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. Curr. Protoc. Cell Biol. 61:14.14.1‐14.14.24. © 2013 by John Wiley & Sons, Inc.

F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling

“…Prism-based TIRF microscopy was performed as previously described ( Schneider and Haugh, 2004 , 2005 ; Johnson and Haugh, 2013 ). The microscope uses a modified microscope (Axioskop 2 FS; Carl Zeiss) equipped with 40×, 0.8 NA and 60×, 1.0 NA Achroplan water dipping objectives (Carl Zeiss) and a charge-coupled device camera (ORCA ER; Hamamatsu Photonics).…”

“…All experiments were performed at 37°C. ImageJ (National Institutes of Health) was used for basic image preparation for presentation to crop, convert, adjust contrast, or smooth images for clarity as described in detail previously ( Johnson and Haugh, 2013 ).…”

“…Images were segmented either automatically by k -means clustering or manually via thresholding. Spatiotemporal mapping and the identification of morphological extensions were performed as described in detail previously ( Welf et al, 2012 ; Johnson and Haugh, 2013 ). In brief, protrusion/retraction for the spatiotemporal maps was calculated by the difference in overlap between consecutive frames.…”

Single-Virus Tracking: From Imaging Methodologies to Virological Applications

“…Later, combining with site-specific labeling strategies, such as peptide tag-mediated labeling and oligonucleotide-guided labeling, host cell-assisted methods have been extensively developed in recent years, which are an alternative approach to label the internal and external components of viruses during virus assembly. − For virus labeling, the distinct advantage of host cell-assisted methods is that it is possible to avoid modification of the viral surface, thus minimizing the influence of labeling on the virus–receptor interactions. To date, host cell-assisted methods have played an important role in visualizing the infection behaviors in different infection stages of a variety of viruses including SV40, HIV, and PV. ,,, …”

“…To date, host cell-assisted methods have played an important role in visualizing the infection behaviors in different infection stages of a variety of viruses including SV40, HIV, and PV. 131,290,307,308 4.2.1. Peptide Tag-Mediated Labeling.…”