The relationship between endomembranes and the plant cytoskeleton.

Abstract

Basic knowledge on the mechanisms governing protein transport within the endoplasmic reticulum (ER) and between ER and the Golgi apparatus is scarce in plants. In mammalian cells, microtubule integrity is essential for ER dynamics and structural maintenance of the Golgi apparatus. Moreover, a dynamic and yet organised interaction of microtubules and associated dyneins orchestrates anterograde and retrograde protein transport between ER and Golgi apparatus. Plant cells are characterised by a pronounced physical organisation, which is achieved through the main cytoskeletal elements, microtubules and actin filaments. However, the involvement of the cytoskeleton in the structural dynamics and protein transport between heterologous organelles of the secretory pathway is yet to be fully characterised. The main objectives of the work reported in this article were to investigate the dynamic relationship between the cytoskeleton and the ER/Golgi continuum, and to quantify the flow of material between ER and Golgi membranes in tobacco epidermal cells and BY-2 cells (Brandizzi et al., 2002; Saint-Jore et al., 2002). We have taken an approach based on the expression of the green fluorescent protein and its spectral derivatives to visualise, in vivo, cytoskeletal elements and secretory organelles (Fig. 1). We have available a bank of probes based on fluorescent proteins that highlight ER, Golgi or both (Boevink et al., 1998; Saint-Jore et al., 2002). Similarly, to visualise actin and microtubules, we have used fluorescent protein chimaeras based on proteins of the GFP family (Brandizzi et al., 2002; Ueda et al., 1999). The involvement of the cytoskeleton in protein transport between ER and Golgi was initially investigated with the fungal metabolite brefeldin A (BFA) (Saint-Jore et al., 2002). The research then developed on analyses based on fluorescence recovery after photobleaching (FRAP) technology (Brandizzi et al., 2002) to measure biomolecular kinetic rates of protein movement within the secretory system of living plant cells. We show that the Golgi and ER associate preferentially with the actin cytoskeleton and the basic dynamics of the two organelles depend on the integrity of the actin network. In the absence of actin, Golgi bodies clump together and stop moving. Similarly, the movement of the ER tubules is arrested. However, when the microtubule cytoskeleton is depolymerised, there is little or no effect on the ER and Golgi. Exposure of tissue to the secretory inhibitor BFA resulted in the redistribution of the GFP-constructs into the ER in both leaf epidermal cells and BY2 cells. Treatment of the cells with the protein synthesis inhibitor cycloheximide did not prevent this effect, indicating that building up of ER fluorescence was not due to new synthesis of GFP during the experimental period. Depolymerisation of both the actin and microtubule cytoskeletons had no effect on the BFA-induced redistribution of Golgi targeted GFP-constructs. Upon removal of BFA, the Golgi stacks reformed independently from either protein synthesis or the cytoskeleton. These results indicated that observed transport from ER to Golgi in these cell types does not require cytoskeletalbased mechanochemical motor systems. To further investigate these transport processes, we used photobleaching techniques to selectively bleach individual Golgi, and we measured the rate of * Corresponding author. Tel.: +44-1865-483290; fax: +44-1865-483955. E-mail address: fbrandizzi@brookes.ac.uk (F. Brandizzi). Cell Biology International 27 (2003) 177–179 Cell Biology International