Toward atomic force microscopy and mass spectrometry to visualize and identify lipid rafts in plasmodesmata

Abstract

Plant cell-to-cell communication is mediated by nanopores called plasmodesmata (PDs) which are complex structures comprising plasma membrane (PM), highly packed endoplasmic reticulum and numerous membrane proteins. Although recent advances on proteomics have led to insights into mechanisms of transport, there is still an inadequate characterization of the lipidic composition of the PM where membrane proteins are inserted. It has been postulated that PDs could be formed by lipid rafts, however no structural evidence has shown to visualize and analyse their lipid components. In this perspective article, we discuss proposed experiments to characterize lipid rafts and proteins in the PDs. By using atomic force microscopy (AFM) and mass spectrometry (MS) of purified PD vesicles it is possible to determine the presence of lipid rafts, specific bound proteins and the lipidomic profile of the PD under physiological conditions and after changing transport permeability. In addition, MS can determine the stoichiometry of intact membrane proteins inserted in lipid rafts. This will give novel insights into the role of membrane proteins and lipid rafts on the PD structure.

Reticulomics: Protein-protein interaction studies with two plasmodesmata-localised reticulon family proteins identify binding partners enriched at plasmodesmata, ER and the plasma membrane

“…This finding is in agreement with the view that PD are rich in lipid components (Naulin et al, 2014;Grison et al, 2015) and may function as unique lipid rafts (Mongrand et al, 2010), perhaps involved in receptor-mediated signaling (Faulkner et al, 2013). The PM within PD is rich in sterols and sphingolipids relative to the general PM (Naulin et al, 2014).…”

“…This finding is in agreement with the view that PD are rich in lipid components (Naulin et al, 2014;Grison et al, 2015) and may function as unique lipid rafts (Mongrand et al, 2010), perhaps involved in receptor-mediated signaling (Faulkner et al, 2013). The PM within PD is rich in sterols and sphingolipids relative to the general PM (Naulin et al, 2014). The PD-localized protein STEROL METHYLTRANSFERASE1 (AT5G13710) controls cholesterol levels (Diener et al, 2000), while the remorin proteins that interact with RTNLB3 and RTNLB6 are components of lipid rafts (Mongrand et al, 2010).…”

Dynamic pattern generation in cell membranes: Current insights into membrane organization

“…This biochemical work has since been complemented using an emerging high resolution technique, NanoSIMS, where the authors have shown that accessible cholesterol is enriched in microvilli in Chinese hamster ovary cells [80]. Similarly, atomic force microscopy, a well-characterized experimental method to determine membrane properties has been combined with emerging methods such as STED-FCS, mass-spectrometry and X-ray techniques which has greatly expanded the applicability of each of the different methods [81, 82]. Other novel non-fluorescent methods such as high-resolution mass spectrometry, interferometric scattering microscopy, Raman microscopy have gained prominence in determining various membrane properties and detecting lipid clusters although given the specialized nature of these methods, wide use of these methods has currently been rather limited [83–88].…”

Specialized membrane domains of plasmodesmata, plant intercellular nanopores

“…Although the involvement of lipids in PD functionality has been suggested, we actually know very little about PD lipid constituents. As a way to determine whether sterol- and sphingolipid-enriched microdomains contribute to PD membranes as suggested by the presence of raft and tetraspanin protein markers, Naulin et al ( 2014 ) propose an original approach based on Mass Spectrometry (MS) and Atomic Force Microscopy (AFM) directly on purified PD membranes. Whereas AFM could be applied to identify microdomains based on topological parameters, MS approaches could resolve the PD lipid profile and identify intact membrane proteins and the stoichiometry and nature of lipids bound to them.…”