Figure 5

Endocytic recycling of CI-MPR is impaired in Pompe disease fibroblasts. To study the trafficking of CI-MPR through the endocytic pathway normal and severe fibroblasts grown on coverslip were incubated at 18° C for 1h with 15 ng/ml Alexa-488-anti-CI-MPR antibody. The cells were washed and fixed (T0) or warmed to 37°C for 5, 15, 30, 60 min (T5, T15, T30, T60) before being fixed, permeabilized and stained with Alexa 488-conjugated donkey anti-mouse IgG to amplify the signal. (A) In control fibroblasts the CI-MPR signal reaches its steady-state within 15 minutes. In severe fibroblasts signal intensity is reduced and the correct localization is not reached. (B) Average fluorescence intensity of internalized anti-CI-MPR antibody was quantified within the area concerning each single cell, using LSM 3.2 software (Zeiss). The analysis was performed on 40 cells. The quantitative analysis of fluorescence intensity (time 0-60 min) shows progressive decrease of signal intensity in control fibroblasts. In severe Pompe disease (PD) fibroblasts the amount of fluorescence remains fairly constant, leading to an inversion of the rate between control and PD cells after 15 to 30 minutes. The error bars indicate the standard error. *=p≤0.05, **=p≤0.001. (C) The distribution of transferrin (Tf) and of trasferrin receptor (TR) is normal in PD fibroblasts. The cells were incubated with 50 mg/ml Alexa-488 transferrin at 18° C for 60 min, washed, fixed, permeabilized and stained with a trasferrin receptor antibody and Alexa-fluor 594 anti-rabbit IgG antibody. Stained cells were visualized by confocal fluorescence microscopy. (D) Average fluorescence intensity of A488-Tf was quantified within the area concerning each single cell, using LSM 3.2 software (Zeiss). The analysis was performed on 40 cells. Quantitative analysis of fluorescence intensity of the Tf signal shows comparable results in control and severe PD fibroblasts (p≤0.026). The error bars indicate the standard error.