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

Abstract

SummaryAlthough 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.

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses

“…PtdOH biosensors have indicated that PtdOH is enriched at the apical and sub-apical plasma membranes in pollen tubes (Potocký et al, 2014;Simon et al, 2014). Interestingly, the same manipulations that affected the behaviour of a PtdOH biosensor in pollen tubes also affected the intracellular distribution of D6PK in root cells, i.e.…”

“…Black arrows, biosynthetic steps analysed in this study; grey arrows, biosynthetic steps that could not be selectively manipulated (Simon et al, 2016;Heilmann, 2009;Meijer and Munnik, 2003;Potocký et al, 2014 (Fig. S1).…”

“…As YFP:D6PK strongly bound to PtdOH, we also tested the effects of the modulation of PtdOH abundance with R59022, an inhibitor of DGK-and PI-PLC-dependent PtdOH synthesis (de Chaffoy de Courcelles et al, 1985;Potocký et al, 2014). R59022 led to the occasional accumulation of YFP:D6PK on endosomes, detectable only when YFP:D6PK was expressed from the D6PK promoter ( Fig.…”

“…The following inhibitors were dissolved in DMSO and used as previously described: Wortmaninn (Applichem) (Jaillais et al, 2006); LY294002 (Sigma) (Simon et al, 2016); PAO (Sigma) (Simon et al, 2016); U73343 and U73122 (Sigma) (Andreeva et al, 2010;Zhao et al, 2010); R59022 (Sigma) (Li and Xue, 2007;Potocký et al, 2014); propanolol (Sigma) (Li and Xue, 2007;Potocký et al, 2014); 1-and 2-butanol (Sigma) (Li and Xue, 2007;Potocký et al, 2014); Brefeldin A (Fischer Scientific) . All inhibitor treatments were controlled by solvent control treatments performed in parallel.…”

Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein

“…The subcellular distribution of PA pools in plants is poorly characterized, although a new fluorescent reporter that shows great promise for future work indicates abundant PA at the plasma membrane of pollen tubes (Potocký et al, 2014). Furthermore, native PA binding proteins accumulate at the plasma membrane (Zhang et al, 2004), whereas in mammalian cells, PA is abundant in not only the plasma membrane, but also the ER, Golgi, and endosomal membranes (Rizzo et al, 2000;Baillie et al, 2002;Loewen et al, 2004).…”