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

Abstract

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. γ-H2AX and cleaved caspase 3 are proteins formed during UVC-induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ-H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ-H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ-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 as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate 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.

The apoptotic ring: A novel entity with phosphorylated histones H2AX and H2B, and activated DNA damage response kinases

“…9 These nuclear structures connect sometimes with the nucleolus suggesting a possible nucleolar origin, and they present some similarities with the nuclear stress bodies that contain among others RNA and RNA-splicing factors. 10 γ-H2AX panstaining has also been observed during checkpoint abrogation in cancer cells treated with camptothecin, 11 indenoisoquinolines 12 or gemcitabine.…”

Towards native-state imaging in biological context in the electron microscope

“…Direct imaging of fluorescent molecules in situ in an EM is the ultimate realisation of CLEM. Integrated laser electron microscopy has taken the first steps towards this by feeding a laser line into the goniometer of a TEM [3] to image UVinduced nuclear structures in apoptotic cells [53].…”

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

“…These requirements, which a sample must meet to be imaged in iLEM, have become an impulse to develop new sample preparation techniques or to optimize the ones that exist. It is commonly known that if heavy metal and fluorescent label particles are localized at the distance of ~100 Å from each other, quenching of the fluorescence signal may occur [132]. In order to prevent this, the material must be prepared in such a way that increasing the distance between markers' particles is possible.…”

“…The optical-imaging mode is used to identify fluorescent markers and then the region of interest (ROI) is analysed at high resolution in the TEM mode. In this way, the time-consuming relocation of the ROI across microscopy platforms is avoided (with non-integrated solutions for correlative microscopy the sample needs to be transferred from one imaging set-up to another and specialised procedures are required to correlate the results) [132,133].…”