Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.
- Klíčová slova
- 3D visualization, Image reconstruction, Image segmentation, Morphology, Organoid, SBF-SEM, Scanning electron microscopy, Serial block-face, Spheroid, Stem cell, Ultrastructure,
- MeSH
- buněčné sféroidy ultrastruktura MeSH
- embryonální kmenové buňky ultrastruktura MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací metody MeSH
- počítačové zpracování obrazu MeSH
- zobrazování trojrozměrné metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.
- Klíčová slova
- CARM1, DNA repair, HDAC1, NBS1, PpoI, chromatin, nucleolus,
- MeSH
- acetylace MeSH
- arginin metabolismus MeSH
- buněčné jadérko genetika ultrastruktura MeSH
- buněčné linie MeSH
- dvouřetězcové zlomy DNA * MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- fosfoproteiny metabolismus MeSH
- geny rRNA MeSH
- histondeacetylasa 1 metabolismus MeSH
- histony metabolismus MeSH
- intracelulární signální peptidy a proteiny MeSH
- jaderné proteiny metabolismus MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- proteinarginin-N-methyltransferasy metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- arginin MeSH
- coactivator-associated arginine methyltransferase 1 MeSH Prohlížeč
- fosfoproteiny MeSH
- Hdac1 protein, mouse MeSH Prohlížeč
- histondeacetylasa 1 MeSH
- histony MeSH
- intracelulární signální peptidy a proteiny MeSH
- jaderné proteiny MeSH
- proteinarginin-N-methyltransferasy MeSH
- RNA ribozomální MeSH
- Tcof1 protein, mouse MeSH Prohlížeč
CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.
- MeSH
- buněčné jadérko účinky léků fyziologie ultrastruktura MeSH
- buněčné linie MeSH
- embryonální kmenové buňky účinky léků fyziologie ultrastruktura MeSH
- epigeneze genetická účinky léků fyziologie MeSH
- kyselina ellagová farmakologie MeSH
- lidé MeSH
- myši MeSH
- proteinarginin-N-methyltransferasy antagonisté a inhibitory fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- coactivator-associated arginine methyltransferase 1 MeSH Prohlížeč
- kyselina ellagová MeSH
- proteinarginin-N-methyltransferasy MeSH
Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1 alpha, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc, the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.
- MeSH
- buněčná diferenciace účinky léků genetika MeSH
- buněčné jádro genetika ultrastruktura MeSH
- buněčné linie MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- homolog proteinu s chromoboxem 5 MeSH
- lidé MeSH
- pluripotentní kmenové buňky metabolismus ultrastruktura MeSH
- restrukturace chromatinu * MeSH
- signální transdukce genetika MeSH
- trans-aktivátory účinky léků metabolismus MeSH
- tretinoin farmakologie MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromozomální proteiny, nehistonové MeSH
- homolog proteinu s chromoboxem 5 MeSH
- trans-aktivátory MeSH
- tretinoin MeSH
