Microinjection Techniques in Fly Embryos to Study the Function and Dynamics of SMC Complexes.

Structural maintenance of chromosomes (SMC) proteins are critical to maintain mitotic fidelity in all organisms. Over the last decades, acute inactivation of these complexes, together with the analysis of their dynamic binding to mitotic chromatin, has provided important insights on the molecular mechanism of these complexes as well as into the consequences of their failure at different stages of mitosis.Here, we describe a methodology to study both SMC function and dynamics using Drosophila melanogaster syncytial embryos. This system presents several advantages over canonical inactivation or imaging approaches. Efficient and fast inactivation of SMC complexes can be achieved by the use of tobacco etch virus (TEV) protease in vivo to cleave engineered versions of the SMC complexes. In contrast to genetically encoded TEV protease expression, Drosophila embryos enable prompt delivery of the protease by microinjection techniques, as detailed here, thereby allowing inactivation of the complexes within few minutes. Such an acute inactivation approach, when coupled with real-time imaging, allows for the analysis of the immediate consequences upon protein inactivation. As described here, this system also presents unique advantages to follow the kinetics of the loading of SMC complexes onto mitotic chromatin. We describe the use of Drosophila embryos to study localization and turnover of these molecules through live imaging and fluorescence recovery after photobleaching (FRAP) approaches.