Live Imaging of Drosophila Embryos: Quantifying Protein Numbers and Dynamics at Subcellular Locations

Abstract

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.

Methods for studying planar cell polarity

“…When the embryo is submerged in halocarbon oil, the chorion becomes transparent, and imaging of embryonic structure is possible without dechorination. Nevertheless, removing the chorion improves the imaging quality and is therefore often done (David et al, 2012). The chorion can be removed using bleach, as described in (David et al, 2012) or manually (Reed et al, 2009).…”

“…Nevertheless, removing the chorion improves the imaging quality and is therefore often done (David et al, 2012). The chorion can be removed using bleach, as described in (David et al, 2012) or manually (Reed et al, 2009). Live imaging of embryos has been used to study planar polarity and cytoskeletal dynamics during the growth of denticles (Price et al, 2006).…”

“…Live imaging of embryos has been used to study planar polarity and cytoskeletal dynamics during the growth of denticles (Price et al, 2006). Prince et al mounted embryos in halocarbon oil between a gas permeable membrane and a coverslip, as described in detail in (David et al, 2012). In other studies embryos have been mounted between coverslips and using 70% glycerol (Sanson et al, 1999).…”

“…In both cases the embryos will be slightly compressed which increases the area that can be viewed in the same focal plane. Embryos can also be glued to a coverslip which allows for imaging hanging embryos and embryos in otherwise unstable positions (David et al, 2012; Figard & Sokac, 2011). The glue is made by dissolving the adhesive from clear tape in heptane.…”

Adherens Junction Distribution Mechanisms during Cell-Cell Contact Elongation in Drosophila

“…Final intensities of the photobleached area were then corrected to 1 by dividing each value by the value immediately before photobleaching. Mobile fractions and t½ values were calculated as described [46]. …”