Live imaging of Drosophila embryos: quantifying protein numbers and dynamics at subcellular locations.
Live imaging is critical for understanding the structure and activities of protein interaction networks in cells. By tagging proteins of interest with fluorescent proteins, such as green fluorescent protein (GFP), their localization in cells can be determined and correlated with cellular activities. This can be extended into developmental systems such as Drosophila to understand the molecular and cellular bases of development. In this chapter, we review sample preparation techniques and basic imaging considerations for Drosophila embryos. We then discuss how these techniques can be extended to count absolute protein numbers at specific subcellular locations, and determine their dynamics using fluorescence recovery after photobleaching (FRAP). These techniques can help reveal the structure and dynamics of protein complexes in live cells.
Department of Cell & Systems Biology, University of Toronto, Toronto, ON, Canada.
Daryl J V
DJ
David
Melanie A
MA
McGill
R F Andrew
RF
McKinley
Tony J C
TJ
Harris
D000818:Animals; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000166:cytology* / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D042541:Intracellular Space / Q000378:metabolism*; D008567:Membranes, Artificial; D057054:Molecular Imaging / Q000379:methods*; D010539:Permeability; D021381:Protein Transport
D029721:Drosophila Proteins; D008567:Membranes, Artificial