Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.

• Keywords
• Genetically encoded biosensors, lipid manipulation, membrane lipid imaging, microscopy, peripheral membrane proteins, protein–lipid interactions,
• MeSH
• Cell Membrane * metabolism   MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Plant Proteins * metabolism   MeSH
• Plants metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins MeSH
• Plant Proteins * MeSH

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.

• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• Matrix Attachment Regions * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Minor Histocompatibility Antigens genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Browser
• Arabidopsis Proteins MeSH
• Minor Histocompatibility Antigens MeSH

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

• Keywords
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytoplasm metabolism   MeSH
• Exocytosis MeSH
• Phospholipids metabolism   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EXO70A1 protein, Arabidopsis MeSH Browser
• Phospholipids MeSH
• Arabidopsis Proteins MeSH

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.

• Keywords
• EXO70, cell wall, exocyst complex, phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, phospholipids, plasma membrane domains, polar exocytosis, trichome,
• MeSH
• Arabidopsis chemistry   genetics   MeSH
• Cell Membrane chemistry   genetics   MeSH
• Exocytosis genetics   MeSH
• Phosphatidylinositol 4,5-Diphosphate chemistry   metabolism   MeSH
• Phosphatidylserines chemistry   genetics   MeSH
• Membrane Lipids genetics   metabolism   MeSH
• Arabidopsis Proteins chemistry   genetics   MeSH
• Trichomes chemistry   genetics   MeSH
• Vesicular Transport Proteins chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EXO70A1 protein, Arabidopsis MeSH Browser
• EXO70H4 protein, Arabidopsis MeSH Browser
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Phosphatidylserines MeSH
• Membrane Lipids MeSH
• Arabidopsis Proteins MeSH
• Vesicular Transport Proteins MeSH

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.

• MeSH
• Cell Membrane metabolism   MeSH
• Euglenozoa genetics   metabolism   MeSH
• Phylogeny * MeSH
• Euglenozoa Infections parasitology   MeSH
• Carcinogenesis genetics   metabolism   MeSH
• Humans MeSH
• Evolution, Molecular MeSH
• Ras Homolog Enriched in Brain Protein chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ras Homolog Enriched in Brain Protein MeSH
• RHEB protein, human MeSH Browser

The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.

• Keywords
• Arabidopsis thaliana, development, endosomes, interaction, root hair, spinning disc microscopy, structured illumination microscopy, trafficking,
• Publication type
• Journal Article MeSH