title: Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor.
abstract: Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics.
authors.affiliation: Institute of Experimental Botany, v. v. i., Academy of Sciences of the Czech Republic, 16502, Prague 6, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Potocký
authors.affiliation: Institute of Experimental Botany, v. v. i., Academy of Sciences of the Czech Republic, 16502, Prague 6, Czech Republic.
authors.forename: Roman
authors.initials: R
authors.lastname: Pleskot
authors.affiliation: Institute of Experimental Botany, v. v. i., Academy of Sciences of the Czech Republic, 16502, Prague 6, Czech Republic.
authors.forename: Přemysl
authors.initials: P
authors.lastname: Pejchar
authors.affiliation: Institut des Neurosciences Cellulaires et Integratives, CNRS UPR3212 and Université de Strasbourg, Strasbourg, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vitale
authors.affiliation: Department of Biology, University of Erlangen-Nuremberg, 91058, Erlangen, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Kost
authors.affiliation: Institute of Experimental Botany, v. v. i., Academy of Sciences of the Czech Republic, 16502, Prague 6, Czech Republic.|Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, 128 44, Prague 2, Czech Republic.
authors.forename: Viktor
authors.initials: V
authors.lastname: Žárský
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted; D010712:Phosphatidic Acids / Q000032:analysis / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D038761:Photobleaching; D053205:Pollen Tube / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D050766:Qb-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism
chemical_list: D004075:Diglycerides; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C497084:Spo20 protein, S cerevisiae; D010739:Phospholipase D
keywords: Nicotiana tabacum (tobacco); Spo20p; live-cell microscopy; phosphatidic acid (PA); phospholipase D; pollen tube
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