Imaging biochemistry inside cells

Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed bioch emical activities connected by rapidly diffusing second messengers, metabol ic intermediates, ions or other proteins. It will probably remain within th e realm of genetics to identify the ensemble of proteins that constitute th ese functional units and to establish the first-order connectivity. The dyn amics of interactions within these protein machines can be assessed in livi ng cells by the application of fluorescence spectroscopy on a microscopic l evel, using fluorescent proteins that are introduced within these functiona l units. Fluorescence is sensitive, specific and non-invasive, and the spec troscopic properties of a fluorescent probe can be analysed to obtain infor mation on its molecular environment. The development and use of sensors bas ed on the genetically encoded variants of green-fluorescent proteins has fa cilitated the observation of 'live' biochemistry on a microscopic level, wi th the advantage of preserving the cellular context of biochemical connecti vity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing co rrelation with phenomena such as the cell cycle, migration and morphogenesi s.