STACKS ON TRACKS - Targeting GFP to the Plant Golgi Apparatus

It would therefore be predicted to reside predominantly in the Golgi and cycle to and from the ER. Segments of infected leaves were observed by confocal microscopy and movies made using Zeiss time resolved software. The relationship between the Golgi apparatus, ER and the cytoskeleton was observed in aERD2-GFP expressing leaf cells after paraformaldehyde fixation, permeabilisation and staining with rhodamine conjugated phalloidin.

Expression of rat sialyl transferase signal anchor sequence-GFP in leaf epidermal cells

Immunogold electron microscopy with an anti-GFP serum (Molecular Probes) showed the construct to be predominantly located towards the trans-face of the Golgi stacks. This result indicate that the mechanism employed by mammalian cells for locating Golgi transferases also functions in plant cells, suggesting the evolution of a pan kingdom targeting strategy for such processing enzymes. Epidermal leaf cells expressing the aERD2-GFP protein again exhibited a population of highly flourescent mobile organelles, but also a cortical reticular network of ER extending into trans-vacuolar strands.

Expression of aERD2-GFP in a leaf epidermal cell

We have previously described this cortical ER network which can be revealed by GFP-HDEL fusions. Immuno-electron microscopy showed the location of the ERD2-GFP protein was also in the Golgi stacks which as in the TMD-GFP fusion were again highly mobile but now could be observed to be exclusively assocated with the ER and travelled along the cortical ribbons of ER membrane whilst occasionally jumping onto rapidly streaming strands of ER. This movement of Golgi is both uni- and bi-directional along a strand of ER and often saltatory for several seconds before accelerating down a strand. This novel close association of Golgi and ER can be viewed in the movies- showing tracking of Golgi on cortical ER. aERD2-GFP fusion. Movie 1 (avi file) Movie 2 (avi file)

Epidermal cell cortical actin network (red)
in a cell expressing aERD2-GFP.
By reducing the green signal and removing
ER fluorescence the Golgi can be seen aligned
on actin cables

Rhodamine phalloidin staining of the epidermal cells revealed a cortical network of actin filaments which were overlaid by the cortical ER network. By reducing the contrast of the ER in green image of these double labelled preparations it could be seen that the Golgi bodies were also associated with this actin network.