Single molecule microscopy reveals key physical features of repair foci in living cells

Abstract

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.

Physical observables to determine the nature of membrane-less cellular sub-compartments

“…The experimental findings of single Rad52 molecules in yeast repair foci [21] suggest that the movement inside the focus are consistent with normal diffusion (Fig. 2C).…”

“…In recent experimental work [21], we used single particle tracking to follow the movement of Rad52 molecules, following a double-strand break in S. cerevisiae yeast cells, which causes the formation of a focus. These experiments show that temporal traces of Rad52 molecules concentrate inside the focus, as shown for a representative cell in Fig.…”

“…2B). To mimick the data, we only record and show traces in two dimensions and added detection noise corresponding to the level reported in the experiments [21]. Based on these simulations, we gather the statistics of the particle motion to create a displacement histogram representing the probability distribution of the observed step sizes between two successive measurements.…”

“…Then the probability distribution of the particle's position is given by: [21]. D0 and A are model parameters in the LPM, but also effective observables in the PBM.…”

“…Coarse-grained theoretical models of the LPM exist [19,20], but predictions of microscale behaviour that can be combined with a statistical analysis of high resolution microscopy data to discriminate between the hypotheses has not yet been formulated. Previously, we analyzed in detail single-particle tracking data in the context of yeast DSB foci, and discussed their comptability with each model [21]. Here, we build a general physical framework for understanding and predicting the behaviour of each model under different regimes.…”