Conventional epifluorescence microscope, laser‐scanning confocal microscope (preferred), or equivalent, with appropriate filters (e.g., standard FITC filter block)
  • Confocal‐imaging time‐lapse software (Zeiss, Leica, Biorad)
  • Additional reagents and equipment for vector construction, transformation, and cell culture (see appendix 3A) adapted for plants
CAUTION: All solutions used to culture and wash bacteria should be treated with a suitable disinfectant before discarding in an autoclavable waste container. Resulting plant material should be treated as biological hazard and handled accordingly.NOTE: Sterile conditions are required for culture and handling of Agrobacterium, but not during tobacco leaf infiltration and subsequent incubation of the plant. All equipment coming into contact with bacteria should be autoclaved.

Alternate Protocol 1: Virus Mediated Expression of GFP Constructs

  • GFP chimera construct
  • PVX vector (pTXS.P3C2) with multiple cloning site and T7 promoter (available from various laboratories)
  • Midiprep kit, without RNase (Qiagen)
  • T7 RNA polymerase transcription kit (e.g., Ambion T7 message in machine kit)
  • SpeI or SphI restriction enzymes (see appendix 3A)
  • Aluminum oxide
  • Aluminum oxide dispenser: small glass flask containing abrasive, sealed with miracloth
  • Additional equipment and reagents for restriction digestion, phenol/chloroform extraction, and ethanol precipitation (see appendix 3A), and quantification of DNA concentration by spectroscopy (see appendix 3D)
IMPORTANT NOTE: In some countries in order to handle plant viruses as vectors, a license must be obtained from the appropriate authorities.IMPORTANT NOTE: All solutions used for virus work must be RNase‐free.
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Figures

Videos

Literature Cited

Literature Cited
   Baulcombe, D.C., Chapman, S.N., and Santa Cruz, S. 1995. Jellyfish green fluorescent protein as a reporter for virus infection. Plant J. 7:1045‐1053.
   Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12:8711‐8721.
   Blackman, L.M., Boevink, P., Santa Cruz, S., Palukaitis, P., and Oparka, K.J. 1998. The movement of cucumber mosaic virus traffics into sieve elements in minor veins of Nicotiana clevelandii. Plant Cell 10:525‐537.
   Boevink, P., Oparka, K., Santa Cruz, S., Martin, B., Betteridge, A., and Hawes, C. 1998. Stacks on tracks: The plant Golgi apparatus traffics on an actin/ER network. Plant J. 15:441‐447.
   Boevink, P., Martin, B., Oparka, K., Santa Cruz, S., and Hawes, C. 1999. Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A. Planta 208:392‐400.
   Chapman, S.N., Kavanagh, T., and Baulcombe, D.C. 1992. Potato‐Virus X as a vector for gene expression in plants. Plant J. 2:549‐557.
   Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20:448‐455.
   Cubitt, A.B., Woolenweber, L.A., and Heim, R. 1999. Understanding structure‐function relationships in the Aequoria victoria green fluorescent protein. In Green Fluorescent Proteins (K.F. Sullivan and S.A. Kay eds.) pp. 19‐30. Academic Press, San Diego.
   Di Sansebastiano, G.‐P., Paris, N., Marc‐Martin, S., and Neuhaus, J.‐M. 1998. Specific accumulation of GFP in a non acidic vacuolar compartment via a C‐terminal propeptide‐mediated sorting pathway. Plant J. 15:449‐457.
   Gadella, T.W.J., van der Krogt, G.N.M., and Bisseling, T. 1999. GFP‐based FRET microscopy in living plant cells. Trends Plant Sci. 4:287‐291.
   Grebenok, R.J., Pierson, E., Lambert, G.M., Gong, F.‐C., Afonso, C.L., Haldeman‐Cahill, R., Carrington, J.C., and Galbraith, D.W. 1997. Green‐fluorescent protein fusions for efficient characterisation of nuclear targeting. Plant J. 11:573‐586.
   Gu, X.J. and Verma, D.P.S. 1997. Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 9:157‐169.
   Haseloff, J., Siemering, K.R., Prasher, D.C., and Hodge, S. 1997. Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. U.S.A. 94:2122‐2127.
   Hawes, C., Boevink, P., and Moore, I. 2000. Green fluorescent protein in plants. In Protein Localization by Fluorescence Microscopy: A Practical Approach (V.J. Allen, ed.) pp. 163‐177. Oxford University Press, Oxford.
   Itaya, A., Hickman, H., Bao, Y.M., Nelson, R., and Ding, B. 1997. Cell‐to‐cell trafficking of cucumber mosaic virus movement protein: Green fluorescent protein fusion produced by biolistic bombardment in tobacco. Plant J. 12:1223‐1230.
   Köhler, R.H. and Hanson, M.R. 2000. Plastid tubules of higher plants are tissue‐specific and developmentally regulated. J. Cell Sci. 113:81‐89.
   Köhler, R.H., Zipfel, W.R., Webb, W.W., and Hanson, M.R. 1997. The green fluorescent protein as a marker to visualise plant mitochondria in vivo. Plant J. 11:613‐621.
   Köst, B., Spielhofer, P., and Chua, N.‐H. 1998. A GFP mouse talin fusion protein labels plant actin filaments in vivo and visualises the actin cytoskeleton in growing pollen tubes. Plant J. 16:393‐401.
   Marc, J., Granger, C.L., Brincat, J., Fisher, D.D., Kao, T.‐H., McCubbin, A.G., and Cyr, R.J. 1998. A GFP‐MAP4 reporter gene for visualising cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927‐1939.
   Nebenführ, A., Gallagher, L.A., Dunahay, T.G., Frohlick, J.A., Mazurkiewicz, A.M., Meehl, J.B., and Staehelin, L.A. 1999. Stop‐and‐go movements of plant Golgi stacks are mediated by the acto‐myosin system. Plant Physiol. 121:1127‐1141.
   Oparka, K.J., Roberts, A.G., Prior, D.A.M., Baulcombe, D., and Santa Cruz, S. 1995. Imaging the green fluorescent protein in plants–Viruses carry the torch. Protoplasma 189:133‐141.
   Rakousky, S., Kocabek, T., Vincenciova, R., and Ondrej, M. 1998. Transient beta‐glucuronidase activity after infiltration of Arabidopsis thaliana by Agrobacterium tumefaciens. Biologia Plantarum 40:33‐41.
   Rossi, L., Escudero, J., Hohn, B., and Tinland, B. 1993. Efficient and sensitive assay for T‐DNA‐dependent transient gene expression. Plant Mol. Biol. Reporter 11:220‐229.
   VandenBosch, K.A. 1991. Immunogold labelling. In Electron Microscopy of Plant Cells (J.L. Hall and C. Hawes, eds.) pp. 181‐218. Academic Press, London.
Key References
   Boevink, P., Santa Cruz, S., Hawes, C., Harris, N., and Oparka, K.J. 1996. Virus‐mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. Plant J. 10:935‐941.
  This paper illustrates the production of GFP targeted to the ER of living plant cells using a PVX‐based expression system.
   Boevink et al. 1998. See above.
  The authors describe the fusion of the wild‐type GFP to the transmembrane domain of a rat sialyl transferase in Nicotiana cells and its localization at the GA. Moreover, they describe the splicing of the wild‐type GFP to the C‐terminus of the Arabidopsis homologue of the yeast HDEL receptor, aERD2 and the localization of the protein chimera at the ER and GA, using the potato virus X expression system.
   Haseloff et al. 1997. See above.
  In this paper the authors describe how the wild‐type GFP was engineered to be expressed in plants in a non‐virus mediated system and to be targeted to the ER.
   Nebenführ et al. 1999. See above.
  The authors describe the fusion of a soybean Gm‐Man1, encoding the resident Golgi protein α‐1,2 mannosidase‐1, to the green fluorescent protein, and its targeting to the Golgi of Bright Yellow 2 suspension‐cultured cells.
Internet Resources
  http://www.brookes.uk/schools/bms/research/molcell/hawes/gfp/gfpold.html
  Web site for movies obtained at the confocal laser microscope in Nicotiana clevelandii epidermal cells expressing GFP fused to the C‐terminus of the trans‐membrane domain of a rat sialyl transferase (localized at the GA) and the GFP spliced to the C‐terminus of the Arabidopsis homologue of the yeast HDEL receptor, aERD2 (localized at the ER and GA).
  http://www.plantsci.cam.ac.uk/Haseloff/Home.html
  Web site for movies of GFP expressed in Arabidopsis root tips showing different patterns of GFP expression generated by enhancer detection.
  http://www.mbg.cornell.edu/kohler/kohler_Trends.html
  Web site with movies showing GFP targeting to plastids and mitochondria.
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