Irene Lavagi
PhD student

Research

Despite recent advances in the characterisation of the secretory pathway, transport to the plasma membrane is poorly understood in any eukaryotic system.
Plant cells also synthesise and secrete polysaccharides that are used to form the cell wall. Transport of cell wall polysaccharides is unknown beyond the Golgi apparatus. The project aims to explore trafficking pathways of both proteins and polysaccharides between the late Golgi and the cell surface.
In order to do so, a method to monitor secretion of cell wall polysaccharides was required. A biochemical assay using suspensions of tobacco protoplasts was developed. This could then be used to compare secretion of polysaccharides with secretion of soluble proteins (for which a biochemical assay had already been developed), and possibly also membrane spanning proteins and GPI-anchored proteins. By doing so, it could be explored whether different cargo molecules are sorted and secreted via the same machinery. In fact, it remains to be determined which type of vesicles delivers proteins to the cell surface, whether there are different vesicles for different classes of proteins (i.e. soluble proteins, membrane spanning proteins, GPI-anchored proteins) and whether cell wall polysaccharides follow a distinct secretory pathway or are incorporated into one of these protein-containing vesicles that shuffle between the trans Golgi network and the cell surface.
Moreover, as for soluble proteins, recruitment of GPI-anchored proteins requires adaptor proteins, which are unknown to date. GFP-fusions of a GPI-anchored protein were produced in order to observe both their subcellular localisation and the effect of different drugs by confocal scanning microscopy.
Sorting and traffic of different cargo molecules beyond the Golgi apparatus was explored by different immmunohistochemical techniques: confocal scanning microscopy and transmission electron microscopy. The monoclonal antibodies for cell wall components that had been selected and chosen with the biochemical assay were also tested by immunofluorescence on whole mounts of Tobacco roots by confocal scanning microscopy. Immunogold single and double labellings were performed to observe where 1) secretory proteins 2) polysaccharides (pectins) 3) GPI-anchored proteins (arabinogalactan proteins) could be found in Tobacco root cells.

Education

October 2004 -	PhD title “Transport of proteins and polysaccharides between the late Golgi and  the plasma membrane ” University of Leeds, University of Oxford Brookes and University of Warwick Sponsored by the EU consortium Pharmaplanta, Framework VI
October 2001-June 2004	BSc Biological Sciences with Molecular Genetics - Warwick University

Contact

University of Leeds
Faculty of Biological Sciences
L C Miall Building
Room 9.13-9.14
Clarendon Way
Leeds - LS2 9JT
West Yorkshire
0113 3437031

bgyil@leeds.ac.uk