Nanodomain-mediated lateral sorting drives polarization of the small GTPase ROP2 in the plasma membrane of root hair cells

Formation of root hairs involves the targeted recruitment of the cellular growth machinery to the root hair initiation domain (RHID), a specialized site at the plasma membrane (PM) of trichoblast cells. Early determinants in RHID establishment are small GTPases of the Rho-of-plants (ROP) protein family, which are required for polarization of downstream effectors, membrane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselves are polarized. To investigate the mechanism underlying ROP2 recruitment, we employed Variable Angle Epifluorescence Microscopy (VAEM) and exploited mCitrine fluorophore blinking for single molecule localization, particle tracking and super-resolved imaging of the trichoblast plasma membrane. We observed the association of mCit-ROP2 within distinct membrane nanodomains, whose polar occurrence at the RHID was dependent on the presence of the RopGEF GEF3, and found a gradual, localized decrease of mCit-ROP2 protein mobility that preceded polarization. We provide evidence for a step-wise model of ROP2 polarization that involves (i) an initial non-polar recruitment to the plasma membrane via interactions with anionic phospholipids, (ii) ROP2 assembly into membrane nanodomains independent of nucleotide-binding state and, sub-sequently, (iii) lateral sorting into the RHID, driven by GEF3-mediated localized reduction of ROP2 mobility.