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Quantifying Protein Mobility in Living Drosophila Embryos Using Fluorescence Recovery After Photobleaching (FRAP)

William Dempsey, Shima Hajimirza

Supervised by: Willy Supatto and Laki Pantazis

The Fraser lab

Morphogenesis in developing embryos relies on diffusion of various proteins in the mother's egg sack. Drosophila embryos, for example, start out as one big sack without proper separation between cells since the cell membranes have not been formed yet. In this project, we were interested in the question of how nuclear matter gets partitioned in Drosophila embryos. Specifically, the idea is to measure in living embryos the diffusion coefficients for two proteins related to nuclear localization: H2A and Nuclear Localization Signal (NLS). Two transgenic Drosophila types were used: wild-type embryos ubiquitously expressing histone (H2A-GFP) and wild-type embryos ubiquitously expressing nuclear localization signal (NLS-GFP). William and Shima demonstrated that H2A-GFP diffuses at a rate of ~0.01 μm2/sec while the temporal resolution was not high enough to determine the NLS-GFP diffusion constant (recover occured in less than 1 second). The free diffusion coefficient GFP for comparison is 87 μm2/sec. Single cell control indicates that H2A can migrate between nuclei, however multiple cell control suggests that production is unlikely. In the figure below we see a Drosophila embryo at the 14-th division cycle ubiquitously expressing H2A-GFP.






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