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Organization of membrane topologies in plants has so far been mainly attributed to the cell wall and the cytoskeleton. Taking rhizobial infections of legume root cells, where plasma membranes undergo dynamic and large-scale topology changes, as an initial model, we challenged this paradigm and tested whether additional scaffolds such as plant-specific remorins that accumulate on highly curved and often wall-less plasma membrane domains, control local membrane dynamics. Indeed, loss-of-function mutants of the remorin protein SYMREM1 failed to develop stabilized membrane tubes as found in colonized cells in wild-type plants, but released empty membrane spheres instead. Expression of this and other remorins in wall-less protoplasts allowed engineering different membrane topologies ranging from membrane blebs to long membrane tubes. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. This function is likely supported by remorin oligomerization into antiparallel dimers and the formation of higher order membrane scaffolding structures. Taken together we describe an evolutionary confined mechanism that allows the stabilization of large-scale membrane conformations and curvatures in plants.One-sentence summaryThe remorin SYMREM1 evolved as structural membrane scaffold that stabilizes membrane tubulation and curvature during symbiotic intracellular infections.

Plants are able to sense their mechanical environment. This mechanical signal is used by the plant to determine its phenotypic features. This is true also at a smaller scale. Morphogenesis, both at the cell and tissue level, involves mechanical signals that influence specific patterns of gene expression and trigger signaling pathways. How a mechanical stress is perceived and how this signal is transduced into the cell remains a challenging question in the plant community. Among the structural components of plant cells, the plasma membrane has received very little attention. Yet, its position at the interface between the cell wall and the interior of the cell makes it a key factor at the nexus between biochemical and mechanical cues. So far, most of the key players that are described to perceive and maintain mechanical cell status and to respond to a mechanical stress are localized at or close to the plasma membrane. In this review, we will focus on the importance of the plasma membrane in mechano-sensing and try to illustrate how the composition of this dynamic compartment is involved in the regulatory processes of a cell to respond to mechanical stress.

International audience; Root hairs are precisely positioned close to the rootward end of epidermal cells. A new study shows that the successful production of root hairs is a two-step process with different molecular players driving the initial polarization and subsequent hair outgrowth.

International audience; Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

International audience; The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation., Summary: YPT-interacting proteins 4a and 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In all eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidic acidic (PA), phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of cellular organelles not only defines the plant electrostatic territory but also distinguishes different compartments within this territory by specifying their varying surface charges.

The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity., Summary: Planar polarity in Arabidopsis is shaped by ACTIN-INTERACTING PROTEIN1-2, which is under the control of WEREWOLF-dependent patterning and ethylene signalling.

International audience; Although many molecular regulators of morphogenesis have been identified in plants, it remains largely unknown how the molecular networks influence local cell shape and how cell growth, form, and position are coordinated during tissue and organ formations. So far, analyses of gene function in morphogenesis have mainly focused on the qualitative analysis of phenotypes, often providing limited mechanistic insight into how particular factors act. For this reason, there has been a growing interest in mathematical and computational models to formalize and test hypotheses. These require much more rigorous, quantitative approaches; in parallel, new quantitative and correlative imaging pipelines have been developed to study morphogenesis. Here, we describe a number of such methods, focusing on live imaging.

SPE Pôle IPM; International audience; Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana.

L'article original est publié par The American Society of Plant Biologists; International audience; In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.

Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana.

Development of diverse multicellular organisms relies on coordination of single-cell polarities within the plane of the tissue layer (planar polarity). Cell polarity often involves plasma membrane heterogeneity generated by accumulation of specific lipids and proteins into membrane subdomains. Coordinated hair positioning along Arabidopsis root epidermal cells provides a planar polarity model in plants, but knowledge about the functions of proteo-lipid domains in planar polarity signalling remains limited. Here we show that Rho-of-plant (ROP) 2 and 6, phosphatidylinositol-4-phosphate 5-kinase 3 (PIP5K3), DYNAMIN-RELATED PROTEIN (DRP) 1A and DRP2B accumulate in a sterol-enriched, polar membrane domain during root hair initiation. DRP1A, DRP2B, PIP5K3 and sterols are required for planar polarity and the AGCVIII kinase D6 PROTEIN KINASE (D6PK) is a modulator of this process. D6PK undergoes phosphatidylinositol-4,5-bisphosphate- and sterol-dependent basal-to-planar polarity switching into the polar, lipid-enriched domain just before hair formation, unravelling lipid-dependent D6PK localization during late planar polarity signalling.

International audience; Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. Each membrane compartment has a unique identity and thereby recruits a specific set of proteins 1-3. It has been established for decades that these identities are acquired by the combined presence of specific lipid and protein molecules that act as biochemical landmark on each membrane. For example, small GTPases from the Rab and ADP ribosylation factor (ARF) family as well as Soluble N-ethylmaleimide-sensitive-factor Attachment protein Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsReprints and permissions information is available at www.nature.com/reprints.

Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking.

Membranes of eukaryotic cells contain high lipid-order sterol-rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher-plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell-plate formation in Arabidopsis relies on sterol- and DYNAMIN-RELATED PROTEIN1A (DRP1A)-dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order-sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid-order membrane domain. The cell-plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co-localize with DRP1A at the cell plate, and DRP1A accumulates in detergent-resistant membrane fractions. Modifications of sterol concentration or composition reduce cell-plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid-order domain, and pave the way to explore potential feedback between lipid order and function of dynamin-related proteins during cytokinesis.

Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-beta-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking.

International audience; Over the past five years, the structure, composition and possible functions of membrane raft-like domains on plant plasma membranes (PM) have been described. Proteomic analyses have indicated that a high proportion of proteins associated with detergent-insoluble membranes (DIMs), supposed to contain raft-like domains isolated from the PM, might be involved in signalling pathways. Recently, the dynamic association of specific proteins with the DIM fraction upon environmental stress has been reported. Innovative imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level in the plant PM, correlating detergent insolubility and membrane-domain localization of presumptive raft proteins. These data suggest a role for plant rafts as signal transduction platforms, similar to those documented for mammalian cells.

International audience; A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used 14N/15N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.