GFP imaging: methodology and application to investigate cellular compartmentation in plants.
The cloning of the jellyfish gfp (green fluorescent protein) gene and its alteration for expression in subcellular locations in transformed plant cells have resulted in new views of intracellular organization and dynamics. Fusions of GFP with entire proteins of known or unknown function have shown where the proteins are located and whether the proteins move from one compartment 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 variants can discern protein/ protein interactions. GFP has been used as a sensor to 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 GFP within or between compartments. This review covers past applications of these methods as well as promising developments in GFP imaging for understanding the functional organization of plant cells.
Hanson
M R
MR
Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY 14853, USA. mrh5@cornell.edu
Köhler
R H
RH
D002451:Cell Compartmentation; D002478:Cells, Cultured; D003952:Diagnostic Imaging / Q000379:methods; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000493:pharmacokinetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000493:pharmacokinetics*; D018613:Microscopy, Confocal; D008967:Molecular Biology / Q000379:methods; D015388:Organelles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D059828:Plant Cells; D019441:Plant Epidermis / Q000166:cytology / Q000187:drug effects; D010940:Plant Proteins / Q000502:physiology*; D030821:Plants, Genetically Modified; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013379:Substrate Specificity
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010940:Plant Proteins; D049452:Green Fluorescent Proteins