Alexandre Martinière

PIN polarity maintenance by the cell wall in Arabidopsis.

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.

Cell wall constrains lateral diffusion of plant plasma-membrane proteins

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.

In Vivo Intracellular pH Measurements in Tobacco and Arabidopsis Reveal an Unexpected pH Gradient in the Endomembrane System

This work combines biochemistry and live-cell imaging to perform in vivo pH measurements of different compartments of the endomembrane system in plant cells. It reports that the prevacuolar compartments are more alkaline than the trans-Golgi network and provides evidence that endosomal proton pumps and proton efflux transporters contribute to pH homeostasis. The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH −1.5) and Arabidopsis thaliana root cells (ΔpH −2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H+ ATPase–dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

A virus responds instantly to the presence of the vector on the host and forms transmission morphs

Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle. DOI: http://dx.doi.org/10.7554/eLife.00183.001

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus.

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.

Homeostasis of plasma membrane viscosity in fluctuating temperatures

Temperature has a direct effect at the cellular level on an organism. For instance, in the case of biomembranes, cooling causes lipids to lose entropy and pack closely together. Reducing temperature should, in the absence of other factors, increase the viscosity of a lipid membrane. We have investigated the effect of temperature variation on plasma membrane (PM) viscosity. We used dispersion tracking of photoactivated green fluorescent protein (GFP) and fluorescence recovery after photobleaching in wild-type and desaturase mutant Arabidopsis thaliana plants along with membrane lipid saturation analysis to monitor the effect of temperature and membrane lipid composition on PM viscosity. Plasma membrane viscosity in A. thaliana is negatively correlated with ambient temperature only under constant-temperature conditions. In the more natural environment of temperature cycles, plants actively manage PM viscosity to counteract the direct effects of temperature. Plasma membrane viscosity is regulated by altering the proportion of desaturated fatty acids. In cold conditions, cell membranes accumulate desaturated fatty acids, which decreases membrane viscosity and vice versa. Moreover, we show that control of fatty acid desaturase 2 (FAD2)-dependent lipid desaturation is essential for this homeostasis of membrane viscosity. Finally, a lack of FAD2 function results in aberrant temperature responses.

Development and properties of genetically encoded pH sensors in plants

Fluorescent proteins (FPs) have given access to a large choice of live imaging techniques and have thereby profoundly modified our view of plant cells. Together with technological improvements in imaging, they have opened the possibility to monitor physico-chemical changes within cells. For this purpose, a new generation of FPs has been engineered. For instance, pHluorin, a point mutated version of green fluorescent protein, allows to get local pH estimates. In this paper, we will describe how genetically encoded sensors can be used to measure pH in the microenvironment of living tissues and subsequently discuss the role of pH in (i) exocytosis, (ii) ion uptake by plant roots, (iii) cell growth, and (iv) protein trafficking.

Salt stress triggers enhanced cycling of Arabidopsis root plasma-membrane aquaporins.

Aquaporins of the plasma membrane intrinsic protein (PIP) subfamily are channels which facilitate the diffusion of water across the plant plasma membrane (PM). Although PIPs have been considered as canonical protein markers of this compartment, their endomembrane trafficking is still not well documented. We recently obtained insights into the constitutive cycling of PIPs in Arabidopsis root cells by means of fluorescence recovery after photobleaching (FRAP). This work also uncovered the behavior of the model isoform AtPIP2;1 in response to NaCl. The present addendum connects these findings to another recent work which describes the dynamic properties of AtPIP2;1 in the PM in normal and salt stress conditions by means of single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS). The results suggest that membrane rafts play an important role in the partitioning of AtPIP2;1 in normal conditions and that clathrin-mediated endocytosis is predominant. In salt stress conditions, the rate of AtPIP2;1 cycling was enhanced and endocytosis was cooperated by a membrane raft-associated salt-induced pathway and a clathrin-dependent pathway.