PMEL Mutations Make Functional Amyloid Pathogenic Figure 3. TMD mutations do not affect PMEL trafficking, maturation, or processing. HeLa cells were transiently transfected with low (A and C–D) or high (B) DNA levels of pCI plasmids encoding wild-type, TMinsWAP or TMR625C hPMEL, and processed for analysis 48 hrs post-transfection. A. IFM analysis. HeLa cells were fixed and co-stained with antibodies against hPMEL (red; panels a, d and g) and LAMP1 (green; panels b, e, and h). Right panels (c, f, i) show the merged images, and insets show a 4X magnification of the boxed regions. Notice the similar pattern of labeling of wild- type (a–c), TMinsWAP (d–f) and TMR625C hPMEL within LAMP1-labelled late endosomes/lysosomes. Bar, 20 mm. B. Immunoblot analysis. Transfected HeLa cells were lysed and fractionated into detergent soluble and insoluble fractions. Detergent soluble fractions (top three panels) were probed with antibodies to either the hPMEL N-terminus to detect P1 and Ma (top), or the C-terminus to detect Mb and CTF (middle panels). Detergent insoluble, fibril-enriched fractions were probed with HMB45 to detect the PMEL-derived MaC fragments (lower panel). Left, molecular weight markers; right, relevant bands are indicated. C. Metabolic pulse chase/ immunoprecipitation analysis. Transfected HeLa cells were metabolically labeled with 35S methionine/ cysteine, chased for the indicated times, and lysed in Triton X-100. PMEL was immunoprecipitated from detergent soluble fractions using an antibody to the C-terminus. Left, molecular weight markers; right, relevant bands are indicated. D. Quantification of abundance of relevant PMEL fragments from at least three pulse chase experiments as shown in C.; the band intensity of each fragment was normalized to that of P1 at time zero [P1(t0)]. Bars, standard error. doi:10.1371/journal.pgen.1002286.g003 TMinsWAP or TMR625C were detected within organelles that often contained internal membrane vesicles or sheets, but not in secretory or early endocytic compartments. Immunoelectron microscopy (IEM) analysis using immunogold labeling of ultrathin cryosections further showed that all of the hPMEL variants were incorporated into the fibrils (data not shown, but see below for incorporation of variants into melanosome fibrils in melanocytic cells). These data indicate that the TMinsWAP and TMR625C mutations do not impair the intrinsic ability of hPMEL to form fibrils within endosomal compartments. TMinsWAP and TMR625C mutations induce altered morphology and melanization of hPMEL fibrils in pigmented cells Unlike in non-pigment cells, in which fibril maturation is inefficient and occurs within late endosomes [13], PMEL protofibrils in pigment cells mature efficiently into sheets [11] that accumulate in stage II melanosomes and that then serve as sites of melanin deposition during melanosome maturation [24]. Figure 4. PMEL TMD mutants can form fibrils when overex- pressed in non-pigmented cells. HeLa cells transiently overexpress- ing wild-type, TMinsWAP or TMR625C hPMEL were fixed and embedded in epon resin for conventional electron microscopy analysis. Note that elongated fibrils in the lumen of multivesicular endocytic compart- ments (labeled with asterisks) are observed in cells expressing all three variants of hPMEL. Bars, 500 nm. doi:10.1371/journal.pgen.1002286.g004 To determine whether the TMinsWAP and TMR625C mutations influence fibril maturation or downstream pigmentation, we analyzed the behavior of hPMEL with or without these mutations expressed transiently or stably in pigment cells. In order to distinguish the transgene from the endogenous mouse PMEL, we expressed the hPMEL variants in ‘‘wild-type’’ mouse melanocyte cell lines, and exploited antibodies (NKI-beteb and HMB-50) that only detect hPMEL. By IFM, wild-type, TMinsWAP and TMR625C hPMEL each localized to punctate structures that partially overlapped with late endocytic compartments marked by LAMP2 and that did not overlap with mature pigmented eumelanosomes (Figure 5 and Figure S3). These data suggest that the wild-type, TMinsWAP and TMR625C hPMEL variants localize similarly in melanocytes. Endogenous mouse PMEL does not significantly co- localize with late endocytic markers (not shown) and thus the partial co-localization of ectopic hPMEL with LAMP2 might reflect less efficient delivery of this isoform to early stage melanosomes, likely due to the low expression levels attained by infection (see below) and the inability of hPMEL to interact with the mouse isoform as detected by coimmunoprecipitation (data not shown). We next tested whether expression of the TMinsWAP and TMR625C hPMEL variants affect the morphology or degree of pigmentation of individual melanosomes. In mouse melan-Ink4a melanocyte stable transfectants that expressed very low levels of the dominant TMinsWAP and TMR625C hPMEL variants (,10– 20% of endogenous mouse PMEL as assessed by immunoblotting), no changes in overall pigmentation were observed relative to cells expressing wild type hPMEL (bright field images in Figure S3, with melanosomes pseudocolored blue in the insets), but this is likely due to the low expression levels (see below). Nevertheless, by IEM of ultrathin cryosections from each of the stable cell lines, thin and disorganized immature protofibrils (arrowheads) that were densely immunogold labeled with anti-hPMEL antibodies were observed within Stage I melanosomes (Figure 6a–c). However, upon maturation of the protofibrils into elongated fibrillar sheets, wild-type and mutant hPMEL showed strikingly different characteristics (compare Figure 6d to 6e–f). In cells expressing wild-type hPMEL, only very sparse immunogold labeling was observed on the parallel sheets of organized fibrils, and most of the organelles with thickened fibrillar sheets, corresponding to Stage III or IV melanosomes, were densely pigmented (Figure 6d) as has been previously reported [24]. This is consistent with the notion that although a fraction of hPMEL is delivered to the late endocytic pathway in mouse melanocytes, sufficient protein is properly trafficked to melanosomes to form fibrils. The lack of labeling on the pigmented fibrils likely reflects epitope sequestration as pigmentation proceeds [13,14,42]. In contrast to cells expressing wild-type hPMEL, compartments with tightly packed fibrils that were densely immunogold labeled for PLoS Genetics | www.plosgenetics.org 6 September 2011 | Volume 7 | Issue 9 | e1002286