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doi: 10.1242/10.1242/jcs.00598

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Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4

Gertrude-E. Costin1, Julio C. Valencia2, Wilfred D. Vieira1, M. Lynn Lamoreux3 and Vincent J. Hearing1,*

1 Pigment Cell Biology Section, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
2 Pathology Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
3 Department of Veterinary Pathobiology, Texas A and M University, College Station, TX 77843, USA



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  		Fig. 1. Characteristics of wild-type and uw-mutant primary murine melanocytes in culture. (A,B) Phase contrast photomicrographs of wild-type and uw-mutant melanocytes. (C,D) Bright-field microphotographs emphasizing the pigmentation of wild-type and uw-mutant melanocytes. (E,F) Bright-field microphotographs showing the pigmented vesicles secreted by uw-mutant melanocytes into the medium (arrows) and their absence in the medium of wild-type melanocytes. All photomicrographs are shown at the same magnification. (G) Media were collected from wild-type and from uw-mutant melanocytes, and were centrifuged to show the similar appearance of those samples (i.e. no soluble melanin was present). (H) Pigmented vesicles released into the medium by uw-mutant melanocytes can be readily sedimented by centrifugation, a process not seen in wild-type melanocytes. (I) Melanin content of extracts of wild-type and uw-mutant melanocytes.

 


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  		Fig. 2. Metabolic pulse-chase labeling of wild-type and uw-mutant melanocytes. Wild-type, uw-mutant and Tyrc albino primary melanocytes were labeled for 1 hour with an [35S]-methionine and cysteine mixture, harvested at the chase times indicated, solubilized and immunoprecipitated using specific antibodies for tyrosinase and Tyrp1, respectively. Immune complexes were separated by SDS-PAGE, and immunoprecipitated bands were visualized as detailed in the Materials and Methods section.

 


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  		Fig. 3. Western blot analysis of melanogenic protein glycosylation in subcellular fractions of wild-type and of uw-mutant melanocytes. Equal amounts of protein (0.1 µg/lane) from cell extracts, melanosomes and secreted vesicles of wild-type or uw-mutant melanocytes were electrophoretically separated by 8% SDS-PAGE. Tyrosinase or Tyrp1 were then detected immunochemically with {alpha}PEP7 or {alpha}PEP1 antibodies, respectively. Glycosylation of melanosomal proteins was determined following digestion with buffer (–), EndoH (E) or PNGaseF (P), as indicated.

 


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  		Fig. 4. Subcellular distribution of melanogenic proteins in wild-type and uw-mutant primary melanocytes. Wild-type and uw-mutant melanocytes were stained with antibodies and fluorescent probes as noted in the Materials and Methods section, and localization of tyrosinase, Tyrp1 or Dct (red) with KDEL or HMB-45 (green) was analyzed by confocal microscopy. Colocalization of antibodies is indicated by the yellow color. Nuclei are identified by the blue DAPI stain. Bar, 20 µm.

 


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  		Fig. 5. Ultrastructure of primary melanocytes established from wild-type and from uw-mutant mice. (A) Wild-type melanocytes showing numerous stage III and IV melanosomes. (B) uw-mutant melanocytes displaying primarily stage I and II melanosomes. (C) uw-mutant melanocytes incubated with DOPA show a dark staining in stage I and II melanosomes (arrows), which proves the presence of active tyrosinase. (D) Secreted vesicle fraction from uw-mutant melanocytes where stage I and II melanosomes, similar to those found in (B), are identified clearly. Bar, 20 µm.

 


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  		Fig. 6. Melanosomal protein trafficking and aberrant processing seen in various types of OCA. The disruption of tyrosinase trafficking occurs at the level of the ER in OCA1 and OCA3, and at the immediate post-Golgi level in OCA2. The results of this study show that in OCA4, tyrosinase processing and trafficking is disrupted and that the enzyme is abnormally secreted from the cells within vesicles before delivery to early melanosomes.

 

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© The Company of Biologists Ltd 2003