Application of green fluorescent protein technology in the study of membrane-trafficking in living neurons

Abstract

Whole body optical imaging using bioluminescence or fluorescence is one of the most rapidly emerging technologies to non-invasively follow all kinds of molecular and cellular processes in small animals. Using tomographic approaches it is now also possible to get better quantitative data. Due to its sensitivity and simplicity it is now also widely used in drug development and drug screening. Finally, using near infrared fluorescent probes that have much deeper penetration also opens up new exciting applications such as intra-operative image guided surgery for sentinel lymph node mapping and radical resection of tumours.Recent advances in imaging strategies that reveal cellular and molecular biological events in real-time facilitate our understanding of biological processes occurring in living animals. The development of molecular tags, such as green fluorescent protein (GFP) from the jellyfish Aequorea victoria, red fluorescent proteins (RFP) from the Discosoma species (dsRed2) and luciferase (Luc) from the firefly Photinus pyralis (fLuc) and the sea pansy Renilla (rLuc), has revolutionised research over the past decade, allowing complex biochemical processes to be associated with the functioning of proteins in living cells.Optical technologies, both microscopic and macroscopic, are developing fast. Recent technical advances for imaging weak visible light sources using cooled charged coupled device (CCCD) cameras, peltier cooled detectors and micro-plate channel intensifiers allow detection of photon emission from inside the tissues of small animals. Whole body fluorescent imaging (FLI) and bioluminescent imaging (BLI) are now applied to study cell- and tissue-specific gene promoter activity and also to follow trafficking, differentiation and fate of i.e. GFP or RFP and/or luciferase expressing cells, or biological processes like apoptosis, protein–protein interaction, angiogenesis, proteolysis and gene-transfer. Optical imaging (OI) and optical reporter systems are also very cost-effective and time-efficient and they are particularly well suited for small animal imaging and for in vitro assays to validate different reporter systems.