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.
Key wordsDrosophila Live imaging Protein counting Fluorescence recovery after photobleaching Adherens junctions Epithelia
Work in our lab is supported by a CIHR operating grant and an NSERC operating grant. A. McKinley holds an Ontario Graduate Scholarship in Science and Technology. T. Harris holds a Tier 2 Canada Research Chair.
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