Chapter 2: Molecular sensors based on fluorescence resonance energy transfer to visualize cellular dynamics.
Visualizing a variety of signaling events in the native cellular environment is now possible with the advent of genetically encodable fluorescent labels like green fluorescent proteins made de novo by living cells themselves. The focus of this method chapter is on genetically encodable molecular sensors based on fluorescence resonance energy transfer (FRET) for visualization of cellular dynamics. This chapter discusses the process of developing a molecular sensor, from choosing donor-acceptor pairs to designing the protein modules that actually sense the signaling events. A few examples of biosensors are discussed to showcase the designs of such FRET-based sensors for live-cell imaging of signaling events. Subsequently, Section III covers the experimental procedure of DNA work, microscope instrumentation, data collection through imaging acquisition, data comprehension, and evaluation. Furthermore, a case study of the PI3K/Akt signaling pathway using a series of FRET sensors highlights the tremendous potential of the method in exploring relevant biological systems.
Ananthanarayanan
Bharath
B
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Ni
Qiang
Q
Zhang
Jin
J
D000818:Animals; D015374:Biosensing Techniques; D002468:Cell Physiological Phenomena; D002477:Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis; D038761:Photobleaching; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt