Hrs-2 overexpression induces enlarged early endosomes.

 

The average area of the 5 largest EGFP/hrs-2 positive organelles in each of 30 transfected cells was quantified and plotted against the level of EGFP/hrs-2 expression as measured by the total fluorescence intensity within the cell. As shown here, increasing levels of expression of EGFP/hrs-2 correspond with increasing size of labeled organelles.

 

Control primary human fibroblasts were transfected with pDNA encoding EGFP/hrs-2, plated onto coverslips and fixed 3 days post-transfection. Cells were viewed with a 100X oil objective lens on a Zeiss Axioplan microscope. Images were captured with an AxioCam digital camera and the AxioVision 2.0 imaging system (Zeiss). Images were analyzed using the Analytical Imaging Station (Imaging Research Inc.). Intensity (expressed as the mean of all the pixels contained within the sample outline) was calculated for transfected cells from a two second exposure and used as a measure of pEGFP/hrs-2 expression level. The area of individual organelles was measured from images captured at exposure times appropriate to pEGFP/hrs-2 expression level for each cell and calculated as pixels inside target outline borders.