Guidelines for the Use of Protein Domains in Acidic Phospholipid Imaging

Abstract

Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach.

Keywords: Biosensor; Genetically encoded probe s; Lipid binding domain; Lipid signaling; Live imaging; Phosphatidic acid; Phosphatidylinositol phosphate; Phosphatidylserine; Phospholipase; PtdIns.

Figure 1. Summary of the subcellular localization of anionic phospholipids in yeast (A), animal (B) and plant (C) cells

Note that the reported localization are not exhaustive and might vary depending on cell types or signaling activities. The cartoon representing the cell in panel B is adapted from Jean and Kiger 2012.

Figure 2. General principle of genetically encoded lipid biosensors

A) A lipid-binding domain (LBD) from a multidomain protein (p40^phox in this example) is fused with a fluorescent protein (FP). This protein fusion acts as a biosensor for PI3P. B) Some LBDs require binding to both a lipid and another molecules (i.e., Ca2+, proteins). This coincidence binding specifies the localization of the corresponding biosensor to a subset of the lipid-enriched membrane, which also contains the target protein. In this example, the PH domain of FAPP1 binds PI4P and ARF1, hereby restricting its localization to the Golgi/TGN. C) Ratiometric FRET sensors are targeted to membranes independently of lipid binding (e.g., via a lipid anchor or a transmembrane segment) and report on the presence of the lipid based on the conformational changes induced in the sensor when the LBD binds its lipid (which increases or decreases the proximity between the two FPs and therefore their FRET ratio).

Figure 3. LBD affinities influence the subcellular localization of the sensors

When several pools of the same lipid exist within the cell, low or high affinity sensors will behave differently with respect to these pools. A) A low affinity sensor (e.g., 1xLBD) will localize to both membranes with slightly more sensor molecules at the compartment with the highest lipid concentration, while (B) a high affinity sensor (e.g., 2xLBD) will localize preferentially to the compartment with the highest lipid concentration. C) Example of low (1xPH^FAPP1) and high (2xPH^FAPP1) affinity sensor localization in Arabidopsis root cell (image from Simon et al., 2014 Plant Journal).