Application of fluorescent protein tags as reporters in live-cell imaging studies.

The advent of the "GFP technology" together with advances in digital imaging and microscopic techniques has revolutionized our view of the living cell. Genetically encoded fluorescent proteins have become widely used as markers in living cells. The application of these fluorescent proteins as noninvasive tags revealed new aspects of protein dynamics and the biological processes they regulate. The modification of naturally occurring fluorescent proteins as well as the identification of new fluorescent proteins now provide researchers with a variety of useful fluorescent markers suitable for all kind of investigations in live-cell imaging studies. This chapter provides an overview of the most widely used fluorescent proteins for the labeling of either individual proteins or compartments of Dictyostelium discoideum cells. Furthermore, aspects such as how to design a fusion protein as well as advantages and disadvantages of specific fluorescent proteins are discussed. Finally, a protocol from transformation to identification of positive clones is provided.