Imaging membrane nanodomains
There has been a long lasting debate about the actual size of membrane nanodomains in vivo. While the original lipid raft model suggests sizes of up to 20nm, most data obtained from visualizing nanodomains in living cells point towards larger structures. Some time ago we reported on a number of nanodomains with a mean width of more than 300nm (for ore details see Ott (2017)). These structures can be resolved by standard Confocal Laser-Scanning Microscopy (CLSM). However, we also demonstrated that even though the majority of investigated membrane domains were laterally immobile, their spatio-temporal appearance may vary upon environmental changes. We are using a number of other techniques to visualize and resolve domain dynamics such as Total Internal Reflection Microscopy (TIRF), 3D Structured Illumination Microscopy (3D-SIM), Fluorescence Lifetime Imaging Microscopy (FLIM) as well as photoactivatable/ -convertible fluorophores.
Remorin proteins in membrane domains
Remorin proteins are one of the few widely accepted marker proteins for membrane nanodomains in plants. We and other labs have shown that these proteins indeed localize to large domain clusters in wild-type tissues (Lefebvre et al., 2010; Jarsch et al., 2014). Such domains were also observed when studying the interactions between the Remorin SYMREM1 and the Nod Factor Receptor NFR1 from Lotus japonicus using Fluorescence-Lifetime Imaging Microscopy (FLIM) (Tóth et al., 2012; Konrad et al., 2014; Jarsch et al., 2014) or when assessing the localization patterns of the Arabidopsis receptors FLS2 and BRI1 (Bücherl et al., 2017). Interestingly, localization of Remorin proteins to membrane micro-domains is not solely determined by their intrinsic properties (e.g. S-acylation) but most likely by another, yet unknown, protein-protein interaction (Konrad et al., 2014).
To investigate the diversity of membrane nanodomains in living cells, we cloned 20 different domain marker proteins belonging to the Remorin and the Flotillin protein families and performed extensive co-localization experiments. Indeed plant plasma membranes are covered with a variety of different, co-existing micro-domains (Jarsch et al., 2014). This analysis now provides us with valuable marker set to assess the functional specification of individual membrane nanodomains.