Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching

Abstract

Regulation and structural requirements of vital nuclear processes such as DNA replication, transcription, RNA processing and DNA repair inside the eukaryote nucleus are as yet poorly understood. Although a wealth of evidence exists pointing to a considerable degree of spatial organisation of chromatin and nuclear processes, there are still questions concerning the dynamics and interaction of nuclear proteins that remain unanswered. The cloning of the gene encoding the green fluorescent protein (GFP) has revolutionised the study of proteins in living cells. The expression of recombinant cDNA fusion plasmids of GFP and proteins of interest currently enables the investigation of those proteins in living cells. Time-lapse confocal microscopy as well as quantitative fluorescence methods such as fluorescence redistribution after photobleaching (FRAP) and fluorescence resonance energy transfer are widely applied to living cells expressing GFP fusion proteins. This review gives an overview of the current state of knowledge of nuclear structure and function. In particular, the different applications of FRAP technology to study the dynamics of GFP-tagged nuclear proteins will be summarised.