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 tri- choblast 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, mem- brane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselves are polarized. To investigate the mechanism un- derlying ROP2 recruitment, we employed Variable Angle Epi- fluorescence Microscopy (VAEM) and exploited mCitrine fluo- rophore blinking for single molecule localization, particle track- ing and super-resolved imaging of the trichoblast plasma mem- brane. We observed the association of mCit-ROP2 within dis- tinct 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.     «

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 tri- choblast 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, mem- brane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselv...     »