Discovery of a new RNA containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Introduction: Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. gamma-H2AX and cleaved caspase-3 are proteins formed during UVC-induced DNA damage and apoptosis, respectively. This research sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy: integrated laser electron microscopy (ILEM). Cleaved caspase-3 and gamma-H2AX were immuno-fluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analyzed at high resolution with transmission electron microscopy. Results: Following the treatment of human umbilical cord vein endothelial cells (HUVECs) with UVC radiation in the majority of the cells gamma-H2AX was formed, whereas only in a subset caspase-3 was activated. In severely damaged cells with high levels of gamma-H2AX a round, electron dense nuclear structure was found, which was hitherto not identified in UV-stressed cells. This structure exists only in nuclei of cells containing cleaved caspase-3 and is present during all stages of the apoptotic process. Energy-loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV-induced nuclear structure contains a high amount of RNA. Conclusions: Because the UV-induced nuclear structure was only found in cells labelled for cleaved caspase-3 it is proposed for an electron microscopic marker for all stages of apoptosis. Such a marker will facilitate especially the screening for early apoptotic cells, which lack the well-known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV-induced apoptotic pathway.