Time-lapse, photoactivation, and photobleaching imaging of nucleolar assembly after mitosis

Nucleolus assembly starts in telophase with the benefit of building blocks passing through mitosis and lasts until cytokinesis generating the two independent interphasic cells. Several approaches make it possible to follow the dynamics of fluorescent molecules in live cells. Here, three complementary approaches are described to measure the dynamics of proteins during nucleolar assembly after mitosis: (1) rapid two-color 4-D imaging time-lapse microscopy that demonstrates the relative localization and movement of two proteins, (2) photoactivation that reveals the directionality of migration from the activated area, and (3) fluorescence recovery after photobleaching (FRAP) that measures the renewing of proteins in the bleached area. We demonstrate that the order of recruitment of the processing machineries into nucleoli results from differential sorting of intermediate structures assembled during telophase, the prenucleolar bodies.