RPE Cells, but Not Choroidal Melanocytes, Are Severely Depigmented in the Absence of Functional Rab38
In contrast with skin melanocytes (Wasmeier et al., 2006
), the RPE of 4m cht/cht
mice exhibit a much reduced number of melanosomes, compared with wild-type controls (). The few melanosomes that are present in the RPE are mature, and they show a normal distribution, clearly having access to the apical processes. The morphology of melanosome in homozygous cht
RPE is near normal, although they are slightly less spindle-shaped than heterozygous controls (width/length ratio = 0.50 ± 0.14 for homozygous cht
versus 0.46±0.17 for heterozygous control). The heterozygous cht
RPE shows a greatly increased number of melanosomes compared with homozygous cht
RPE, although there is a small reduction in melanosome number compared with wild type (). Conversely, homozygous cht
choroidal melanocytes exhibit a much more subtle reduction in melanosome number versus wild-type controls ().
Figure 1. Pigmentation in the eye in wild-type and cht mice. Conventional EM of wild-type (+/+), heterozygous cht (−/+), and homozygous cht (−/−) RPE and choroid at 4 mo and 2 y, showing severe depigmentation in the cht RPE. Bar, 1.0 μm. (more ...)
Analysis of the RPE of 2-y-old (2y) mice shows that there are still very few melanosomes in the homozygous, compared with the heterozygous cht mice (). In fact, the number of melanosomes in the RPE in 2y mice seems similar to the number at 4 mo, such that the differences between wild type, heterozygous and homozygous cht are retained. In contrast, the number of melanosomes/melanocyte in the choroidal melanocytes clearly increases in all mice, and by 2 y there is no clear difference in pigmentation between wild type, heterozygous and homozygous cht.
Rab38 and Rab32 Are Expressed in RPE Cells
Rab32 has been shown to partially compensate for Rab38 function in melanocytes of the skin (Wasmeier et al., 2006
). To determine whether Rab32 is expressed in pigmented cells of the eye, we subjected protein extracts derived from primary cultures of RPE cells isolated from 3-wk-old wild-type mice, whole mouse eyes, eye cup (containing RPE and choroid), and retina (devoid of RPE and choroid) to immunoblot analysis using anti-Rab antibodies. Rab38 and Rab32 were present in primary RPE cells, in the eye, and eye cup, but they were undetectable in the neural retina ().
Figure 2. Ocular expression of Rab38 and Rab32. Proteins (25 μg/lane) from cultured cell lines (melanocytes or AtT20; left) or from different tissue preparations obtained from 3-wk-old wild-type mouse eyes (right) were separated by SDS-PAGE. Samples were (more ...)
The Majority of Melanosomes Are Synthesized before Birth in the RPE
It was not possible to determine the nature of the defect in melanosome biogenesis from the phenotype of the adult cht/cht RPE. Therefore, we examined the effect of loss of functional Rab38 on melanosome biogenesis at time points when melanin synthesis normally occurs in the RPE. This entailed examining mice of embryonic and early postnatal age. Therefore, heterozygous and homozygous cht mice were crossed so that heterozygous and homozygous cht littermates (readily distinguishable on the basis of coat color) could be compared. Although there seems to be a small reduction in melanosome number in heterozygous cht mice, there is still a clear difference between heterozygous and homozygous mice.
Melanosome biogenesis was first characterized in control (heterozygous) RPE. The majority of melanin synthesis occurs between E10 and E15 in mouse RPE (Bodenstein and Sidman, 1987
; Beermann et al., 1992
). Consistent with this timing, conventional EM showed both immature and mature melanosomes in the RPE of E14 control mice, but most melanosomes had apparently reached maturity (stage IV) by P1 (). Although very few immature melanosomes could be observed at this age by conventional EM, cryoimmuno-EM showed that ~50% of the melanosomes at E14, P1, and P8 stained positive with anti-pmel17 antibody (HMB45) ( and A for quantitation). This antibody has previously been found to stain predominantly immature melanosomes in melanocytes (Raposo et al., 2001
). In the RPE, some apparently mature melanosomes stain positive with HMB45, but in those melanosomes the pmel17 staining is predominantly around the perimeter. The number of pmel17 gold particles/melanosome gradually declines from E14 to P8, suggesting either a gradual antigen masking or gradual removal of the protein (B). In the adult (4m) RPE, pmel17 staining was virtually absent.
Melanosome biogenesis in RPE. Conventional EM (CEM) and cryoimmuno-EM showing pmel17 and tyrosinase staining of heterozygous cht RPE at E14, P1, P8, and 4 mo. Bars for conventional EM, 0.5 μm and for cryoimmuno-EM, 0.2 μm.
Figure 4. Melanosome biogenesis in choroidal melanocytes. The percentage of melanosomes (both immature and mature) in heterozygous cht RPE and choroidal melanocytes that were pmel17 positive and tyrosinase positive (at least 5 gold particles/melanosome) was quantitated (more ...)
We also studied tyrosinase expression and localization in control mice at the same time points. At E14 and P1, there was strong staining of tyrosinase in the RPE, but this was considerably reduced by 8 d after birth and it was virtually undetectable in the adult ( and A for quantitation). Interestingly, at E14 all melanin-containing melanosomes stained strongly for tyrosinase, whereas in 1- and 8-d-old mice, some melanosomes had largely lost tyrosinase staining. Quantitation confirmed that there was a decline in the percentage of tyrosinase positive melanosomes from E14 to P8 (A), resulting in a decline in the mean number of tyrosinase gold particles/melanosome (B). Overall, these results indicate that melanosome biogenesis occurs predominantly before birth in the RPE.
The Majority of Melanosomes Are Synthesized after Birth in Choroidal Melanocytes
Melanosome formation was also followed in choroidal melanocytes. Choroidal melanocytes could not be identified at E14, but both immature and mature melanosomes were present in choroidal melanocytes at P1 and P8 (C). The number of pigmented melanocytes in the choroid at P1 was too low to allow quantitative cryoimmuno-EM, but at P8 the majority (>80%) of melanosomes stained positive for tyrosinase, and ~20% were positive for pmel17 (A). Most mature melanosomes in choroidal melanocytes stained poorly for pmel17 (C). This is in contrast to the RPE where some mature melanosomes stained for pmel17, but it is similar to skin melanocytes where pmel17 staining is largely confined to immature melanosomes (Raposo et al., 2001
). In the adult, the majority of melanosomes in choroidal melanocytes were mature, but occasional pmel17-positive vacuoles could be observed by cryoimmuno-EM (, A and D). In many melanocytes of the adult choroid, tyrosinase staining was virtually undetectable, but in others it was present, although they had fewer tyrosinase gold particles than on melanosomes at early time points in either the RPE or choroidal melanocytes (, B and D).
These results indicate that the majority of melanosomes are synthesized after birth in choroidal melanocytes. High levels of melanosome production continue for at least 8 d after birth, indicating a longer time window for the peak of melanosome synthesis than in the RPE. Our results are also consistent with the idea that a low level of melanosome synthesis is maintained in the adult choroid.
Premelanosomes Are Formed in the cht/cht RPE but Fail to Deposit Melanin
Conventional EM of the RPE of E14, 1- and 8-d-old cht/cht mice showed that the cytoplasm contained many striated vacuoles with the morphological appearance of immature (stage II) melanosomes with no melanin (). Cryoimmuno-EM showed that these vacuoles stained strongly for pmel17, confirming their identity as immature melanosomes (). In contrast, RPE from adult cht/cht mice contained very few morphologically distinguishable immature melanosomes, and there was no detectable pmel17 staining (). Quantitation of the numbers of melanosomes of different stages by conventional EM showed that in the cht/cht RPE at P1, the majority (60%) of melanosomes were stage II and only 13% were stage IV (A). In contrast, 71% of melanosomes in RPE from the control mice were mature (stage IV) at 1 d old (A). There was a small reduction in total number of melanosomes at P1 in mutant compared with control RPE, suggesting that any deficiency in the biogenesis of stage II melanosomes is small. Strikingly, there were very few immature melanosomes in adult homozygous or heterozygous mice (A), despite the reduction in the total number of melanosomes in the cht RPE ( and A). It is not possible to determine the precise number of melanosomes that are formed or lost between P1 and adult by counting melanosome number/area of RPE, because the size of the RPE increases during this time. Nevertheless the size of the RPE was approximately the same in heterozygous and homozygous mice and so the apparent loss of melanosomes between P1 and 4 mo in the homozygous, compared with the apparently constant numbers of melanosomes in the heterozygous mice, indicates that immature melanosomes are lost in the mutant cht RPE. Quantitation of pmel17 staining in the homozygous RPE showed that there was a higher percentage of pmel17-positive melanosomes than in control RPE at E14, P1, and P8 (B), together with more gold particles/melanosome (C). The enhanced pmel17 gold labeling in the homozygous cht RPE is likely to reflect reduced melanin deposition and hence reduced antigen masking. Therefore, it is clear that the loss of functional Rab38 causes a major reduction in the ability of immature melanosomes to deposit melanin in the RPE, and those that fail to deposit melanin seem to be degraded in the adult mice.
Figure 5. Premelanosome formation in cht RPE. CEM and cryoimmuno-EM showing pmel17 staining of homozygous cht RPE at E14, P1, P8, and 4 mo. Examples of stage II melanosomes are indicated (II). Bars for conventional EM, 0.5 μm and for cryoimmuno-EM, 0.2 (more ...)
Figure 6. Premelanosome formation in cht choroidal melanocytes. The number of stage II, III, and IV melanosomes per area of RPE (A) in 1-d- and 4-mo-old homozygous and heterozygous cht mice was determined from conventional EM images. The percentage of melanosomes (more ...)
A Low Level of Melanosome Synthesis in Adult cht Choroidal Melanocytes Allows Melanosome Numbers to Recover
Melanocytes of the choroid at P1 contained many immature (stage II) melanosomes in cht/cht mice, but by P8 and 4 mo the majority of melanosomes in the choroid were mature (D). However, pmel17 staining identified a similar number of immature melanosomes at P8 to that found in control mice (, B and E) with similar levels of pmel17 labeling (, C and E). Thus, the defect in melanin deposition (and hence antigen masking of pmel17) is less in choroidal melanocytes than in the RPE. Quantitative analysis showed small numbers of pmel17-positive melanosomes (2%) in the adult homozygous choroid, which was less than that observed in heterozygous mice. This could indicate that some immature melanosomes in the cht/cht adult choroid fail to deposit melanin and that they are lost even though the continued low levels of melanosome synthesis and tyrosinase delivery allow melanosome numbers to eventually recover in the adult.
Delivery of Tyrosinase to Premelanosomes Is Impaired in the Absence of Rab38
So, what could be the mechanism of action of Rab38 in RPE cells? As a first step toward this goal, we localized endogenous Rab38 and Rab32. Rab32 staining gave a better signal at the cryoimmuno-EM level, and it was detected mainly on the perimeter membrane of mature melanosomes in control E14 RPE (A). Low levels of staining could also be observed in the cytoplasm. Rab32 labeling was strongest at E14 and P1, but it declined in the adult (unpublished observations). The signal obtained with anti-Rab38 antibody was lower than with anti-Rab32, but it showed a similar localization (primarily on the perimeter membrane of melanosomes) and a similar expression pattern over time (highest at E14 and low in the adult) (B).
Figure 7. Distribution of Rab32, Rab38, and tyrosinase in the RPE. (A and B) Cryoimmuno-EM showing Rab32 (A) and Rab38 (B) staining of heterozygous cht RPE at E14. Bars, 0.2 μm. (C) Cryoimmuno-EM of tyrosinase (10-nm gold; arrows) and pmel17 (15-nm gold) (more ...)
The localization of Rab32/38 to mature melanosomes has also been observed in skin melanocytes (Wasmeier et al., 2006
), but it is unlikely to represent their only site of action, because they are required for the deposition of melanin in immature melanosomes. A reduction in melanin deposition in melanosomes could reflect an inhibition of delivery of melanin-synthesizing enzymes to the immature melanosome or the environment within the immature melanosome could be unsuitable for their maximum activity. Therefore, the effect of loss of Rab38 on tyrosinase localization was analyzed. RPE cells from P1 mice were cryoimmunolabeled for tyrosinase and pmel17 (to clearly identify immature melanosomes). In cht/cht
mice, the small number of melanosomes that contained melanin stained strongly for tyrosinase (C), as expected, because tyrosinase is required for melanin synthesis. The large numbers of pmel17-positive immature melanosomes contained very little or no tyrosinase staining, suggesting a defect in delivery of tyrosinase to the immature melanosome (C). Quantitative analysis of tyrosinase staining (i.e., the mean number of tyrosinase gold particles on both immature and mature melanosomes) showed that the number of tyrosinase gold particles is reduced in both the RPE and the melanocytes of the choroid of the cht/cht
mice at all time points, compared with heterozygous mice, except in the adult choroid where very few positive melanosomes were available for counting (D). The reduction in tyrosinase labeling at P1 in cht
RPE is greatest on the immature melanosomes, consistent with C. However, all time points from E14 to P8 were likewise affected. We noted also some tyrosinase label on tubular and vesicular structures in the cytoplasm, a finding supported by quantitative comparison of relative tyrosinase labeling on melanosomal versus tubulovesicular membranes in cht
and control RPE at P1. This showed an increase in the relative amount of tyrosinase on tubulovesicular structures in cht
, compared with control RPE (E). Together, these results indicate a defect in the delivery of tyrosinase to immature melanosomes in cht
Immature Melanosomes Formed in cht/cht RPE Contain Large Amounts of Cathepsin D
The observation that immature melanosomes were being lost from the RPE to a greater extent in the homozygous mice suggested that immature melanosomes could be degraded in the cht/cht mouse. To begin to assess the degradative potential of the maturing melanosomes, homozygous cht RPE at P8 was double-stained for cathepsin D and pmel17 to identify immature melanosomes. A small number of cathepsin D-positive vacuoles that lacked pmel17 were presumably lysosomes, although they did not have the electron-dense appearance and multiple lumenal membranes present in lysosomes in many cell types. Another population of vacuoles that contained an approximately equal density of cathepsin D staining colabeled for pmel17, and they were thus identified as immature melanosomes (, A and B). Clearly, immature melanosomes that transiently accumulate in the cht/cht RPE contain a significant proportion of the cellular content of cathepsin D, indicating that they may have degradative potential. To determine whether the presence of cathepsin D in immature melanosomes was due to the loss of functional Rab38, double labeling of cathepsin D and pmel17 was performed in control RPE. Because there are very few immature melanosomes at P8, we analyzed E14 RPE and we observed that the immature pmel17-positive melanosomes also contained significant quantities of cathepsin D (, A and B).
Cathepsin D localization in immature melanosomes in the RPE. Cryoimmuno-EM showing cathepsin D (10-nm gold; arrows) and pmel17 (15-nm gold) staining of homozygous cht P8 RPE (A) and heterozygous cht E14 RPE (B). Bars, 0.2 μm.
Premelanosomes Formed in the RPE of albino Mice Lacking Tyrosinase Are Also Unstable
Instability of immature melanosomes in the RPE of cht/cht mice could be due to immature melanosomes that fail to deposit melanin being inherently unstable or to a specific loss of function of Rab38. We therefore examined albino mice, which fail to deposit melanin in immature melanosomes due to the lack of expression of functional tyrosinase. Although at P1 many immature melanosomes were present in the cytoplasm, as identified by lumenal striations and pmel17 staining, these structures were absent from the adult albino mice (). Strikingly, there was no detectable pmel17 staining (), confirming that, as observed in cht mice, the immature melanosomes that fail to deposit melanin seem to be unstable.
Figure 9. Premelanosome formation in albino RPE and choroidal melanocytes. Conventional EM and cryoimmuno-EM showing pmel17 staining of albino RPE and choroidal melanocytes (chor) at P1 and 4 mo. Bars for conventional EM, 0.5 μm and for cryoimmuno-EM, 0.2 (more ...)
As described above, melanosome production in the melanocytes of the choroid begins later than in the RPE and extends after birth, being present at a low rate during adulthood. Choroidal melanocytes of the homozygous cht mouse have immature melanosomes, which could be readily identified by pmel17 staining despite the prevalence of mature melanosomes (). In adult albino mice, we could observe many immature melanosomes in choroidal melanocytes, a finding that was very clear given the absence of mature melanosomes ().