261 Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch, France, l-3 July 1998

Green fluorescent protein to study endomembrane dynamics in plant cells

Chris Hawes*, Karl Oparkae, Simon Santa Cruz§ and Petra Boevinks

*Research School of Biological & Molecular Sciences, Oxford Brookes University, Oxford OX3 OBP, UK

5 Scottish Crop Research Institute, hergowrie Dundee, DD2 5DA, UK

Introduction The inter-relationships between the major organelles of the secretory pathways of yeast and mammalian cells are well documented and both the anterograde and retrograde pathways of protein, membrane and vesicle flow between the endoplasmic reticulum (ER) and Golgi apparatus (GA) have been dissected morphologically, biochemically and with molecular techniques (Farquhar MG, Palade GE (1998) Trends CelI &I. 8, 2-10). In contrast the relationship between the ER and GA in higher plants is less clear. With the development of the jellyfish green fluorescent protein (GFP) as a vital marker, it is now possible to label components of the secretory system and to study their dynamics in living cells (Cole NB et aI. (1996) Science 273,797-801). We have investigated the relationship between tobacco leaf ER and GA using GFP targeted to the two organelles. To target the ER the signal peptide of sporamine, a vacuolar storage protein was spliced to the N- terminus of GFP and the HDEL tetrapeptide was spliced to the carboxy terminus and expressed in tobacco cells leaf cells using the potato virus X expression system (Boevink P et al. (1996) The Plant Journal, 10, 935-941). For GA targeting, the trans-membrane domain (signal anchor sequence) of a rat sialyl transferase was spliced to GFP (STtmd-GFP), whilst both the ER and GA were labelled with a chimeric GFP-aERD2 protein (the arabidopsis H/KDEL receptor homologue) was expressed by the same system.

Results and Discussion In leaf cells of Nicotiana cledandii infected with the virus construct expressing GFP with the N- terminal signal peptide and C-terminal HDEL tetrapeptide the protein was targeted to the cortical network of ER. Confocal microscopy of leaf epidermal cells revealed that this network was labile in nature with tubules of ER being attached to rapidly streaming fruns-vacuolar strands. In contrast, the chimeric protein STtmd- GFP when expressed in leaves was located in numerous small fluorescent bodies in the cytoplasm. These were highly motile showing unidirectional movement akin to cytoplasmic

streaming and less organised saltatory movements at the cell cortex. Immunogold labelling of these cells prepared by the progressive lowering of temperature technique and embedded in LR White resin, with an anti-GEE serum, demonstrated labelling of the GA predominantly over the trans-half of the stacks. As there is no evidence for sialyl-transferases in plant cells, this result demonstrates that an animal Golgi targeting sequence can function in plant cells and target a protein to a similar location. This suggests that plant Golgi may utilise targeting mechanisms similar to those employed by mammalian cells for compartmentalising glycan transferases. Confocal microscopy of aERD2-GFP expression in leaves again showed that the probe was located in highly fluorescent spherical bodies, but also at a low intensity in the cortical network of ER tubules which were morphologically identical to the network labelled with GEE-HDEL. Immunogold detection of GFP also showed labelling over the GA. In these cells the Golgi bodies were also highly motile but now could be seen to be associated with the ER and moving over the surface of the cortical network of ER tubules. Such movement was tracked using the multiple frame averaging facility of the confocal microscope.

Confocal microscopy of fixed ERD2-GFP expressing leaf epidermal cells after rhodamine phalloidin staining showed that the cortical ER network was associated with an underlying actin network and that the Golgi bodies were also associated with this actin network. Treatment of leaves with N-ethylmaleimide froze all Golgi movement over the ER network, whilst depolymerisation of the actin network with cytochalasin resulted in an inhibition of Golgi movement and clustering of the fluorescent bodies on small islands of lamellar ER within the cortical tubular network. This data indicated the presence of an actin/myosin motor system which is most likely responsible for Golgi movement.

To further investigate the close spatial relationship between the cortical ER network and the Golgi stacks, leaves expressing ERD2-GFl’ or STtmd-GFP were treated with brefeldin A (BFA). In both cases treatment with this secretory inhibitor at 10 l&ml and SOpg/ml resulted in disappearance of the fluorescent Go@. This was

262 Biology of the Cell (1998) 90, 247-290

accompanied by an increase in brightness of the GFP fluorescence associated with the ER in ERD2- GFP expressing leaves, but in STtmd-GFP expressing cells with no ER fluorescence Golgi disappearance was accompanied by a concomitant build up of fluorescence in the ER. This data suggests that BFA has induced a retrograde transport of Golgi protein to the ER similar to that reported for mammalian cells (Lippincott- Schwartz J et al. (1990) Cell 60,821-836). The BFA induced deconstruction of the Golgi proved to be reversible as within five hours of floating BFA treated leaf segments on water fluorescent Golgi could again be observed which were also highly motile over the ER network.

This putative reabsorption of leaf Golgi into the nearby ER network is in contrast to the previously reported BFA induced vesiculation of

Golgi in meristematic root cells where there was no evidence of retrograde transport back to the ER (Satiat-Jeunemaitre B, Hawes C (1992) J. Cell Sci. 103,1153-1166). However, in the root system there is to date no equivalent evidence for the intimate ER-Go&i relationship as reported here in leaves. Due to the extremely close proximity of the two organelles, the possibility exists for a novel form of membrane and/or protein transfer between the ER and the &face of the plant Golgi in which the Golgi stacks travel over the surface of the ER collecting products packaged in vesicles or even in tubules from exit sites on the ER.

Acknowledgement This work was supported by a grant from the Leverhulme Trust