Background

Drosophila melanogaster embryogenesis is a well-developed model system for studying mechanisms that drive plasma membrane shape changes during development. The Drosophila embryo is maternally loaded with machinery in the form of subcellular organelles, proteins, and mRNA, to drive the early stages of development. It is of interest to elucidate the function of organelle dynamics during early morphogenesis events in embryogenesis.

Drosophila embryogenesis begins as a syncytial cell, where nuclear cycles 1–13 occur in a common cytoplasm (Foe and Alberts, 1983). Nuclear division cycles 10–13 occur at the cortex. Complete cells that are epithelial in nature form in the interphase of syncytial division cycle 14. The plasma membrane extends around each nucleus and closes off at the bottom. Subcellular organelles, such as the endoplasmic reticulum (ER) and Golgi complex, are associated with each nuclear-cytoplasmic domain in the cortical syncytial division cycles (Frescas et al., 2006). Mitochondria are also present, interspersed within the ER and Golgi network (Chowdhary et al., 2017). Mitochondria are small, fragmented, and abundant in the syncytial blastoderm embryo (Chowdhary et al., 2017). Microtubule motors regulate their distribution in the syncytial division cycles and cellularization (Chowdhary et al., 2017 and 2020).

Genetically encoded fluorescently tagged transgenes distributed to the ER, Golgi complex, and mitochondria have helped ascertain their distribution in living embryos (Cox and Spradling, 2003; Frescas et al., 2006; Chowdhary et al., 2017). Diffusion dynamics of such fluorescently tagged molecules and organelles have been described using live imaging and photobleaching techniques, such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). These techniques depend on bleaching of existing fluorescence and enable an analysis of local exchange rates of tagged molecules, at given regions of interest in cells (Frescas et al., 2006). Photoactivation involves the use of photo-convertible fluorophores that can switch their fluorescence properties upon photoactivation (Patterson, 2011). PA-GFP needs to be activated with a high-power light of approximately 400 nm wavelength, to show fluorescence at approximately 520 nm. Thus, photoactivation allows highlighting subcellular organelles during distinct stages of development in a relatively non-invasive manner, by activation of PA-GFP at a very specific location using the 400 nm light and tracking this marked population against the non-activated, non-fluorescent background (Patterson and Lippincott-Schwartz, 2002). Mito-PA-GFP has been used to study mitochondria dynamics in distinct stages of Drosophila oogenesis and embryogenesis (Chowdhary et al., 2017 and 2020; Lieber et al., 2019).

Cell shape change seen in cell migration, cytokinesis, and embryo development is accompanied by the mitochondrial movement to the location of shape transition (Denisenko et al., 2019). The presence of mitochondria locally in the Drosophila embryo is necessary for driving morphogenesis in embryo development (Chowdhary et al., 2017 and 2020). Visualization of mitochondria in Drosophila embryos using mito-GFP shows that they are abundant, and that their movements cannot be ascertained with precision due to their density (Chowdhary et al., 2017 and 2020). Highlighting mitochondria in specific regions via photoactivation, and then imaging their movement, is a powerful method to follow their dynamics during distinct morphogenetic transitions in embryo development. In this protocol, we describe photoactivation as a method for highlighting mitochondria in distinct regions in Drosophila embryo development. We provide a stepwise description of the photoactivation procedure followed by image analysis, to decipher the distribution of mitochondria during syncytial division cycles and cellularization. Using this protocol, we observed that photoactivation of mitochondria in mito-PA-GFP expressing embryos in one nuclear-cytoplasmic domain shows a lack of movement into adjacent domains (Chowdhary et al., 2017). Photoactivation of mitochondria basally during cellularization shows translocation to apical locations (Chowdhary et al., 2020).