Abstract

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating gamma H2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (similar to x 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of gamma H2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to gamma H2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoi tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside gamma H2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, gamma H2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 mu m(2)), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (similar to 60 nanofoci/mu m(2)). Scattered gamma H2AX nanofoci were equally present (similar to 3 nanofoci/mu m(2)), suggesting an endogenous origin. BLEO-treated cells were challenged with specific inhibitors of canonical H2AX kinases, namely: KU-55933, VE-821 and NU-7026 for ATM, ATR and DNA-PK, respectively. Under treatment with pooled inhibitors, clustered nanofoci vanished from super-resolution images while scattered nanofoci decreased (similar to 50%) in density. Residual scattered nanofoci could reflect, among other alternatives, H2AX phosphorylation mediated by VRK1, a recently described non-canonical H2AX kinase. In addition to H2AX findings, an analytical approach to quantify clusters of highly differing density from SMLM data is put forward.