Dataset Information

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.

ABSTRACT: Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (L_o) and liquid-disordered, cholesterol-poor (L_d) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich L_o phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor L_d phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank L_o domains and enlarged L_d domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged L_o and shrank L_d Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P ₂] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both L_o and L_d domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P ₂ and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in L_o than in L_d Further, PtdIns(4,5)P ₂ was restored more rapidly in L_o than in L_d Thus destruction and restoration of PtdIns(4,5)P ₂ are faster in L_o than in L_d This suggests that L_o is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

SUBMITTER: Myeong J

PROVIDER: S-EPMC7936302 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundPhosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P2 also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P2 levels in living cells is critical to understanding the biology of PtdIns(4,5)P2. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCdelta1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P2 imaging tools.ResultsAll of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P2. None of these domains showed any advantage over the PLCdelta1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)P2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P3. Detailed comparison of the Tubby and PLCdelta1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.ConclusionThese results showed that both the PLCdelta1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCdelta1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P3 binding may compromise its accuracy to measure PtdIns(4,5)P2 changes. The Tubby domain is more accurate to report on PtdIns(4,5)P2 but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)P2 levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P2.

Dataset Information

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets