Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Abstract

This article is available online at http://www.jlr.org enzymes is associated with disorders such as diabetes and cancer, which suggests that cellular concentrations of each PI species is usually under strict control ( 4,5 ).A number of PI-binding domains have been identifi ed, including pleckstrin homology (PH); Phox (PX); Fab1, YOTB, Vac1p, and EEA1 (FYVE); Epsin N-terminal homology (ENTH); and Glucosyltransferase, Rab-like GTPase activator, and myotubularin (GRAM) domains ( 6, 7 ). Although most PH domains show little specifi city in the PIs they bind, several show relatively high specifi city; these include the PH domains of phospholipase C ␦ 1 (PLC ␦ 1) for PtdIns(4,5)P 2 and the general receptor for phosphoinositides-1 (GRP1) for PtdIns(3,4,5)P 3 ( 8, 9 ). In addition, the tandem-PH domain-containing protein-1 (TAPP1) and the four phosphate-adaptor protein-1 (FAPP1) PH domains specifi cally bind PtdIns(3,4)P 2 and PtdIns4P, respectively ( 10, 11 ). Other domains that bind specifi c PIs have also been described. The FYVE domains of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and early endosome antigen-1 (EEA1) specifi cally recognize PtdIns3P, which is primarily found in endosomes, multivesicular bodies, and phagosomes ( 12 ). The GRAM domains of myotubularin and myotubularin-related protein 2 (MTMR2) are involved in endosomal and vacuolar targeting through interaction with PtdIns(3,5)P 2 ( 13 ). Moreover, some PI-binding domains have functions beyond simply recruiting host proteins to the membrane surface. Such domains, including ENTH, Bin Amphiphysin Rvs (BAR), and FCH and BAR (F-BAR), bind PIs with little specifi city and participate in endocytosis, cytokinesis, and Press, February 3, 2012 DOI 10.1194 Quantifi cation and visualization of phosphoinositides by quantum dot-labeled specifi c binding-domain probes Abbreviations: CHO-IR , Chinese hamster ovary cells expressing the wild-type human insulin receptor; CHO-IR, GFP, green fl uorescent protein; GRP1, general receptor for phosphoinositides-1; PC, phosphatidylcholine; PDGF, platelet-derived growth factor; PE, phosphatidylethanolamine; PH, pleckstrin homology; PI, phosphoinositide; PI3K, PI 3-kinase; PLC ␦ 1, phospholipase C ␦ 1; Qdot, quantum dot; ROI, region of interest; TAPP-1, tandem-PH domain-containing protein-1. Published, JLR Papers in

Polyphosphoinositide binding domains: Key to inositol lipid biology

“…Because the main source of PtdIns(3,4) P 2 is PtdIns(3,4,5) P 3 made by Class I PI3Ks in the plasma membrane and dephosphorylated by SHIP1/2 5-phosphatases, this lipid was assumed to be localized to the PM. This was confirmed with the use of the TAPP1 PH domain as a probe in live [147] or fixed cells [146] and the lipid was found especially enriched in lamellopodia [148,149]. EM analysis using the TAPP1 PH domain as a probe found some of the lipid in the ER and endosomes, especially in the lumen of MVBs [57].…”

“…The largest elevation in PtdIns(3,4) P 2 is achieved by treatment with H 2 O 2 , which, through inhibition of PTEN and probably the PtdIns(3,4) P 2 phosphatases, INPP4A/B, slowly converts almost the entire PtdIns(4,5) P 2 pool to PtdIns(3,4) P 2 [144,145]. However, in a normal stimulated cell PtdIns(3,4) P 2 levels reach only 2–5 % of those of PtdIns(4,5) P 2 [146]. Because the main source of PtdIns(3,4) P 2 is PtdIns(3,4,5) P 3 made by Class I PI3Ks in the plasma membrane and dephosphorylated by SHIP1/2 5-phosphatases, this lipid was assumed to be localized to the PM.…”

Network Analysis of the Focal Adhesion to Invadopodia Transition Identifies a PI3K-PKCα Invasive Signaling Axis

“…p85-WT-FLAG and p85-S361/652A-FLAG (56) were a kind gift of Dr. Lew Cantley (Harvard University) and were cloned into pLenti6/V5 (Invitrogen). GST-6Cys-PLCδ1-PH and GST-6Cys-GRP1-PH (57) were a kind gift from Dr. Tadaomi Takenawa (Kobe University).…”

“…Cells were fixed and permeabilized at 37°C for 1h with 4% PFA, 0.1% glutaraldehyde and 1.5 mg/ml saponin in cytoskeleton buffer, as described (57). Cells were stained with Qdot-6Cys-GRP1-PH and Qdot-6Cys-PLCδ1-PH domains in 1%BSA at 4 °C overnight and imaged by confocal microscopy.…”

PI3K‐C2α: One enzyme for two products coupling vesicle trafficking and signal transduction

“…1). PtdIns(3,4) P 2 is enriched in the plasma membrane but its levels reaches only 2–5% of those of PtdIns(4,5) P 2 [40].…”