Characterisation of actin cytoskeletal functions and interactions during planar polarity formation in Arabidopsis

Sammanfattning: The establishment of cell and tissue polarity is essential for the development of eukaryotic organisms including plants and animals. When cell polarity is uniformly organised within a plane of a single tissue layer, it is referred to as planar polarity. In the model plant Arabidopsis thaliana, the polarly organised formation of root hairs towards the basal (root tip-oriented) ends of epidermal cells provides an ideal model system to study planar polarity formation. The instructive cue which uniformly organises tissue polarity in the Arabidopsis root epidermis is a concentration gradient of the plant hormone auxin. This concentration gradient leads to the polar recruitment of Rho-of-plant proteins to membrane sites where root hairs will form, making them the earliest cellular markers of planar polarity. Despite having identified several of the components required for planar polarity formation and the cellular read-out of it, little is known about how planar polarity is perceived and stably executed on the single cell level. Recently, a role for ACTIN2 in polar hair positioning was described, suggesting that actin may be involved in the cellular read-out of planar polarity.In the work presented here, we further investigate the contribution of the actin cytoskeleton to polar hair positioning. We identify a role for actin upstream of polar Rho-of-plant (ROP) positioning and describe ACTIN7 and ACTIN-INTERACTING PROTEIN1-2 (AIP1-2) as components required for planar polarity formation which interact directly in yeast and in vitro and genetically during planar polarity formation. AIP1-2 expression proves hair cell file-specific and depends on WEREWOLF function, revealing a link between planar polarity and cell fate patterning in the root. In addition we find that ACT7 genetically interacts with SABRE (SAB) and that actin and SAB co-localise at the cell plate and plasma membrane domains during cell division. Furthermore, we show that actin is not only involved in the positioning, but also in the organisation of the polar membrane domain which marks where a root hair will form. Among the components which localise at this site are the phosphoinositide-biosynthetic enzyme PHOSPHATIDYLINOSITOL 4-PHOSPHATE 5-KINASE3 (PIP5K3), the DYNAMIN-RELATED PROTEINs (DRPs) DRP1A and DRP2B, the D6 PROTEIN KINASE (D6PK) and membrane sterols. For several of these components, we report a function in planar polarity formation and in the organisation of the hair initiation site.With this work, we provide deeper insight into the function of the actin cytoskeleton and its interactions during planar polarity formation and identify additional components that contribute to the process. Moreover, we report AIP1-2 placement under control of the cell fate patterning system during establishment of planar polarity. Our results provide stepping stones for future studies aimed at investigating the mechanistic processes that contribute to planar polarity formation in more molecular and cellular detail.