Quantifying lipid changes in various membrane compartments using lipid binding protein domains

Abstract

SUMMARY One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms.

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

“…This methodology relies on a single-plasmid design that incorporates the self-cleaving viral 2A peptide from Thosea asigna (T2A; Donnelly et al, 2001; Szymczak et al, 2004; Liu et al, 2017). The internal T2A site facilitates the split of the translated polypeptide into two proteins and allows for the uniform and roughly equimolar expression of both the organelle-anchored BRET acceptor (mVenus) and Luciferase-tagged lipid-binding probe, which serves as the BRET donor (Varnai et al, 2017). Due to the prominent localization of the Bc PI-PLC H82A probe within the outer mitochondrial membrane, we chose to validate the design of the BRET-based biosensors by attempting to monitor mitochondrial DAG levels.…”

“…While subcellular fractionation studies have provided the first detailed information regarding the lipid composition of specific organelles (van Meer and de Kroon, 2011; Klose et al, 2013; Brügger, 2014), it is becoming increasingly evident that even the most efficient isolation methods cannot work quickly enough to preserve the lipid composition of cellular membranes and also suffer from the unavoidable cross-contamination of organelles (van Meer, 2005; Satori et al, 2013; Kappler et al, 2016). Outside of these in vitro biochemical studies, imaging breakthroughs using endogenous membrane-binding protein domains have allowed for the visualization of embedded lipid species with high spatial and temporal resolution in live cells (Varnai et al, 2017; Wills et al, 2018). The refinement and rational design of lipid biosensors has been aided by detailed structural and biophysical studies that have established sequence features that directly contribute to the specificity and affinity of peripheral membrane-binding protein domains based on the recognition of general membrane features as well as individual lipid components (Lee, 2003; Cho and Stahelin, 2005; Pogozheva et al, 2013; Whited and Johs, 2015; Pasenkiewicz-Gierula et al, 2016).…”

“…Due to the expansive cellular roles of PPIn lipids, both as substrates for signaling enzymes and sites for protein-membrane interactions, it is not surprising that many human pathologies are caused by perturbations in PPIn production or turnover (Pendaries et al, 2003; Wymann and Schneiter, 2008; McCrea and De Camilli, 2009; Bunney and Katan, 2010; Dyson et al, 2012; Altan-Bonnet, 2017). The interest in PPIn metabolism has resulted in significant efforts to validate selective biosensors to define the distribution and inter-conversion pathways that modulate localized PPIn levels (Indevall-Hagren and De Camilli, 2014; Varnai et al, 2017; Wills et al, 2018). However, as the complex processes governing PPIn metabolism must begin with PtdIns synthesis, investigations of PPIn metabolism continue to be limited by the lack of understanding of how PtdIns production and transport are controlled.…”

A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

“…To achieve an equal ratio of coexpression, the luciferase-fused probe and the Rab7-tagged Venus were expressed from a single plasmid separated by the viral T2A peptide sequence. The presence of PI4P in Rab7 endosomes brings the luciferase in proximity to the Venus protein and, in the presence of the coelenterazine substrate, energy-transfer takes place that can be monitored in a plate reader giving a measurement from whole cell population (46). To increase the availability of the P4M2x-SLuc component in the cytosol, we used an inhibitor of the PI4KA enzyme, which causes elimination of the large PM pool of PI4P, making more of the lipid probe available in the cytosol to find the endosomal PI4P pools (Fig.…”

Phosphoinositide Diversity, Distribution, and Effector Function: Stepping Out of the Box

“…The assignment of PtdInsP location described above is mostly based on the use of PtdInsP specific antibodies and/or the expression of functional binding domains derived from bona fide effector proteins fused to a fluorescent tag. For example, the PH domain of PLCδ1 is commonly used to detect PtdIns(4,5) P 2 , while the tandem FYVE domain from EEA1 or Hrs is used to identify PtdIns(3) P . However, it is increasingly clear that the classical view that each PtdInsP has a narrow subcellular distribution does not capture reality (Figure ).…”