Intra-Nuclear Single-Particle Tracking (I-SPT) to Reveal the Functional Architecture of Chromosomes
Chromosome architecture needs to be investigated in relation with the chemical function of DNA. The kinetics of gene expression, DNA replication, and repair are driven by the mechanisms by which a functional nuclear protein finds its substrate in the nucleus. Single-particle tracking (SPT) is a method to quantify fluorescent molecules dynamics from the tracks of the single molecules recorded by high-resolution microscopes. SPT offers direct observation of the movement and single-molecule resolution. Usually SPT is performed on membranes because of higher contrast. Here, we introduce a novel method to record the trajectories of weakly fluorescent molecules in the nucleus of living cells. I-SPT uses some specific detection and analysis tools to enable the computation of reliable statistics on nuclear particle movement.
Key wordsChromatin functional architecture Single-particle tracking Photoactivatable proteins Molecule diffusion
The author would like to thank Ignacio Izeddin and Xavier Darzacq who co-designed this protocol, Mark Leake and Roman Sedlak for critical reading of the manuscript. This research was supported by the Fondartion pour la Recherche Médicale (FRM) and Marie Curie Action.
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