PIN2 Turnover in Arabidopsis Root Epidermal Cells Explored by the Photoconvertible Protein Dendra2

Abstract

The steady state level of integral membrane proteins is dependent on a strictly controlled delivery and removal. Here we show that Dendra2, a green-to-red photoconvertible fluorescent protein, is a suitable tool to study protein turnover in plants. We characterized the fluorescence properties of Dendra2 expressed either as a free protein or as a tag in Arabidopsis thaliana roots and optimized photoconversion settings to study protein turnover. Dendra2 was fused to the PIN2 protein, an auxin transporter in the root tip, and by time-lapse imaging and assessment of red and green signal intensities in the membrane after photoconversion we quantified directly and simultaneously the rate of PIN2 delivery of the newly synthesized protein into the plasma membrane as well as the disappearance of the protein from the plasma membrane due to degradation. Additionally we have verified several factors which are expected to affect PIN2 protein turnover and therefore potentially regulate root growth.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis

“…The finding that 1-NAA is not able to inhibit endocytosis of PIN2, was confirmed in a modified photoconversion experiment. In this case, we firstly photoconverted PIN2-Dendra2 in roots and after that, we kept seedlings under standard cultivation conditions for 2 h. As expected in the context of our previous studies (Jásik et al, 2013;Jásik and Schmelzer, 2014), after 2 h, a significant part of the PM PIN2 population was already replaced by the newly synthesized green fluorescing form (Fig. 3).…”

“…The abovementioned discrepancies prompted us to perform a more complex study on the influence of different auxins and auxin transport inhibitors on PIN2 intracellular dynamics using the photoconvertible fluorescence protein method. In our earlier study (Jásik et al, 2013), we demonstrated that Dendra2 as a tag is not photoconverted under standard confocal imaging conditions using the 488 nm line of the Argon laser for excitation. Therefore, Dendra2 can be used as a classical fluorescence protein tag similar to GFP.…”

“…It is worth noting that GFP tagging or immunocytochemistry methodologies, which were used in earlier studies in combination with application of pharmacological compounds, including BFA, showed only net protein abundance in the cellular compartments, but intracellular dynamics of internalized and newly synthesized protein populations were hidden. Recently we used an approach employing the green-to-red photoconvertible Dendra2 fluorescent protein developed by Gurskaya et al (2006) that allows the simultaneous quantification of the abundance and dynamics of the currently present and the newly synthesized PIN2 populations in cell compartments (Jásik et al, 2013). We have demonstrated that the appearance of PIN2 protein in BFACs is an inappropriate way to study protein endocytosis (Jásik and Schmelzer, 2014).…”

“…Because photoconversion of Dendra2 is an irreversible process (Chudakov et al, 2007), this fluorescent protein is a suitable tool for quantitative monitoring of the two PIN2 populations in the PM and in BFACs (Jásik et al, 2013;Jásik and Schmelzer, 2014). The speed of PIN2-Dendra2 fusion protein internalization can be estimated by measuring the red signal intensity in the PM and, vice versa, the degree of the PIN2 PM pool recovery can be determined by evaluating the green fluorescence intensity in the PM.…”

“…3). However, the red fluorescing population was still predominant because the PM half-life time of PIN2 is approximately 3.5 h (Jásik et al, 2013). These seedlings were treated with 10 mM 1-NAA for 15 min, then imaged and cultivated on medium with 50 mM BFA.…”