Mutations in or near the Transmembrane Domain Alter PMEL Amyloid Formation from Functional to Pathogenic
Figure 7
Reduced melanosome pigmentation in melanocytes expressing PMEL TMD mutants.
Immortalized melanMu:MuHA pigmented melanocytes were transiently infected with retroviruses that encode wild-type, TMinsWAP or TMR625C hPMEL-and co-expressed EGFP. Four days post-infection, cells expressing high levels of hPMEL transgene were selected by flow cytometric sorting for high EGFP expression, then fixed, embedded in epon resin, and processed for conventional thin section EM. A. Cells expressing wild-type hPMEL (left panels) show predominantly pigmented stage III and IV (III, IV) melanosomes and few unpigmented Stage I and II melanosomes (I, II). By contrast, cells expressing either TMinsWAP (middle panels) or TMR625C (right panels) hPMEL variants harbor many fewer Stage IV melanosomes and many more non-pigmented, Stage I-II melanosomes. M, mitochondria. B. The total number of melanosomes of each stage in 6–7 whole cells from each set of samples was quantified relative to the total number of melanosomes per cell. The mean, median, maximum (max) and minimum (min) values, and 25th and 75th quartile values for distance moved in each experimental set are shown. C. Quantification of the number of each stage melanosome divided by the number of melanosomes in that field; at least 60 fields of equal size/magnification were analyzed, containing more than 400 melanosomes of different maturation stages. *, p <0.05; **, p <0.01 as determined by ANOVA and Student's t-test. D. Quantification of the number of pigmented granules (Stage III-IV) per µm2 of cell area in cells expressing wild-type, TMinsWAP or TMR625C hPMEL variants or cells transduced with virus that did not co-express hPMEL. Each measurement is shown individually; median values are indicated. **, p<0.01 as determined by ANOVA and Student's t-test.