title: Isolation of Plasmodesmata.
abstract: 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.
authors.affiliation: John Innes Centre, Norwich Research Park, Norwich, UK.
authors.forename: Christine
authors.initials: C
authors.lastname: Faulkner
authors.affiliation: Laboratory of Membrane Biogenesis, CNRS-UMR 5200, Université Bordeaux, Segalen Bâtiment A3, INRA Bordeaux Aquitaine, 71 Avenue Edouard Bourlaux CS 20032, F-33140, Villenave d'Ornon, France. emmanuelle.bayer@u-bordeaux.fr.
authors.forename: Emmanuelle M F
authors.initials: EM
authors.lastname: Bayer
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002473:Cell Wall / Q000737:chemistry* / Q000648:ultrastructure; D002480:Cellulase / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000737:chemistry* / Q000648:ultrastructure; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D059828:Plant Cells / Q000737:chemistry*; D031425:Plasmodesmata / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D029681:Arabidopsis Proteins; D015415:Biomarkers; D002352:Carrier Proteins; D003470:Culture Media; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C542004:PDCB1 protein, Arabidopsis; C528450:PDLP1a protein, Arabidopsis; D002480:Cellulase
keywords: Arabidopsis thaliana; Cell wall; Cellulase; Lipidomic; Membrane; Plasmodesmata; Proteomic; Suspension cell