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Characterization of Melanosomes in Murine Hermansky–Pudlak Syndrome: Mechanisms of Hypopigmentation

Thuyen Nguyen and Maria L. Wei Department of Dermatology, Veterans Affairs Medical Center, University of California, San Francisco, California, USA

The Hermansky–Pudlak syndrome is a genetically heterogeneous autosomal recessive disorder affecting mice and humans, which causes oculocutaneous albinism, prolonged bleeding, and in some cases, pulmonary fibrosis or granulomatous colitis. We previously demonstrated that the gene defects causing murine Hermansky–Pudlak syndrome cause blocks in melanosome biogenesis and/or trafficking in 10 Hermansky–Pudlak syndrome strains. Here, we report an in vivo quantitative analysis on five additional murine models of the Hermansky–Pudlak syndrome. We demonstrate that all strains examined here except for ashen have defects in morphogenesis, the most severely affected is sandy, muted, and buff followed by subtle gray. The ashen strain only has a defect in secretion, as indicated by retention of melanosomes in melanocytes. We document three cellular mechanisms contributing to the hypopigmentation seen in the Hermansky–Pudlak syndrome: (1) exocytosis of immature hypopigmented melanosomes from melanocytes with subsequent keratinocyte uptake; (2) decreased intramela- nocyte steady-state numbers of melanosomes available for transfer to keratinocytes; and (3) accumulation of melanosomes within melanocytes due to defective exocytosis, as seen in ashen. We also report that melanosomes in the DBA/2J strain, the parental strain of the Hermansky–Pudlak syndrome strain sandy, are abnormal, indicating that aberrant biogenesis of melanosomes may play a part in the pathogenesis of pigmentary glaucoma observed in these mice. Key words: melanocytes/organelle biogenesis/pigmentary glaucoma/pigmentation. J Invest Dermatol 122:452 –460, 2004

The cellular regulation of organelle biogenesis is a tightly Melanosomes, organelles in which the pigment melanin controlled process and dysregulation can cause significant is packaged and secreted by skin melanocytes, provide an disease. One disease of organelle biogenesis is the ideal model system for studying the defects in organelle Hermansky–Pudlak syndrome (HPS), described in humans biogenesis seen in HPS (Marks and Seabra, 2001). The and mice (Swank et al, 1998; Huizing et al, 2000). In normal morphology of melanosomes has been very well humans, six HPS genes have been identified, HPS1, studied and described on an ultrastructural level. The AP3B1, HPS3, HPS4, HPS5, and HPS6 (Oh et al, 1996; maturation process of the melanosome proceeds through Dell’Angelica et al, 1999; Anikster et al, 2001; Suzuki et al, four morphologically distinct phases (Seiji et al, 1963): type I 2002; Zhang et al, 2003). In mice, at least 16 HPS genes melanosomes share characteristics with late endosomes in have been noted, including six that are orthologous to the having intralumenal vesicles; type II forms are elongated human genes (Gardner et al, 1997; Feng et al, 1999; Suzuki with intralumenal striations; type III forms have pigment et al, 2001, 2002). The genes in HPS for which putative deposited upon the striations; and type IV are completely functions have been ascribed appear to be defective in filled with pigment. A number of melanocyte-specific vesicle and organelle production and transport (Huizing proteins (tyrosinase, TYRP1, DCT/TRP2, gp100/Pmel, et al, 2001; Marks and Seabra, 2001). The major pathologic MART-1/melan-a, P protein) are targeted to the melano- conditions associated with HPS (cellular accumulation of some and their normal trafficking patterns have been ceroid-like pigment, oculocutaneous albinism, prolonged studied to varying extents. Recent studies have shed light bleeding, and pulmonary fibrosis), implicate HPS genes in on early melanosome maturation steps, demonstrating that the biogenesis of lysosomes and lysosome-related cell- the gp100/Pmel protein resides in the limiting membrane of specific organelles such as melanosomes (found in pigment type I melanosomes, becomes internalized via incorpora- cells in skin, eye, ear, and meninges), platelet dense tion into intralumenal vesicles and subsequently plays a granules, and lung lamellar bodies (Marks and Seabra, role in the formation of the internal striations (Berson et al, 2001; Lyerla et al, 2003). 2001, 2003; Raposo and Marks, 2002). Subsequent to melanosome biogenesis and transport to the melanocyte periphery, the organelle or its contents are transferred to neighboring keratinocytes. Abbreviations: DCT, dopachrome tautomerase; HPS, Hermansky– Pudlak Syndrome; TEM, transmission electron microscopy; TYRP- Previously, we demonstrated that melanosomes in 10 1, tyrosinase-related protein 1; TRP-2, tyrosinase-related protein 2. HPS murine strains exhibit a range of biogenesis defects,

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 452 122 : 2 FEBRUARY 2004 MECHANISMS OF HYPOPIGMENTATION 453 resulting in morphologically abnormal organelles (Nguyen limiting membrane and is an enzyme functioning in pigment et al, 2002). We showed that each defective HPS gene synthesis (Sarangarajan and Boissy, 2001). It can be seen introduced a block or rate-limiting step that resulted in the that in the sandy strain, however, the additional HPS gene accumulation of immature melanosomal forms and were mutation is a cause of further marked pigment loss. The able to propose a map of where each gene affected the muted strain exhibits a small but significant measurable biogenesis pathway. Here we examine five additional HPS pigmentation difference from its heterozygote littermate strains, and show that all but one strain have defects in control. melanosome biogenesis. We characterize the defects and propose that multiple mechanisms contribute to the Melanosome morphology To determine if the HPS gene hypopigmentation seen in HPS. mutations in the strains studied here caused defective morphogenesis, melanosome morphology in skin tissue was examined by transmission electron microscopy. Three Results of the control strains (C57BL/6, C3H/HeSnJ, mu/ þ ) had numerous, completely pigmented, ellipsoidal type IV mel- Varying degrees of hypopigmentation in HPS strains All anosomes (Fig 2). The fourth control strain, DBA/2J, had of the HPS strains studied here were initially identified on abnormal melanosomal morphology. Whereas there were the basis of abnormal coat color and thus are by definition many fully pigmented melanosomes in DBA/2J, these pigment diluted; however, only one strain, the buff strain, is were slightly irregularly shaped, surrounded by ‘‘wavy’’ or on the black C57BL/6J genetic background. So whereas a ‘‘ruffled’’ limiting membranes that also contained peripheral direct comparison of the pigment in all of the mutant strains granular material. No normally striated melanosomal type II studied here was not feasible, the pigment dilution of each or III forms were observed in DBA2/J. Rather, intermediate mutant strain relative to its parental strain could be forms that had intralumenal short, truncated curvilinear compared (Fig 1). The buff strain had a significant degree segments, suggesting incomplete or abnormal striations, of pigment dilution, when compared with the control were seen. Melanosomes in the initial stages of pigmenta- C57BL/6J strain. The ashen and subtle gray strains exhibit tion had pigment accumulated like ‘‘beads on a string’’ slight changes in pigmentation compared with the control along these abnormal linear segments. Several melano- C3H/HeSnJ strain. The sandy strain arose from DBA/2J. somes in this strain appeared to be two mature melano- These two strains are interesting in that the control DBA/2J somes that had undergone fusion in that they shared a strain is homozygous for two mutations known to affect common limiting membrane but had two separate elliptical melanosome function: the MYO5a/dilute (Jenkins et al, fully pigmented cores (Fig 2c). 1981; Copeland et al, 1983) and Tyrp1 (Moyer, 1963; Two of the mutant strains, ashen and subtle gray, also Rittenhouse, 1968; Zdarsky et al, 1990; Jackson et al, had numerous mature melanosomal forms, similar to those 1990; Corrigan, 2002) genes, each of which cause pigment seen in the control C57BL/6 and C3H/HeSnJ strains, and dilution compared with the black C57BL/6 strain, which is these two strains seemed to have fairly normal melanoso- wild type at these loci. The MYO5a/dilute gene product is mal morphology overall (Fig 2b). the myosin Va motor protein (Mercer et al, 1991) and In contrast, the buff, sandy, and muted strains had a mutations in the dilute gene cause retention of melano- marked absence of fully pigmented elongated forms. The somes in the cell body (Rittenhouse, 1968; Provance et al, sandy strain had the most morphologically abnormal 1996; Wu et al, 1998). The TYRP1 protein is restricted in appearing melanosomes (Fig 2c). Few abnormally striated expression to pigment cells, is targeted to the melanosome forms were observed, and very few fully pigmented structures or recognizable type I structures were observed. Instead, there were numerous abnormal vesicular forms with internal amorphous grainy material, with shapes that were unevenly spherical or very irregular; some of these abnormal vesicular forms contained foci of pigment. The muted strain was similar to sandy in having an accumulation of abnormal vesicular forms, but also had many striated melanosomal forms (Fig 2c). Both the buff and muted strains had fully pigmented round forms but buff melano- somes appeared smaller than those observed in its control strain (Fig 2a).

Melanosome size is abnormal in the buff strain Because of the observation that buff appeared to have lost the regulation of melanosome size, the average size of mature melanosomes in the cell body of melanocytes was Figure 1 measured to quantify any differences with control strains. Pigmentation in HPS mutant strains. The dorsal back coat color was The buff melanosomes had a significantly diminished size measured using a Mexameter. Percent pigmentation for each mutant strain was calculated compared with its respective control strain, as (Fig 3). The other mutant strains did not show a significant detailed in the Materials and Methods. difference in fully pigmented melanosome size when 454 NGUYEN AND WEI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2 Abnormalities in HPS melanosome morphology. Dorsal back skin tissue was examined by transmission electron microscopy. a, C57, C57BL/6 control strain. G, Golgi. bf, buff. b, C3H, C3H/HeSnJ control strain. ash, ashen. sut, subtle gray. c, DBA/2J, control strain. Note the short curvilinear intraluminal segments in the DBA/2J melanosome indicated by the white arrowhead. sdy, sandy. mu/ þ , control strain. mu, muted. Black arrows, type I melanosomes. White arrowheads, type II melanosomes. Black arrowheads, type III melanosomes. White arrows, abnormal vesicular forms. Stars, mitochondria. Double arrows, two foci of pigment with common limiting membrane. Scale bars ¼ 0.2 mm. 122 : 2 FEBRUARY 2004 MECHANISMS OF HYPOPIGMENTATION 455

Figure 3 Defective size regulation in buff melanosomes. The major axis of fully pigmented elliptical forms was measured at a magnification of 50,000. Error bars indicate SEM.

Figure 5 HPS melanosome immaturity. Each melanosome was classified by morphology, and the percentage of each form was plotted for each mutant strain. Brackets above the graph indicate a control strain (leftmost strain) with its respective mutant strain(s).

IV, as occurs in the C57BL/6 strain (Nguyen et al, 2002). Size progression is also evident in the C3H/HeSnJ control strain, but less so in the DBA/2J control strain (Fig 4), in which abnormal melanosomal morphology was noted above. In order to assess the extent of melanosomal maturation in each of the mutant strains, the sizes of the different melanosomal forms were measured and compared. The size progression is preserved in the buff, ashen, and subtle gray strains, but is absent in the sandy and muted strains, which suggests an impaired maturation process in these two strains. Examination of the intramelanocyte steady-state distribu- tion of the different melanosomal types can detect if a block in morphogenesis has occurred or if a rate-limiting step is introduced into the biogenetic pathway, because immature precursors will accumulate and mature forms will either be Figure 4 absent or relatively decreased in number. So, to assess at Maturation of sandy and muted melanosomes is blocked. The what step melanosomal biogenesis was impaired in each of major axis of each melanosomal form was measured at 50,000 these strains, the percentage of types I, II/III, and IV forms magnification. Brackets above the graph indicate a control strain were determined for each strain (Fig 5). Three strains (buff, (leftmost strain) with its respective mutant strain(s). Asterisk indicates a mutant strain with no significant change in size from striated to fully sandy, and muted) had evidence of significant blocks in pigmented forms. For the DBA/2J strain, the abnormally striated forms melanosome biogenesis. These three strains showed a were classified as striated melanosomes; no multivesicular forms were decrease in the percentage of mature melanosomes observed. For sandy, abnormally striated forms were classified as striated melanosomes. Error bars indicate SEM. present and a concomitant increase in the proportion of abnormal vesicular forms that could not be identified as a compared with their respective control strains (data not normal melanosomal stage. These abnormal vesicular shown). forms were also present as a minor proportion of organelles in the C57BL/6, C3H/HeSnJ and muted heterozygote control strains, and to a greater extent in the DBA/2J HPS strains except ashen show defects in melanosome control strain. The abnormal vesicular forms are detailed in maturation One marker of melanosomal maturation is a Table II and Figure 6. Another strain, subtle gray, is aptly size increase as melanosomes progress from type I to type named in that the effect on melanosome biogenesis is mild, 456 NGUYEN AND WEI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and a relatively small accumulation of immature type I and 1998). To confirm that this is true in vivo as well, the number II/III forms is seen in comparison with the control CH3/ of melanosomes per unit area of melanocyte cytoplasm was HeSnJ strain with a concomitant small decrease in the determined (Fig 7). The number of ashen melanosomes per proportion of fully pigmented forms. Melanosomes in the unit area was increased by 72% compared with in the ashen strain are predominantly mature, with an increase in control strain, consistent with melanosomes being retained the proportion of mature forms compared with in the control within the cell body. In contrast, in the subtle gray and strain, indicating the absence of any impairment of biogenesis in this strain.

Intracellular numbers of melanosomes in melanocytes of selected HPS strains differ from those in control melanocytes Previous studies of cultured ashen melano- cytes showed that as a result of the mutation in the Rab27a gene, melanosomes are not transported to the dendrites but remain in a perinuclear distribution, and are not efficiently exocytosed (Provance et al, 1996; Wu et al,

Table I. Control and HPS mutant strains in this study

Control strain HPS strain Gene Function C57BL/6 buff VPS33A Vacuole biogenesis in yeast

C3H/HeSnJ ashen Rab27a GTPase, melanosome Figure 7 transport Abnormal steady-state melanosome numbers in ashen, subtle gray, and muted strains. For each strain, the number of melanosomes subtle gray ? ? per unit area was determined. Then the value of each control strain was DBA/2J sandy ? ? normalized to 1, and the values for the respective mutants were normalized accordingly. Brackets above the graph indicate a control mu/ þ muted mu ? strain (leftmost strain) with its respective mutant strain(s). Asterisks indicates a mutant strain that differs significantly from parental strain.

Table II. Features of abnormal vesicular structures accumulated in melanocytes

DBA/2J Sandy Muted Buff dark gray grainy lumen(a) dark gray grainy lumen dark gray grainy lumen dark gray grainy lumen multiple pigmented cores(b) multiple pigmented cores dark gray lumen with black core multiple pigmented cores dark gray lumen, ILVÃ dark gray lumen, ILV dark gray lumen, ILV(c) partial pigment, no ILV or striations(d) dark gray lumen with black core(e) dark gray lumen with multiple gray cores (f) ÃILV: intralumental vesicles a–fSee Figure 6.

Figure 6 122 : 2 FEBRUARY 2004 MECHANISMS OF HYPOPIGMENTATION 457

Figure 8 Immature forms are secreted and taken up by keratinocytes. (a) A type III mela- nosome in the process of being exocy- tosed from the periphery of a melanocyte in the muted strain. Star, site of type III melanosome exocytosis. Arrows, plasma membrane (PM). Nuc, nucleus. Mel, mel- anocyte. Ker, keratinocyte. (b) Melano- somes in keratinocytes. sut, subtle gray. mu, muted. bf, buff. sdy, sandy. ash, ashen. pa, pallid. cno, cappuccino. rp, reduced pigmentation. White arrowhead, type II melanosome. Black arrowhead, type III melanosome. Star, mitochondrion. Black arrow, keratin filament. White arrow, aberrant vesicular form. Scale bars ¼ 0.2 mm.

muted strains, the number of melanosomes per unit area cytoplasm of keratinocytes. In the muted and buff strains, was decreased compared with in control strains, suggest- type II and III melanosomes were observed in keratinocytes ing that either the rate of biogenesis was slowed or that surrounding melanocytes (Fig 8b) and/or in the hair shaft there was an ongoing destruction of abnormal organelles. (data not shown). Similar findings were observed in the Another alternative, that an increased rate of exocytosis pallid, cappuccino, and reduced pigmentation HPS strains. of the melanosomes was occurring, was unlikely, as an In the control C57BL/6 and C3H/HeSnJ control strains, only increase in the number of melanosomes in the neighboring type IV forms comprised the many melanosomes observed keratinocytes was not observed (data not shown). in keratinocytes surrounding melanocytes (data not shown). Similarly, in the mu/ þ control keratinocytes, the majority of Immature melanosome forms are exocytosed for uptake melanosomes were type IV, with very few type III forms by keratinocytes We previously observed that pigmenta- observed. Notably, in the subtle gray and ashen mutants, tion in HPS strains varied inversely with the percentage of only fully pigmented melanosomes were observed in hypopigmented melanosomal forms present in melanocytes keratinocytes. Interestingly, melanosomes in ashen kerati- and suggested that hypopigmentation may result from the nocytes were present singly, whereas in the other mutant exocytosis of an increased proportion of unpigmented or strains, melanosomes in keratinocytes were grouped in hypopigmented melanosomal forms (Nguyen et al, 2002). In membrane limited structures, likely phagolysosomes. this study, we observed evidence of immature melanosomal Very few melanosomes were seen in keratinocytes sur- forms in the process of exocytosis from melanocytes (Fig rounding DBA/2J melanocytes, due to the mutant MYO5a/ 8a). The ability of these exocytosed immature melanosomal dilute gene, which causes retention of melanosomes in forms to be internalized by neighboring keratinocytes was melanocytes (Rittenhouse, 1968; Provance et al, 1996; Wu indicated by the appearance of these forms in the et al, 1998). In sandy, the mutant strain derived from DBA/ 458 NGUYEN AND WEI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

2J, melanosomes of the same abnormal morphology as small spherical melanosomal structures that are similar to seen in melanocytes (not identifiable as types I–IV) were the small vesicular structures that accumulate in yeast cells seen in keratinocytes indicating that the ability to be that have mutations in vps class B and C genes, which secreted was not completely abolished despite the abnor- function in lysosomal biogenesis (Banta et al, 1988; mal morphology. Raymond et al, 1992; Wada et al, 1992); recently the defective gene in buff was identified as the VPS33A gene, Discussion and smaller melanosomes were also noted in the choroid of buff animals (Suzuki et al, 2003). This in vivo quantitative study demonstrates that the murine One strain, subtle gray, demonstrated a lesser effect on HPS strains, muted, sandy, buff, and subtle gray all have melanosome biogenesis: a sizeable proportion of normal defects in melanosomal maturation, whereas the ashen type IV melanosomes was observed but an increase in the strain has no evidence of defective biogenesis, but is proportion of striated forms suggested the introduction of a characterized by retention of melanosomes within the rate-limiting step in the pathway of melanosome develop- melanocyte cell body. This study also documents the ment. The ashen strain was unique in having no significant exocytosis of immature melanosome forms and uptake of detectable defect in melanosome biogenesis, and was those forms by keratinocytes. notable for having an accumulation of melanosomes A striking feature of HPS gene products is that many of intracellularly in melanocytes, consistent with findings in them form protein complexes with one another. These cultured cells (Provance et al, 1996). This suggests that the complexes have been termed BLOC-1 (pallid, cappuccino, Rab27a mutation in ashen (Wilson et al, 2000) only had an muted proteins, and possibly reduced pigmentation pro- effect on melanosome transport, unlike the gunmetal strain, tein), BLOC-2 (ruby eye and ruby eye-2 proteins), and in which the defective gene is a rab geranylgeranyl BLOC-3 and BLOC-4 (HPS1/pale ear and HPS4/light ear transferase (Detter et al, 2000) and in which we previously proteins) (Falcon-Perez et al, 2002; Moriyama and Bonifa- observed that melanosomes were smaller in size compared cino, 2002; Chiang et al, 2003; Ciciotte et al, 2003; Martina with in the control strain, as well as an intramelanocytic et al, 2003; Zhang et al, 2003). Our results here together accumulation of melanosomes (Nguyen et al, 2002). The with our previously reported results (Nguyen et al, 2002) gunmetal mutation is likely to affect a number of rab reveal that: (1) the melanosome morphology of the BLOC-3/ molecules, one or more of which may act on melanosome 4 mutants pale ear and light ear are indistinguishable biogenesis, explaining the pleiotropic effect of the gunmetal qualitatively and quantitatively; (2) the BLOC-1 mutants mutation, whereas the ashen mutation is specific for pallid and cappuccino also have very similar morphologies, Rab27a, which only appears to affect melanosome trans- both qualitatively and quantitatively; (3) the morphology of port. The block in transport and transfer to keratinocytes in the BLOC-2 mutants ruby eye and ruby eye-2 are similar ashen and gunmetal strains is not complete, as both strains except that the melanosome size is larger on average in have melanosomes in the hair shaft (M.L.W. and T.N., ruby eye. The BLOC-1 mutant muted can be distinguished unpublished observations) and both have a pigmented from the BLOC-1 pallid and cappuccino mutants by the (albeit diluted) phenotype. presence of an increased proportion of pigmented melano- The findings in this study suggest that the cause of the somal forms, but the phenotype in muted is very similar to hypopigmentation seen in HPS is likely due to a number of that of the reduced pigmentation mutant, predicted to be a contributing factors. The steady-state number of melano- possible component of BLOC-1 (Falcon-Perez et al, 2002; somes in melanocytes was decreased in subtle gray and Moriyama and Bonifacino, 2002). Thus the melanosome muted, whereas the surrounding keratinocytes did not have morphologies in HPS mutants appear to reflect defects in an increased number of melanosomes. As the steady-state functional protein associations. number is determined by the rates of biosynthesis, The three most markedly affected HPS strains studied degradation, and exocytosis, it is likely that the decreased here were sandy, muted, and buff. The most severely intramelanocyte number is due to either a slowed rate of abnormal melanosomes were observed in the sandy strain, biogenesis or an increase in destruction due to structural likely due to the combined effect of the mutant sandy and defects, possibly through autophagy. This decreased Tyrp1 genes, as early studies suggest that mutations in the number could result in fewer melanosomes transferred to MYO5a/dilute gene have minimal effect on melanosome the hair shaft, leading to hypopigmentation. morphology (Rittenhouse, 1968) and melanosomes in the Another factor contributing to producing the hypopig- brown strain of mice (homozygous for a mutation in the mented phenotype is the transfer of immature hypo- Tyrp1 gene) have morphologies very similar to melano- pigmented melanosomal forms to keratinocytes; the somes in DBA/2J (M.W. and T.N., unpublished observa- subsequent incorporation of these forms into the hair shaft tions). It is likely that the sandy gene product functions at a likely contributes to a pigment dilution phenotype. It is very early point in the pathway of melanosome biogenesis, possible that keratinocytes may not internalize immature as few recognizable early melanosomal forms are observed. forms efficiently, or that internalized immature melano- The scarcity of intramelanosomal striations in sandy somes are more susceptible to degradation, further con- melanosomes suggests that the function of the Pmel/ tributing to pigment dilution. In addition, the quality of gp100 protein is lost. As the Pmel/gp100 protein is delivered pigment produced in HPS mutant strains may differ from the to the melanosome via endosomal trafficking (Raposo et al, pigment in control strains. Differences could arise due to 2001), the sandy mutation may disrupt endosomal to abnormal pigment synthesis or to defective assembly of melanosome trafficking. The buff strain is characterized by protein and pigment components within the lumen of the 122 : 2 FEBRUARY 2004 MECHANISMS OF HYPOPIGMENTATION 459 melanosome, which could lead to a final polymer with Future studies in HPS mutant cells studying membrane altered optical properties. trafficking to the melanosome and assembly of the The observation that immature melanosome forms can melanosome should shed considerable light on the specific be secreted from HPS melanocytes and taken up by functions of these diverse and intriguing gene products, and keratinocytes has interesting implications for the mechan- potentially lead to specific therapies for this disease. ism of melanin secretion. In this study, the muted, buff, pallid, sandy, cappuccino, and reduced pigmentation strains were observed to have immature forms in keratino- Materials and Methods cytes, indicating that melanosomes may have all the Mice The control and HPS strains used in this study are listed in molecular components necessary for exocytosis at an early Table I. Samples from the dorsal back skin of 4 wk old animals stage of biogenesis. Why then are type II/III forms not seen were analyzed as noted below. The protocols used were approved in keratinocytes surrounding melanocytes in control strains by the Institutional Animal Care and Use Committee (IACUC). such as C57BL/6 and C3H/HeSnJ? If in the control strains Pigment measurement The coat color of the dorsal back hair was the rate of maturation from type II to type IV forms is rapid measured using a Mexameter MX 18 (Courage & Khazaka, compared with the rate of secretion, then a secretion of Cologne, Germany), which gives a calculated value of melanin predominantly type IV forms would be expected. In the HPS content based on the absorption of light emitted at 568 nm and mutants where blocks or rate-limiting steps have been 880 nm. To calculate percent pigment as a measurement of dilution introduced into the pathway of maturation, but not into the severity, background was subtracted by subtracting Ptyr, the coat c–2J pathway of transport and secretion, secretion of immature color measurement for the white albino mouse C57BL/6J-Tyr forms would be feasible. (TyrG291T), which is lacking tyrosinase activity and has no pigment production. A comparison with the coat pigment of the control In examining the control strains, a notable finding was strain (Pcon) was done: that the morphology of the melanosomes in the control

DBA/2J strain was abnormal, in agreement with a previous Percent pigmentation ¼ðP PtyrÞ=ðPcon PtyrÞ100 report (Rittenhouse, 1968). DBA/2J is not an HPS strain, as the bleeding time for this strain is normal (Sweeney et al, Electron microscopy The tissue samples were fixed and pro- cessed for electron microscopy as described previously (Nguyen 1990), indicating that melanosomal morphologic abnormal- et al, 2002). Cross-sections of melanosomes were measured at ities can occur independently of HPS. The appearance of 50,000 magnification for major axis and minor axis, in order to the abnormal melanosomes in DBA/2J was unique, and did assess relative size and shape. Images of a calibration grid were not resemble the morphology of any of the HPS affected taken to control for any variability in magnification. Melanocytes melanosomes, including sandy. Interestingly, the Tyrp1 were identified by their organization in hair follicles, as well as their locus in DBA/2J has recently been mapped as a causative lack of keratin filaments and desmosomes (both are present in gene for a murine model of pigmentary glaucoma (Chang keratinocytes). Each melanosome was measured and classified by morphology, i.e.: (1) multiple intralumenal vesicles present, no et al, 1999; Anderson et al, 2002). It was suggested by the pigment or striations seen; (2) striated matrix present, round cross- authors of that study that the mutation in Tyrp1 could cause section; (3) striated matrix, elliptical cross-section; (4) fully a melanosomal morphologic defect, possibly leading to pigmented, round cross-section; and (5) fully pigmented, elliptical fragility of melanosomes, fragmentation, and the formation cross-section. Noted also were any melanosomal features unique of debris, which could cause impaired intraorbital drainage, to any particular strain. For each strain at least 50 melanosomes resulting in glaucoma (Chang et al, 1999). Our observation were examined in each of at least two different animals. Keratinocytes were identified by the presence of desmosomes or of morphologically abnormal melanosomes in DBA/2J is intermediate filaments. consistent with this model of the pathogenesis of murine pigmentary glaucoma. Statistical analysis Statistics were calculated with Stata 7 (Stata In conclusion, the results in this study together with a Corp., College Station, Texas). The melanosome type IV size previous study (Nguyen et al, 2002) demonstrate that in all differences and melanosomes per unit area were assessed using but one of the 15 HPS strains examined to date, HPS gene ANOVA with follow-up contrasts to compare each strain with its products are required at specific steps of melanosomal control. The melanosome size progression data were tested using generalized estimating equations (Diggle et al, 2001) in which biogenesis. Intramelanocyte accumulation of immature melanosome stage was modeled with linear and quadratic terms, hypopigmented melanosomal forms in most HPS strains specific to each strain. Each strain was compared with its control results in exocytosis of these forms and subsequent uptake on the basis of the curvature, linear trend, and level. by keratinocytes, contributing to the hypopigmented HPS phenotype. In addition, decreased numbers of melano- somes in the subtle gray and muted strains may also Many thanks to Corey Largman, for critical reading of the manuscript contribute to hypopigmentation by decreasing the numbers and to Charles McCulloch for assistance with statistical analysis. Many thanks also to Richard Swank and Edward Novak for critical of melanosomes available for transfer to keratinocytes. In reading of the manuscript and for providing the animals for the study. ashen, intramelanocyte retention of melanosomes leads to Thanks also to Mary Kay Ellsworth for animal husbandry. We are inefficient exocytosis, limiting melanin uptake by keratino- grateful to Sandra Huling of the SFVAMC Cell Imaging Laboratory for excellent technical assistance and to Jerelyn Magnusson for able cytes, and results in hypopigmentation in this strain. Of assistance on the manuscript. This work was supported by the note, the classification of the ashen strain as a model of Department of Veterans Administration (Research Career Development HPS is controversial, as one group has reported findings Award, M.L.W.), and by the Helen Heuer Fund, Department of consistent with the classification of ashen as an HPS strain Dermatology, UCSF (T.N.). (Wilson et al, 2000; Novak et al, 2002) and another group has reported conflicting results (Barral et al, 2002). DOI: 10.1046/j.0022-202X.2004.22117.x 460 NGUYEN AND WEI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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