Molecular characterisation of edge-directed trafficking in Arabidopsis

The complex organisation and spatio-temporally variable dynamics of the eukaryotic endomembrane system is underpinned by an equally complex array of molecular machinery. Whilst the organisation and dynamics of eukaryotic endomembranes have been observable for some time, detailed study of the molecular machinery associated with endomembranes has only become possible in recent decades. In plants, the organisation and dynamics of the endomembrane system is intimately connected to their plastic development and unique growth properties. It has been suggested that many of the specific requirements of plant growth and development are supported by unique features of post-Golgi trafficking, which differs substantially from that of mammals. In the model plant species Arabidopsis thaliana, the trans-Golgi network/early endosome functions as a post-Golgi sorting hub on which trafficking pathways to and from the cell surface converge. The function of the trans-Golgi network/early endosome is underpinned by a large number of molecular components, of which the Rab-A GTPases are a significant part. Rab GTPases cycle between membrane-associated and membrane-dissociated states and spatio-temporally organise the recruitment of other essential trafficking machinery to endomembranes. Recent work has shown that a member of the Arabidopsis Rab-A GTPases, RAB-A5c, localises to compartments at the geometric edges of cells in growing tissues as well as the trans-Golgi network/early endosome. Inhibition of RAB-A5c function produces defects in anisotropic growth that can be mimicked in silico through local perturbations in cell wall mechanical properties around cell geometric edges in finite element models of plant cells. This and other observations have led to the hypothesis that RAB-A5c defines a polarised pathway from the trans-Golgi network/early endosome to the cell surface via cell geometric edges, which possibly functions to modify the cell wall in growing tissues specifically around cell edges. The existence of such a trafficking pathway remains uncertain however in the absence of any identified pathway cargoes, which also limits possible further investigation of the organisation and functions of such a pathway.
In this thesis, I investigate possible interactors of RAB-A5c and components of its associated trafficking pathway identified through co-immunoprecipitation. This work identifies two Receptor-Like Proteins as possible direct interactors of RAB-A5c and components of its associated trafficking pathway. This identification confirms the existence of an edge-directed trafficking pathway in growing tissues of Arabidopsis and that cell geometric edges are a discrete spatial domain of the Arabidopsis plasma membrane. Moreover, localisation and trafficking of truncated variants of these Receptor-Like Proteins gives indications as to their possible functions and contributions of different protein domains to their trafficking. Two hypotheses for the function of these Receptor-Like Protein components of edge-directed trafficking are presented and will be the subject of future investigations. Finally, perturbation of the endomembrane and cytoskeletal systems challenges previously-held assumptions about the organisation of edge-directed trafficking and the suitability of techniques commonly used to investigate Rab GTPase function in plants.

Advisor: Moore, I