GFP imaging: methodology and application to investigate cellular compartmentation in plants

The cloning of the jellyfish gfp (green fluorescent protein) gene and its a lteration for expression in subcellular locations in transformed plant cell s have resulted in new views of intracellular organization and dynamics. Fu sions of GFP with entire proteins of known or unknown function have shown w here the proteins are located and whether the proteins move from one compar tment to another. GFP and variants with different spectral properties have been deliberately targeted to separate compartments to determine their size , shape, mobility, and dynamic changes during development or environmental response. Fluorescence Resonance Energy Transfer (FRET) between GFP variant s can discern protein/protein interactions. GFP has been used as a sensor t o detect changes or differences in calcium, pH, voltage, metal, and enzyme activity. Photobleaching and photoactivation of GFP as well as fluorescence correlation spectroscopy can measure rates of diffusion and movement of GF P within or between compartments. This review covers past applications of t hese methods as well as promising developments in GFP imaging for understan ding the functional organization of plant cells.