title: Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.
abstract: This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors.
authors: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.
authors: Heath E
authors: HE
authors: Johnson
authors: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.
authors: Jason M
authors: JM
authors: Haugh
mesh_terms: D000818:Animals; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D020239:Receptor Cross-Talk; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015398:Signal Transduction
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; C541572:PHLPP2 protein, human; D010749:Phosphoprotein Phosphatases
keywords: cell biophysics; cell migration; image analysis; image processing; signal transduction