PMEL Mutations Make Functional Amyloid Pathogenic Figure 5. Wild-type and TMD mutant hPMEL variants localize similarly when expressed in mouse melanocytes. Immortalized melan- Ink4a mouse melanocytes that stably expressed wild-type (a–c), TMinsWAP (d–f) or TMR625C (g–i) variants of hPMEL were fixed and analyzed by IFM relative to the lysosomal marker, LAMP2 (red), and to pigment granules visualized by bright field microscopy (BF; pseudocolored blue). Ectopic hPMEL was detected using NKI-Beteb (green), an antibody that recognizes human PMEL, but not murine PMEL. Shown are overlaps of PMEL relative to LAMP2 (a, d, g) or melanosomes (b, e, h) alone or together (c, f, i). Insets show a 4X magnification of the boxed areas. Note that both mutants show a similar pattern of staining and co-localization as wild-type hPMEL. Individual labels and the original bright field images are shown in Figure S3. doi:10.1371/journal.pgen.1002286.g005 hPMEL were easily observed in cells expressing the TMR625C and especially the TMinsWAP variants (asterisks, Figure 6e–f; more images can be found in Figure S4). The sheets seemed unusually tightly packed with no space between the fibrils (note the spacing between fibrils in organelles that lack labeling for the transgene, indicated by brackets, and the loss of spacing in organelles that are labeled for the TMinsWAP and TMR625C transgenes). The organelles that were immunogold labeled largely showed no overt pigmentation, suggesting that either the fibrils were no longer capable of binding to melanins or that they inhibited melanin production. In some cases, particularly in cells expressing the TMR625C variant, labeling could be observed on the non- pigmented periphery of melanized melanosomes (Figure S4h and i). To better test whether the variant hPMEL isoforms affected overall pigmentation, we transiently expressed them in melan- mu:MuHA, a highly pigmented mouse melanocyte cell line that is less likely than melan-Ink4a to de-differentiate (our unpublished observations), using recombinant retroviruses that coexpress EGFP. The highest hPMEL expressers were enriched by cell sorting for high EGFP expression. By immunoblotting, transgene expression was substantially higher than in the stable transfectants but still less than endogenous mouse PMEL expression (data not shown). Cells were then processed for standard EM analysis. Whereas cells expressing wild-type hPMEL were normally pigmented as compared to cells expressing empty vector (not shown) and harbored few stage II (unpigmented) melanosomes, cells expressing the TMR625C and especially the TMinsWAP variants were hypopigmented, harbored fewer pigmented melanosomes and were enriched in early stage melanosomes (Figure 7A–7B). Quantification showed that cells expressing TMinsWAP hPMEL showed an increase in non-pigmented Stage II (p = 0.006) with a concomitant loss of pigmented Stage III melanosomes (p = 0.014) as compared to wild-type hPMEL (Figure 7B, 7C); cells expressing TMR625C hPMEL likewise showed a decrease in Stage IV melanosomes (p = 0.027) with a concomitant increase in Stage II (p = 0.018) and Stage I melanosomes (p = 0.014). Importantly, mutant PMEL-expressing cells with the highest increase in early stage melanosomes consistently showed the most marked decrease in pigmented organelles, suggesting a defect in melanosome maturation or pigment production that might be associated with higher expression levels of mutant PMEL. Moreover, quantifica- tion of the number of pigmented (Stage III and IV) organelles per unit area was decreased most strikingly in cells expressing TMinsWAP (p = 0.002; Figure 7D). In cells expressing TMR625C there was also a tendency towards a decrease in the number of pigmented organelles (see Figure 7A–7C), but it was not significantly different from cells expressing wild-type PMEL (Figure 7D), likely due to the high variability encountered in these cells (see Figure 7B). These results suggest that even after short PLoS Genetics | www.plosgenetics.org 7 September 2011 | Volume 7 | Issue 9 | e1002286