BackgroundCells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones.ResultsShort-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40–700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin.ConclusionsThese studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0802-2) contains supplementary material, which is available to authorized users.
For receptor tyrosine kinases supramolecular organization on the cell membrane is critical for their function. Super-resolution fluorescence microscopy techniques have offered new opportunities for the analysis of single receptor localization. Here, we analysed the cluster formation of the epidermal growth factor receptor variant III (EGFRvIII), a deletion variant which is expressed in glioblastoma. The constitutively activated variant EGFRvIII is expressed in cells with an egfr gene amplification and is thought to enhance the tumorigenic potential especially of glioblastoma cells. Due to the lack of an adequate model system, it is still unclear how endogenous EGFRvIII expression alters cellular signalling and if it is organized in clusters like the wild type receptor. We have recently described the establishment of two pairs of iso-genetic cell lines (BS153 and DKMG), displaying endogenous EGFRvIII expression or not. Using these cell lines we investigated single receptor localization of EGFRvIII by high precision localization microscopy. Cluster analysis revealed that EGFRvIII is present in clusters on the surface of the cells, with about 60% or even more receptor molecules being assembled in clusters of approximately 100 nm in diameter whereby the cluster definition was iteratively determined. The signal to signal distance may indicate dimer formation while signal quantification indicates 1 × 10-5 × 10 EGFRvIII molecules per cell. Altogether, these data give unique insights into the membrane surface localization of EGFRvIII in glioblastoma cells. These insights will help to unveil the function of this tumour associated receptor variant which might lead to a better understanding of glioblastoma and therefore could lead to improved therapy approaches.
Background: Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones.Results: Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. Conclusions: These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.
Methods, experimental setups and perspectives of three-dimensional deep view imaging microscopy of cell or tissue samples are reported. Preliminary biophysical and clinically relevant examples are presented.
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