Fluorescent Reporter Proteins

Abstract

INTRODUCTION For more than a decade the growing class of f luorescent p roteins (FPs) defined as homologues of A equorea v ictoria g reen FP (avGFP), which are capable of forming an intrinsic chromophore, has almost single-handedly launched and fueled a new era in cell biology. These powerful research tools provide investigators with a means of fusing a genetically encoded optical probe to any one of a practically unlimited variety of protein targets to examine living systems using fluorescence microscopy and related methodology (see Figure 1.1; for recent reviews, see references). The diverse array of practical applications for FPs ranges from targeted markers for organelles and other subcellular structures, to protein fusions designed to monitor mobility and dynamics, to reporters of transcriptional regulation (Figure 1.2). FPs have also opened the door to creating highly specific biosensors for live-cell imaging of numerous intracellular phenomena, including pH and ion concentration fluctuations, protein kinase activity, apoptosis, voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In addition, by applying selected promoters and targeting signals, FP biosensors can be introduced into an intact organism and directed to specific tissues, cell types, and subcellular compartments to enable monitoring a variety of physiological processes using f luorescence r esonance e nergy t ransfer (FRET) techniques. If FPs are the “fuel” for the live-cell imaging revolution, the “engines” are the technical advances in widefield fluorescence and confocal microscopes. Some notable advances include low light level digital charge coupled device (CCD) cameras as well as spinning-disk and swept-field instruments.