Besten, M.; Heesemans, T.; Froeling, R.; Zilliox, M.; Peeters, Y.; Romein, R.; Tian, H.; Lamers, J.; Vorselen, D.; Charrier, B.; Borst, J. W.; Sprakel, J.

The plant plasma membrane is a highly dynamic structure that is crucial for cell compartmentalization, the maintenance of (bio)chemical gradients, signaling and cell growth and responses to stress. In plants, plasma membranes are tightly connected to the cell walls that encase them. These cell walls can act as diffusion barriers and prevent the use of a wide range of synthetic fluorescent probes that have been developed to study animal cell membranes, which lack a cell wall, with live functional imaging. Here, we introduce LipoTag, a minimal chemical motif that, upon chemical conjugation, transforms hydrophobic fluorophores into water-soluble, membrane-targeted probes that can permeate plant cell walls to reach their intended location. LipoTag uses a localized positive charge in combination with a short aliphatic spacer to direct cargo to the plasma membrane. We used LipoTag to develop a suite of membrane-specific fluorescent probes that work in walled organisms beyond the plant kingdom. In addition, we used LipoTag to develop functional reporters for the quantitative imaging of membrane density, lipid order and membrane oxidation in living plant tissues. LipoTag forms a modular platform for exploring the plant plasma membrane with a suite of contemporary imaging modalities.