NET2A: bridging the gap in plant-specific actin-membrane interactions

Abstract

Both metazoa and fungi employ a plethora of adaptor proteins in order to create specialist sites of interaction between the actin cytoskeleton and membranes. Such membrane adaptor proteins are of fundamental importance in the coordination of intercellular signalling and communication, as well as mediating the spatial arrangement of specialized subcellular compartments and the site of endocytosis and exocytosis (Hussey et al., 2006; Moscatelli et al., 2012). Despite the vast assortment of adaptor proteins described in animal cells, including spectrin, ankyrin, α-actinin and members of the FERM domain superfamily, no functional plant homologues have yet been elucidated. Characterizing novel plant-specific ABPs could provide unique interfaces between membranes and actin, however their identities have remained elusive. Work outlined in this thesis identifies the pollen specific ABP known as NET2A, an integral member of the recently recognized Networked (NET) superfamily of actin-binding proteins (Deeks et al., 2012).

NET2A revealed a striking network of filaments decorated with foci in the late stages of A. thaliana microspore development. These prolific filamentous arrays were observed surrounding the vegetative and sperm nuclei during the late stages of microspore development. Furthermore, NET2A is also tethered at the distal pollen tube plasma membrane, forming discreet sub-micron foci that spectacularly decorate actin filaments in a distinctive ‘beads on a string’ orientation. This NET2A-actin association is mediated via conserved tyrosine residues found within a novel actin-binding domain that constitutes a plant-specific superfamily of thirteen Arabidopsis proteins, sub-divided into four distinct phylogenetic clades, each of which label a unique membrane system. Members of the NET1, 3 and 4 clades highlight interfaces at the plasmodesmatal cell junctions, nuclear membrane and tonoplast respectively, thereby emphasizing the significance of this superfamily in arbitrating actin-membrane interactions across a diverse range of tissues.

The unique sub-cellular localization of NET2A is thought to be mediated by the oxysterol binding protein (OSBP)-related proteins (ORPs). These proteins may cooperate with NET2A and function within the PRK-signalling pathway, which constitutes a pathway that is integral to the maintenance of anisotropic growth in pollen tubes.

Inactivation of the NET2A gene resulted in distorted vegetative nuclear morphology and aberrant callose deposition patterning in dehiscent pollen grains. Furthermore, defects were exhibited in the reverse fountain streaming of mitochondria in growing pollen tubes, thus advocating a number of prospective in vivo functions of NET2A. These may include acting as a transducer of external signals originating from the pistil, subsequently relaying them to the actin cytoskeleton; a function thought to occur in a functionally redundant manner, in association with the three other pollen-expressed members of the NET2 sub-clade. Another theory is that NET2A may facilitate the recruitment of longitudinal actin filaments to the distal pollen tube PM, and may function as a tethering protein in developing microspores, protecting the male germ unit (MGU) from myosin-mediated distortion.

The findings of this thesis have defined a unique sub-cellular localization for NET2A in pollen, and a number of statistically significant phenotypes have emerged from its inactivation. Corresponding hypotheses have subsequently been proposed regarding NET2A function, in an attempt to account for these observations. However, additional work must now be undertaken in order to substantiate these ideas.