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Cell-to-cell communication in plants: the possible involvement of membrane rafts in plasmodesmata function – CONNECT

CONNECTING PLANT CELLS

Cell-to-cell communication in plants: the possible involvement of membrane rafts in plasmodesmata function <br />

The role of lipid microdomains in plasmodesmata function

In plants, the intercellular communication through the membrane lined nano-channels called plasmodesmata (PD) plays pivotal roles in the orchestration of development and defence responses. Plant viruses but also fungus can exploit PD transport machinery to establish infection and the emerging view is that PD may well represent a consensus target for pathogens and constitute key element in defence signalling. Understanding of how PD dictate cellular connectivity is dependent on comprehensive knowledge of the composition of PD and functional characterization of their constituents. Although some progress has been made in the identification of PD proteins, the role played by major membrane constituents, e.g. the lipids, remains totally uncovered. Yet PD are primarily membranous structures, defined by specialised membrane domains of the endoplasmic reticulum (desmotubule) and the plasma membrane (PD-PM) and the majority of identified PD proteins are membrane-associated. <br /> <br />The CONNECT project concentrates on the functional relationship that may exist between plasmodesmata and rafts, as well as the implication of lipids in PD function and structure.

Using suspension-cultured cells, to isolate pure PD, we propose for the first time to analyse the lipid composition of these unique membrane structures. Coupling lipidomic, biochemical approaches, spectrometric analyses as well as high-resolution microscopy we would like to establish whether or not raft-like domains are enriched at PD. Is the specialised PM domain lining PD sharing properties similar to rafts; i.e. highly organised and enriched in sterols and sphingolipids? Is lipid segregation along PD membranes fundamental for proper PD function? In particular the role of lipids in PD targeting will be investigated by modifying their sterol composition. Last we propose to examine the involvement of rafts and Remorin in the control of Potato virus X movement via PD. In particular, we make the hypothesis that Remorin interacts with TGBp1 at PD to limit viral cell-to-cell movement and that raft aggregation occurs around PD during viral infection.

we isolated PD-enriched membrane fractions from Arabidopsis suspension cells and analyzed their lipid composition. Extensive characterisation of the PD-enriched fraction revealed that the level of purity required to conduct a reliable and accurate lipidomic analysis has been reached. In particular, we show that the cellular membrane compartments continuous with the PD pores, namely the ER and the PM, are excised during the purification process and do not significantly contaminate the final fraction.
Using MS-based comparative lipidomic analysis, we could show that the PD-PM domain has a distinct lipid profile when compared to the cellular PM. In particular our data indicate that the PD-PM is enriched in sterols, highly glycosylated sphingolipids harbouring very long chain fatty acids (Glycosyl Inositol Phosphoryl Ceramides; GIPCs) and phospholipids containing fatty acids with reduced degree of desaturation. Intriguingly, this lipid profile is reminiscent of “raft” domains and therefore suggests that the PD membranes cluster sterol- and sphingolipid-enriched domains.
We next explore the potential for lipids and especially sterols to organise PD-specialed membrane domains and regulate cell-to-cell connectivity. Employing pharmacological approaches, we were able to demonstrate that alteration of the membrane pool of sterols strongly interferes with the ability of PD-Callose Binding Protein1 and the ß1-3 glucanase PdBG2, which are both GPI-anchored proteins, to associate with primary PD. Inhibition of PD targeting under sterol inhibitor treatment was correlated with a modification of both callose deposition and PD permeability.
Altogether, our data do not only provide a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity possibly by establishing and maintaining positional specificity of callose modifying GPI-proteins at PD.

Determination of the physicochemical properties of PD membranes in relation to their lipid composition and its role in the regulation of cell-to-cell trafficking during viral propagation

• Pérez-Sancho J., Tilsner J., Samuels L.A., Botella M.A., Bayer E.M. and Rosado A. Stitching organelles: Organization and function of plant membrane contact sites. Trends in Cell Biology (accepted for publication).

• Faulkner C and Bayer E.M. * Isolation of plasmodesmata. Methods in Molecular Biology edition “The Isolation of Plant Organelles and Structures: Methods and Protocols” (accepted for publication).

• Tilsner J, Nicolas W. Rosado A and Bayer E.M. * Staying tight : plasmodesmal membrane contact sites and the control of cell-to-cell connectivity in plants. Annual Review of Plant Biology 67:23.1-23.28

• Grison MS, Brocard L, Fouillen L, Nicolas W, Wewer V , Dörmann P, Nacir H, Benitez-Alfonso Y, Claverol S, Germain V, Boutté Y, Mongrand S and Bayer EM* (2015). Specific membrane lipid composition is important for plasmodesmata function. The plant Cell 27(4):1228-50

Plasmodesmata (PD) are nano-scaled membrane-lined channels PD that span the thick cell wall of virtually all plant cells, establishing both cytoplasmic and membrane continuity throughout the entire plant body (Fig.1). In recent years, PD have emerged as key elements of the cell-to-cell communication machinery and as such have been implicated in processes guaranteeing the collaborative functioning of the cells, and controlled developmental events. Plant viruses but also fungus can exploit PD transport machinery to establish infection and the emerging view is that PD may well represent a consensus target for pathogens and constitute key element in defence signalling and plant development. Understanding of how PD dictate cellular connectivity is dependent on comprehensive knowledge of the composition of PD and functional characterization of their constituents. Although some progress has been made in the identification of PD proteins, the role played by major membrane constituents, e.g. the lipids, remains totally uncovered. Yet PD are primarily membranous structures, defined by specialised membrane domains of the endoplasmic reticulum (desmotubule) and the plasma membrane (PD-PM) and the majority of identified PD proteins are membrane-associated. Research in biological membrane over the last decade has unequivocally demonstrated that lipids are functional units that can modulates membrane organisation and cellular function. Just as proteins, lipids are likely to be key elements of PD specialised membrane domains and as such contribute to proper functionality at PD.
In line with that recent data suggests that the PM domain lining PD may share similar properties to membrane rafts, high ordered sterol- and sphingolipid-enriched nanodomains of the PM involved in signalling, protein targeting and lateral segregation of membrane constituents. Hence, several raft markers have been shown not only to localise to microdomains at the PM per se but also accumulates at PD. Moreover recent data suggest that rafts could play a central role in the control of viral cell-to-cell spray through the PD channels. Altogether, this raises questions about the lipidic nature of the PD-PM. Do rafts contribute to the PD-PM? What is the role of lipids in defining PD membrane structurally and functionally?
The CONNECT project concentrates on the functional relationship that may exist between PD and rafts, as well as the implication of lipids in PD function and structure. Using suspension-cultured cells, to isolate pure PD, we propose for the first time to analyse the lipid composition of these unique membrane structures. Coupling lipidomic, biochemical approaches, spectrometric analyses as well as high-resolution microscopy we would like to establish whether or not raft-like domains are enriched at PD. Is the specialised PD-PM domain sharing properties similar to rafts; i.e. highly organised and enriched in sterols and sphingolipids? Is lipid segregation along PD-PM fundamental for proper PD function? In particular the role of lipids in PD targeting will be investigated by modifying their sterol composition. Last we propose to examine the involvement of rafts in the control of Potato virus X movement via PD. In particular, we make the hypothesis that rafts may be involved in the control of PD permeability and that raft aggregation occurs around PD during viral infection.

Project coordinator

Madame Emmanuelle BAYER (Laboratiore de Biogenèse Membranaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LBM Laboratiore de Biogenèse Membranaire

Help of the ANR 228,592 euros
Beginning and duration of the scientific project: September 2014 - 36 Months

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