High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae
Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell’s nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.
Key wordsNucleolus FROS Nuclear organization Live-cell imaging Fluorescence microscopy Chromosome domain dynamics Yeast Saccharomyces cerevisiae
This work was supported by ATS-Nudgene and Emergence-CLEMgene of the Toulouse-IDEX. O.G. and C.N. are supported by Agence Nationale de la Recherche (ANDY). We thank I. Léger-Silvestre for thoughtful discussions and technical advices. Julien Mathon wrote the MTT-based executable files.
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