Exploring the role of lipids in intercellular conduits: breakthroughs in the pipeline

International audienceIt has been known for more than a century that most of the plant cells are connected to their neighbors through membranous pores perforating the cell wall, namely plasmodesmata (PDs). The recent discovery of tunneling nanotubes (TNTs), thin membrane bridges established between distant mammalian cells, suggests that intercellular communication mediated through cytoplasmic continuity could be a conserved feature of eukaryotic organisms. Although TNTs differ from PDs in their formation and architecture, both are characterized by a continuity of the plasma membrane between two cells, delimiting a nanotubular channel supported by actin-based cytoskeleton. Due to this unusual membrane organization, lipids are likely to play critical roles in the formation and stability of intercellular conduits like TNTs and PDs, but also in regulating the transfer through these structures. While it is crucial for a better understanding of those fascinating communication highways, the study of TNT lipid composition and dynamics turned out to be extremely challenging. The present review aims to give an overview of the recent findings in this context. We will also discuss some of the promising imaging approaches, which might be the key for future breakthroughs in the field and could also benefit the research on PDs

Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes.

International audienceTunneling Nanotubes (TNTs) are actin enriched filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens use TNT-like structures as "freeways" to propagate across cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia, differently from TNTs, are not able to mediate transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both these structures. We demonstrate that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and TNTs form through distinct molecular mechanisms. Our results further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion

Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells

The architecture of functional TNTs is still under debate. Here, the authors combine correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structure of TNTs in neuronal cells, showing that they form structures that are distinct form other membrane protrusions

Constitutive salicylic acid accumulation in pi4kIII beta 1 beta 2 Arabidopsis plants stunts rosette but not root growth

International audiencePhospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIII beta 1 beta 2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIII beta 1 beta 2 phenotype. The pi4kIII beta 1 beta 2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIII beta 1 beta 2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIII beta 1 beta 2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIII beta 1 beta 2, sid2 pi4kIII beta 1 beta 2 and NahG pi4kIII beta 1 beta 2 had similar amounts of SA as the wild-type (WT), npr1pi4kIII beta 1 beta 2 had more SA than pi4kIII beta 1 beta 2 despite being less dwarfed. This indicates that PI4KIII beta 1 and PI4KIII beta 2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIII beta 1 beta 2 was not suppressed in any of the triple mutants. The pi4kIII beta 1 beta 2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype

The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase

International audiencePhosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 μM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation

Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

<div><h3>Background</h3><p>Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.</p> <h3>Methodology/Principal findings</h3><p>Potential PLD substrates and products were studied in <em>Arabidopsis thaliana</em> suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of <em>n</em>-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in <em>in vitro</em> assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the <em>in vivo</em> products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.</p> <h3>Conclusions</h3><p>MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products <em>per se</em> is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.</p> </div

PBut profiles analyzed before and after SA addition.

<p>Cell medium was supplemented with 0.1% (v/v) <i>n</i>-butanol. After 45 minutes, 750 µM SA was added, and lipids were extracted 100 min later. (A) Black bars, PBut in untreated cells; white bars, PBut in SA treated cells; striped bars, PC; grey bars, PE. (B) PBut in the presence of SA compared to PC. Black bars, PBut in presence of SA; white bars, PC. Insert: for molecular species representing more than 1% of the species of PBut and PC, we calculated the ratio of the value obtained in PBut to the value obtained in PC. Results are represented on a <i>log2</i> scale. (C) PBut in the presence of SA compared to PE. Black bars, PBut in the presence of SA; white bars, PE. Insert: for molecular species representing more than 1% of the species of PBut and PE, we calculated the ratio of the value obtained in PBut to the value obtained in PE. Results are represented on a <i>log2</i> scale.</p

Profiles of PBut produced <i>in vitro</i> by Arabidopsis microsomal PLDs.

<p>Microsomes were used in an enzymatic assay on lipid vesicles. The substrate used was PE. The reaction assay was defined as α-type, β/γ-type or δ-type. The reactions were performed at 37°C for 20 min in the presence of 0.6% (v/v) <i>n-</i>butanol. White bars, substrate; black bars, PBut.</p