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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