Plasmodesmata Ultrastructure Determination Using Electron Tomography.

Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram.

Isolation of Plasmodesmata Membranes for Lipidomic and Proteomic Analysis.

Plasmodesmata (PD) are membranous intercellular nanochannels crossing the plant cell wall to connect adjacent cells in plants. Our understanding of PD function heavily relies on the identification of their molecular components, these being proteins or lipids. In that regard, proteomic and lipidomic analyses of purified PD represent a crucial strategy in the field. Here we describe a simple two-step purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells suitable for "omic" approaches. The first step of this procedure consists on isolating pure cell walls containing intact PD, followed by a second step which involves an enzymatic degradation of the wall matrix to release PD membranes. The PD-enriched fraction can then serve to identify the lipid and protein composition of PD using lipidomic and proteomic approaches, which we also describe in this method article.

Isolation of Plasmodesmata.

Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology. Here we outline a simple two-step purification procedure that allows isolation of PD-derived membranes from Arabidopsis suspension cells. The method involves isolation of purified cell wall fragments containing intact PD which is followed by enzymatic degradation of the cell wall to release the PD. This membrane-rich fraction can be subjected to protein and lipid extraction for molecular characterization of PD components. The first step of this procedure involves the isolation of cell wall fragments containing intact PD, free from contamination from other cellular compartments. Purified PD membranes are then released from the cell wall matrix by enzymatic degradation. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

In plants, the intercellular communication through the membranous channels called plasmodesmata (PD; singular plasmodesma) plays pivotal roles in the orchestration of development, defence responses, and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialized domains of the endoplasmic reticulum (ER) and the plasma membrane (PM). PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalization, or screening of random cDNAs, only few PD proteins had been conclusively identified and characterized. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on "free" PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic-based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD-associated proteins.

Membrane rafts in plant cells.

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.