Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor

Potocký M.*, Pleskot R.*, Pejchar P.*, Vitale N., Kost B., Žárský V. *These authors contributed equally to this work.
NEW PHYTOLOGIST 203: 483-494, 2014

Klíčová slova: phosphatidic acid, live-cell microscopy, pollen tube, phospholipase D, Spo20p, Nicotiana tabacum (tobacco)
Abstrakt: · 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 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 levels show distinct responses to various lipid signaling inhibitors. FRAP analysis suggests high PA turnover in the plasma membrane. · Our data show that biosensor based on 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.
DOI: 10.1111/nph.12814 Autoři z ÚEB: Přemysl Pejchar, Roman Pleskot, Martin Potocký, Viktor Žárský