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Christian Imhäuser

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Proceedings Article | 18 May 2010 Paper

3D topology and arrangement of proteins inside ceramide-rich domains

Christian Imhäuser, Heike Gulbins, Erich Gulbins, Hans-Gerd Lipinski

Proceedings Volume 7715, 77152N (2010) https://doi.org/10.1117/12.854345

KEYWORDS: Molecules, Proteins, 3D modeling, 3D image processing, Visualization, Receptors, Volume rendering, Image filtering, 3D visualizations, Image processing

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Clustering of receptor and signalling molecules (such as CD95 or CD40) within ceramide-enriched membrane domains results in a very high density of these proteins facilitating activation of associated enzymes, the exclusion of inhibitory molecules and/or the recruitment of further signalling molecules to transmit the signal into the cells. However, at present the mechanisms of receptor clustering and the exact distribution of proteins within the ceramide-enriched domains are unknown. Therefore, we generated digital images from anti-CD95 stimulated JYcells that were stained with FITC-coupled anti-ceramide and Cy3-labelled anti-CD95 antibodies. We developed image processing methods to determine the spatial distribution of proteins in ceramide-enriched membrane domains and visualized them by volume rendering and surface models. After image preprocessing with appropriate filters for contrast enhancement, noise reduction and logarithmic scaling, 3D models were generated using adapted volume and surface reconstruction. To detect the colocalization of CD95 and ceramide molecules we developed several different methods rasterizing 3D data of each channel into cells and counting intensity values above a specified colour threshold value. The colocalization voxel was set either by normalized product of totals (product intensity) or depending on binarization. In addition, a cross-covariance function to quantify the colocalization was determined and embedded as a 3D object. These computerized techniques allowed for a quantitative analysis of the spatial arrangement of proteins in ceramide-rich domains of living cells.

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