Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy
Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy
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DOI：
10.3390/ijms19123713
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发表时间：
2018-12-01
影响因子：
5.6
通讯作者：
Falk, Martin
中科院分区：
文献类型：
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作者：
Bobkova, Elizaveta;Depes, Daniel;Falk, Martin
DNA双链断裂(DSB)是由电离辐射引起的最严重的染色质损伤类型。在DSB修复过程中，细胞以不同的方式将不同的蛋白质招募到受损部位，这种方式取决于局部染色质结构、DSB在细胞核中的位置以及进入的修复途径。53BP1是参与细胞修复途径决定的重要成员之一。虽然已有许多分子生物学的研究，但53BP1修复中心的结构及其在辐射后时间，特别是蛋白质募集期间的发展仍有待阐明。超分辨光学显微镜是一种强大的新工具，可以在3D保守的细胞核中进行此类研究。最近，我们展示了单分子定位显微镜(SMLM)作为这些高分辨率方法之一的适用性，用于分析完整细胞核中动态修复蛋白的分布和修复焦点的内部纳米结构。在本研究中，我们集中研究了不同类型的抗辐射细胞、中等抗辐射的新生儿真皮成纤维细胞(NHDF)和高抗辐射的U87胶质母细胞瘤细胞中的53BP1焦点，这些细胞暴露在高LET N-15离子辐射中。在给定的时间点，直到照射后24小时，以10-20 nm的分辨率定量地评估了荧光标记的53BP1分子的坐标和空间分布。根据SMLM参数确定这些标签的簇作为修复焦点的亚单位。研究了这种团簇的形成和松弛过程。较高的剂量产生了足够数量的DNA断裂，以比较53BP1在DSB处理过程中对所研究细胞类型的辐照后动态。使用垂直(90度)照射方案，剂量为4.0GY，以实现相对较高数量的粒子轨迹的更好分离，这些粒子轨迹通常横跨每个原子核。对于离子径迹分析，剂量降至1.3GY，并结合锐角照射(与细胞平面成10度角)。结果显示，与U87细胞相比，在成纤维细胞中，53BP1蛋白被招募到SMLM定义的簇中的比例更高。此外，不同细胞类型的病灶形成和松弛的速度也不同。在NHDF和U87细胞中，即使在照射后24小时，仍有一定数量的检测到的和功能相关的簇保持不变；然而，这些簇的数量又因细胞类型而异。总之，我们的发现表明，由SMLM和松弛(即，其余的53BP1标签不再满足簇定义)确定的修复簇的形成与细胞类型有关，并可能从功能上解释并与细胞特异性放射敏感性相关。本研究表明，SMLM是一种非常适合研究细胞核中时空蛋白质组织的方法，以及它如何影响给定DSB位点上特定修复途径的细胞决策。
DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET N-15-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10-20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90 degrees) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10 degrees relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.