Fig. 2

Quantification method for plasma membrane localization. High resolution confocal images (1024 × 1024 px with a pixel size of 140 nm) were taken of HeLa cells transfected with: (a) a plasma membrane marker Lck fused to CFP, (b) a protein of interest fused to YFP (wild type TGAT in this example) and (c) a cytoplasmic marker using a soluble RFP. The CFP channel was registered to the YFP channel based on the spatial shift determined with the positive control. The RFP channel was registered to the YFP channel based on the spatial shift determined with the negative control. Background fluorescence was subtracted from each image prior to processing. Using ImageJ many lines (10 px wide and 6–10 μm long) were carefully drawn perpendicular to the plasma membrane in regions with a well-defined cytoplasm-plasma membrane-extracellular space transition (yellow lines in panel a). For each channel line-scans were performed with the same lines using linear interpolation, the profiles were aligned and centered based on the peak in the Lck-CFP channel and placed on the same axis (panel d). The other channels were aligned and centered using the same positional shift and axis (panels e and f). Subsequently the average profiles were calculated (colored lines in panels d-g), and normalized to unity with respect to the cytoplasm fluorescence level (panel g). Because the Lck-CFP marker is also localized in the cytoplasm, the profile was corrected by subtracting the normalized RFP profile (dashed line panel g). In order to extract the cytoplasmic (CP) and plasma membrane (PM) component, the YFP profile was unmixed using the normalized RFP profile and the corrected Lck-CFP profile (panel h). The 95% confidence intervals (thin solid lines above and below the profiles) were estimated using bootstrapping (See material and Methods for details)