A Superfamily of Actin-Binding Proteins at the Actin-Membrane Nexus of Higher Plants

Abstract

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plant-specific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus

“…To gain insights into the cellular sites of IQD function, we studied the subcellular distribution of all 33 Arabidopsis IQD family members after transient expression in Nicotiana benthamiana leaves, which is a convenient system in which to monitor protein localization (Deeks et al, 2012). We expressed GFP-tagged IQDs under the control of the cauliflower mosaic virus (CaMV) 35S promoter to facilitate their detection.…”

Blobs and curves: object-based colocalisation for plant cells

“…Deeks et al 2012) At standard optical resolution the location of callose accumulation is often indistinguishable from the plasmodesmal mouth and channel (e.g. Deeks et al 2012). The majority of publications demonstrating positive colocalisation between known pore components and uncharacterised proteins use transient expression assays in plant tissues where plasmodesmata exist as isolated and distinctive entities; for this reason, complex colocalisation analysis is not required.…”

“…In tissues such as the A. thaliana root meristem the pattern of plasmodesmata is condensed, with neighbouring pores clustering at distances close to the diffraction limit of resolution (e.g. Simpson et al 2009;Deeks et al 2012). The segregation of root meristem cell boundaries into Commented [MJD2]: Reference to sub-resolution removed as we are no longer highlighting capabiltiies to interpret subresolution structures.…”

“…In this study we re-analyse a published colocalisation study of the actin-binding protein NET1A-GFP with labelled root meristem plasmodesmata (Deeks et al 2012) using object-based algorithms. We show that this method provides a quantified overview of the spatial relationship between NET1A-GFP fusion protein and aniline blue signals in units of distance rather than an abstract coefficient.…”

“…Assignment of protein localisation to plasmodesmata using fluorescence microscopy is achieved using co-labelling with either antibodies or fusions to known plasmodesmal components; or the staining of plasmodesmalassociated callose in the surrounding cell wall (e.g. Deeks et al 2012) At standard optical resolution the location of callose accumulation is often indistinguishable from the plasmodesmal mouth and channel (e.g. Deeks et al 2012).…”