Studies on NET4B and associated proteins

Abstract

The cytoskeleton is an essential component of the eukaryotic cell, determining both the cellular architecture and function. In plant cells, the cytoskeleton is composed of two distinct networks of filamentous proteins; microtubules and actin microfilaments, as well as a plethora of associated proteins that organise or regulate the network. Metazoans use a range of adaptor proteins, such as spectrin, α-actinin, filamin, and the FERM-domain proteins, that link actin to membrane structures. However these protein families are absent in plants, despite the predominance of the actin cytoskeleton in organelle and endomembrane trafficking. Recently a novel plant-specific superfamily of actin-binding proteins has been identified, termed the Networked (NET) family. The NET family is composed of thirteen members in Arabidopsis thaliana, divided into four phylogenetic clades, with members of each subclade associating with specific membrane compartments (Deeks et al. 2012; Wang et al. 2014). The NET4s are the only NET subfamily that can be found universally throughout the genomes of the Tracheophytes, and in A. thaliana NET4A associates with actin surrounding the vacuole. NET4B remains a relatively uncharacterised member of the NET family and was thus the focus of this project. Through live cell imaging and an actin cosedimentation assay, NET4B was shown to bind actin filaments in vivo and in vitro. The expression pattern of NET4B in plants was investigated using NET4Bpromoter::GUS lines, demonstrating a high expression in roots and guard cells. Immunogold labelling of plant roots with a NET4B specific antibody revealed its preferential localisation to the tonoplast. The NET4s therefore represent novel actin-vacuole adaptors in plants, and this project investigates the role of these proteins in plant cell growth and identifies key interacting partners that implicate the NET4s in signalling events.