Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes.
Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.
Divisions of Lipid Biochemistry and  Kobe University Graduate School of Medicine, Kobe 650-0017,  Japan.
Yasuhiro
Y
Irino
Emi
E
Tokuda
Junya
J
Hasegawa
Toshiki
T
Itoh
Tadaomi
T
Takenawa
D000143:Acids / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002465:Cell Movement; D006224:Cricetinae; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry*; D041681:NIH 3T3 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000302:isolation & purification; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000302:isolation & purification; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D014162:Transfection
D000143:Acids; D007328:Insulin; D015335:Molecular Probes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010982:Platelet-Derived Growth Factor; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate