Clustered localization of EGFRvIII in glioblastoma cells as detected by high precision localization microscopy

Abstract

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.

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

“…Cluster analysis software was applied to the localization images [46,47]. The algorithm identifies “cluster-points” within the localization data according to user-defined parameters.…”

“…The pointillist localization image can be used for further analyses based on distance and frequency measurements (e.g., [45,46]). Usually, density images (see Figure 1 for the different possible methods to produce images from the localization data) are used.…”

“…Nevertheless, the full resolution is used for further quantification of nano-structure analyses like cluster formation, etc. [46,47]. …”

Nanoscale distribution of TLR4 on primary human macrophages stimulated with LPS and ATI

“…Overall, our findings show that TLR4 molecules are present in a pre-clustered state on unstimulated macrophages, which confirms previous investigations on receptor clustering. [55][56][57] Next, we investigated whether ATI and tetranitromethane (TNM)-nitrated ATI have an effect on the membrane distribution of TLR4. Therefore, macrophages were stimulated with ATI or nitrated ATI for 15 min and 30 min, respectively (ESI Note 2 + 3, Fig.…”

COMBO-FISH: A Versatile Tool Beyond Standard FISH to Study Chromatin Organization by Fluorescence Light Microscopy

“…For each series of SMLM experiments, typically 50 cell nuclei were registered. An in-house software (see [31,39,54,65,77,78] for detailed descriptions) was used for the localization of signal points from blinking events in raw image stacks. The resulting data matrix containing the coordinates of detected signals was subject to further computational analysis.…”