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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.

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.

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.

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.