Liddle Pablo, Jara-Wilde Jorge, Lafon-Hughes Laura, Castro Iván, Härtel Steffen, Folle Gustavo
Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay.
SCIAN-Lab, Biomedical Neuroscience Institute (BNI), Santiago, Chile.
Mol Cell Biochem. 2020 Oct;473(1-2):77-91. doi: 10.1007/s11010-020-03809-4. Epub 2020 Jul 7.
In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoï tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside γH2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, γH2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 µm), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (~ 60 nanofoci/µm). Scattered γH2AX nanofoci were equally present (~ 3 nanofoci/µm), 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 (~ 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.
为响应DNA双链断裂（DSB），组蛋白H2AX通过前馈信号放大环在损伤周围被磷酸化，产生可通过免疫荧光和共聚焦显微镜检测到的γH2AX焦点，表现为强度均匀的椭圆形区域。我们利用单分子定位显微镜（SMLM）提供的显著分辨率提高（约×10），在纳米尺度上研究内源性（对照）或由放射模拟物博来霉素（BLEO）诱导的γH2AX信号在HeLa细胞中的分布。在这两种情况下，均检测到局限于γH2AX焦点的聚集亚结构（纳米焦点）和遍布剩余核区域的分散纳米焦点。SR-Tesseler软件（基于沃罗诺伊镶嵌的分割）与自定义Python脚本相结合，首先将γH2AX焦点内的聚集纳米焦点与分散纳米焦点分离，然后对每种纳米焦点类型进行聚类分析。与对照相比，BLEO处理细胞核中的γH2AX焦点平均面积更大（0.41对0.19µm），每个焦点的纳米焦点更多（22.7对13.2），纳米焦点密度相当（约60个纳米焦点/µm）。分散的γH2AX纳米焦点同样存在（约3个纳米焦点/µm），表明其起源于内源性。用经典H2AX激酶的特异性抑制剂分别处理BLEO处理的细胞，即：用于ATM的KU-55933、用于ATR的VE-821和用于DNA-PK的NU-7026。在用混合抑制剂处理时，聚集的纳米焦点从超分辨率图像中消失，而分散的纳米焦点密度降低（约50%）。残留的分散纳米焦点除其他可能性外，可能反映由VRK1介导的H2AX磷酸化，VRK1是最近描述的一种非经典H2AX激酶。除了H2AX的研究结果外，还提出了一种从SMLM数据中量化密度差异很大的簇的分析方法。
