Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy.

In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.

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

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.

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

Tunneling 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.

Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking.

Tunneling nanotubes (TNTs) are thin membranous channels providing direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication and pathogen transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this unit represent a guideline for the characterization of TNTs (or TNT-like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; and (4) monitor their formation in living cells are provided. Finally, this unit describes how to assess TNT-mediated cell-to-cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs.

Constitutive salicylic acid accumulation in pi4kIIIß1ß2 Arabidopsis plants stunts rosette but not root growth.

Phospholipids 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), pi4kIIIß1ß2, 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 pi4kIIIß1ß2 phenotype. The pi4kIIIß1ß2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIIIß1ß2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIIIß1ß2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIIIß1ß2, sid2 pi4kIIIß1ß2 and NahG pi4kIIIß1ß2 had similar amounts of SA as the wild-type (WT), npr1pi4kIIIß1ß2 had more SA than pi4kIIIß1ß2 despite being less dwarfed. This indicates that PI4KIIIß1 and PI4KIIIß2 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 pi4kIIIß1ß2 was not suppressed in any of the triple mutants. The pi4kIIIß1ß2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.

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

It 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.

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

Phosphoinositide-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.

Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms.

Phosphoinositides are minor constituents of eukaryotic membranes but participate in a wide range of cellular processes. The most abundant and best characterized phosphoinositide species are phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its main precursor, phosphatidylinositol 4-phosphate (PI4P). PI4P and PI(4,5)P2 regulate various structural and developmental functions but are also centrally involved in a plethora of signal transduction pathways in all eukaryotic models. They are not only precursors of second messengers but also directly interact with many protein effectors, thus regulating their localisation and/or activity. Furthermore, the discovery of independent PI(4,5)P2 signalling functions in the nucleus of mammalian cells have open a new perspective in the field. Striking similarities between mammalian, yeast and higher plant phosphoinositide signalling are noticeable, revealing early appearance and evolutionary conservation of this intracellular language. However, major differences have also been highlighted over the years, suggesting that organisms may have evolved different PI4P and PI(4,5)P2 functions over the course of eukaryotic diversification. Comparative studies of the different eukaryotic models is thus crucial for a comprehensive view of this fascinating signalling system. The present review aims to emphasize convergences and divergences between eukaryotic kingdoms in the mechanisms underlying PI4P and PI(4,5)P2 roles in signal transduction, in response to extracellular stimuli.

Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: Potential PLD substrates and products were studied in Arabidopsis thaliana 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 n-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 in vitro 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 in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. CONCLUSIONS: 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 per se 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.

Eat in or take away? How phosphatidylinositol 4-kinases feed the phospholipase C pathway with substrate.

Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first step in the synthesis of phosphoinositide pools hydrolysed by phosphoinositide-dependent phospholipase C (PI-PLC) and thus constitute a potential key regulation point of this pathway. Twelve putative PI4K isoforms, divided as type-II (AtPI4KIIγ1- 8) and type-III PI4Ks (AtPI4KIIIα1- 2 and AtPI4KIIIß1- 2), have been identified in Arabidopsis genome. By a combination of pharmalogical and genetic approaches we recently evidenced that AtPI4KIIIß1 and AtPI4KIIIß2 contribute to supply PI-PLC with substrate and that AtPI4KIIIα1 is probably also involved in this process. Given the current knowledge on PI-PLC and type-III PI4Ks localization in plant cells it raises the question whether type-III PI4Ks produce phosphatidylinositol 4-phosphate at the site of its consumption by the PI-PLC pathway. We therefore discuss the spatial organization of substrate supply to PI-PLC in plant cells with reference to recent data evidenced in mammalian cells.

Arabidopsis type-III phosphatidylinositol 4-kinases ß1 and ß2 are upstream of the phospholipase C pathway triggered by cold exposure.

Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P(2) or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P(2). In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 µM wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIIIα1, ß1 and ß2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIIIß1ß2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIIIß1 and pi4kIIIß2 mutants, indicating that AtPI4KIIIß1 and AtPI4KIIIß2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIIIß1ß2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.