Analysis of nuclear export using photoactivatable GFP fusion proteins and interspecies heterokaryons.
In this chapter, we review protocols for the analysis of nucleocytoplasmic shuttling of transcription factors and nuclear proteins, using two different approaches. The first involves the use of photoactivatable forms of the protein of interest by fusion to photoactivatable green fluorescent protein to follow its movement out of the nucleus by live-cell confocal microscopy. This methodology allows for the kinetic characterization of protein movements as well as measurement of steady-state levels. In a second procedure to assess the ability of a nuclear protein to move into and out of the nucleus, we describe the use of interspecies heterokaryon assays, which provide a measurement of steady-state distribution. These technologies are directly applicable to the analysis of nucleocytoplasmic movements not only of transcription factors, but also other nuclear proteins.
Nakrieko
Kerry-Ann
KA
Department of Physiology, University of Western Ontario, London, ON, Canada.
Ivanova
Iordanka A
IA
Dagnino
Lina
L
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D003094:Collagen / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003094:Collagen