Aberrant pH of Melanosomes in Pink-Eyed Dilution (p) Mutant Melanocytes
2000; Elsevier BV; Volume: 115; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2000.00108.x
ISSN1523-1747
AutoresNeelu Puri, John M. Gardner, Murray H. Brilliant,
Tópico(s)RNA regulation and disease
ResumoIn past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex. In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex. 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine tyrosinase related protein 1 Null mutations of the mouse pink-eyed dilution gene p, and its human homolog P, are defined by hypopigmentation with a near total lack of melanin pigment (Brilliant, 1992Brilliant M.H. The mouse pink-eyed dilution locus: a model for aspects of Prader–Willi syndrome, Angelman syndrome, and a form of hypomelanosis of Ito.Mammalian Genome. 1992; 3: 187-191Crossref PubMed Scopus (33) Google Scholar;Lyon et al., 1992Lyon M.F. King T.R. Gondo Y. Gardner J.M. Nakatsu Y. Eicher E.M. Brilliant M.H. Genetic and molecular analysis of recessive alleles at the pink-eyed dilution locus.Proc Natl Acad Sci USA. 1992; 89: 6968-6972Crossref PubMed Scopus (76) Google Scholar;Oetting et al., 1996Oetting W.S. Brilliant M.H. King R.A. The clinical spectrum of albinism in humans.Mol Med Today. 1996; 2: 330-335Abstract Full Text PDF PubMed Scopus (46) Google Scholar) in melanocytes (Russell, 1949Russell E.S. A quantitative histological study of the pigment found in coat colour mutants of the house-mouse. The nature of genetic effects of five major allelic series.Genetics. 1949; 34: 146-166Google Scholar;Markert and Silvers, 1956Markert C.L. Silvers W.K. Effects of genotype and cellular environment on melanoblast differentiation in the house mouse.Genetics. 1956; 41: 429-450PubMed Google Scholar). The reduction of brown/black eumelanin is greater than the reduction in yellow/red pheomelanin (Ozeki et al., 1995Ozeki H. Ito S. Wakamatsu K. Hirobe T. Chemical characterization of hair melanins in various coat color mutants of mice.J Invest Dermatol. 1995; 105: 361-366Crossref PubMed Scopus (181) Google Scholar;Prota et al., 1995Prota G. Lamoreux M.L. Muller J. et al.Comparative analysis of melanins and melanosomes produced by various coat color mutants.Pigment Cell Res. 1995; 8: 153-163Crossref PubMed Scopus (69) Google Scholar). Previously, we cloned the p gene and found it to encode a protein with 12 predicted membrane-spanning domains (Gardner et al., 1992Gardner J.M. Nakatsu Y. Gondo Y. Lee S. Lyon M.F. King R.A. Brilliant M.H. The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes.Science. 1992; 257: 1121-1124Crossref PubMed Scopus (181) Google Scholar). From this predicted protein structure and the phenotype of melanocytes with p gene mutation, we (Gardner et al., 1992Gardner J.M. Nakatsu Y. Gondo Y. Lee S. Lyon M.F. King R.A. Brilliant M.H. The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes.Science. 1992; 257: 1121-1124Crossref PubMed Scopus (181) Google Scholar;Rosemblat et al., 1994Rosemblat S. Durham-Pierre D. Gardner J.M. Nakatsu Y. Brilliant M.H. Orlow S.J. Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.Proc Natl Acad Sci USA. 1994; 91: 12071-12075Crossref PubMed Scopus (129) Google Scholar) and colleagues (Rinchik et al., 1993Rinchik E.M. Bultman S.J. Horsthemke B. et al.A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism.Nature. 1993; 361: 72-76Crossref PubMed Scopus (310) Google Scholar;Lee et al., 1994Lee S.-T. Nicholls R.D. Bundey S. Laxova R. Musarella M. Spritz R.A. Mutations of the, p. gene in oculocutaneous albinism, and Prader–Willi syndrome plus albinism.N Engl J Med. 1994; 330: 529-534Crossref PubMed Scopus (158) Google Scholar; 1995) hypothesized that the p gene is a transport or pore protein critical to melanocyte function. Our initial results with antibodies against the p protein demonstrated that the p protein is associated with the melanosomal membrane (Rosemblat et al., 1994Rosemblat S. Durham-Pierre D. Gardner J.M. Nakatsu Y. Brilliant M.H. Orlow S.J. Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.Proc Natl Acad Sci USA. 1994; 91: 12071-12075Crossref PubMed Scopus (129) Google Scholar). Therefore, the p protein might transport a critical substance between the cytoplasm and melanosomes.Sidman and Pearlstein, 1965Sidman R.L. Pearlstein R. Pink-eyed dilution (, p.) gene in rodents: increased pigmentation in tissue culture.Dev Biol. 1965; 12: 93-116Crossref PubMed Scopus (49) Google Scholar observed that retinal melanocytes (in organ culture) from p/p mice become melanized in the presence of high concentrations of tyrosine, a precursor for melanin. They speculated that the p protein could be involved in tyrosine uptake or otherwise modulate tyrosinase activity due to an effect on tyrosine-utilizing systems. To assay whether or not the p protein was involved in tyrosine transport, we (Gahl et al., 1995Gahl W.A. Potterf B. Durham-Pierre D. Brilliant M.H. Hearing V.J. Melanosomal tyrosine transport in normal and pink-eyed dilution murine melanocytes.Pigment Cell Res. 1995; 8: 229-233Crossref PubMed Scopus (50) Google Scholar) and colleagues (Potterf et al., 1998Potterf S.B. Furumura M. Sviderskaya E.V. Bennett D.C. Hearing V.J. Normal tyrosine transport and abnormal tyrosinase routing in pink-eyed dilution melanocytes.Exp Cell Res. 1998; 244: 319-326Crossref PubMed Scopus (53) Google Scholar) measured tyrosine transport across the cell and melanosome membranes. The results of those studies indicated that tyrosine transport was the same in both wild-type and homozygous p mutant melanocytes. Thus, tyrosine transport is not mediated by the p protein. The initial and rate limiting step in melanin biosynthesis is catalyzed by tyrosinase (reviewed byPawelek et al., 1998Pawelek J.M. Chakraborty A.K. The enzymology of melanogenesis.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J.P. The Pigmentary System, Physiology and Pathophysiology. Oxford University Press, New York1998Google Scholar). In addition to the substrate tyrosine, maximal in situ tyrosinase activity requires an appropriate ionic environment. The melanosomal lumen is known to be acidic (Moellmann et al., 1988Moellmann G. Slominski A. Kuklinska E. Lerner A.B. Regulation of melanogenesis in melanocytes.Pigment Cell Res. 1988; 1: 79-87Crossref Scopus (58) Google Scholar;Bhatnagar et al., 1993Bhatnagar V. Anjaiah S. Puri N. Darshanam B.N.A. Ramaiah A. pH of melanosomes of B16 murine melanoma is acidic: its physiological importance in the regulation of melanin biosynthesis.Arch Biochem Biophys. 1993; 307: 183-192Crossref PubMed Scopus (95) Google Scholar).Devi et al., 1987Devi C.C. Tripathi R.K. Ramaiah A. pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications.Eur J Biochem. 1987; 166: 705-711Crossref PubMed Scopus (38) Google Scholar have shown that preincubation of murine tyrosinase at an acidic pH causes the enzyme to lose the lag period that occurs when tyrosine is a substrate [in the absence of added L-dihydroxyphenylalanine (L-DOPA)]. Thus, the acidic pH of melanosomes favors optimum tyrosinase activity and the melanization of melanosomes (Ramaiah, 1996Ramaiah A. Lag kinetics of tyrosinase: its physiological implications.Indian J Biochem Biophys. 1996; 33: 349-356PubMed Google Scholar). In this study, we tested the hypothesis that the p protein functions in the acidification of melanosomes, presumably as an ion exchange (or channel) protein in the melanosomal membrane. To test this hypothesis, we assayed the pH of melanosomes and their precursors from both wild-type and p mutant melanocytes in vitro. Melanosomal and premelanosomal compartments were identified by the presence of the melanosomal marker, tyrosinase related protein 1 (Tyrp1) (Vijayasaradhi et al., 1991Vijayasaradhi S. Doskoch P.M. Houghton A.N. Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product.Exp Cell Res. 1991; 196: 233-240Crossref PubMed Scopus (67) Google Scholar). Acidic compartments were identified using 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP, a basic congener of dinitrophenol that accumulates in acidic components, where it can be fixed in situ with aldehydes). DAMP was used in studies byAnderson and Pathak, 1985Anderson R.G.W. Pathak R.K. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments.Cell. 1985; 40: 635-643Abstract Full Text PDF PubMed Scopus (214) Google Scholar to demonstrate the acidic nature of the lysosome, an organelle related to melanosomes (Orlow, 1995Orlow S.J. Melanosomes are specialized members of the lysosomal lineage of organelles.J Invest Dermatol. 1995; 105: 3-7Crossref PubMed Scopus (225) Google Scholar). The results of this study implicate involvement of the p protein in the generation or maintenance of an acidic melanosomal pH. The dorsal skin of a neonatal mouse was removed aseptically and cut into several small pieces, rinsed in Ca2+,Mg2+-free phosphate-buffered saline (PBS), and incubated in 0.25% trypsin at 37°C and 5% CO2 for 2 h. The dermis was then separated away from the epidermis and incubated in 0.05% trypsin containing 0.5 U per ml of collagenase for 30 min. The digested dermis was pipetted repeatedly in 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) until the solution became turbid. The cells were pelleted and resuspended in Ham's F-10 medium. Melanocytes were cultured in Ham's F-10 medium containing 10% fetal bovine serum, 100 U per ml of penicillin, 100 μg per ml of streptomycin, 2 mM L-glutamine, 0.1 mM dibutyryl cyclic adenosine-5′-monophosphate, 100 nM phorbol-12-myristate-13-acetate and 25 μg per ml bovine pituitary extract-protein (Life Technologies, Rockville, MD). Melanocyte lines from C57BL/6+/+, pJ/pJ, and pcp/p6H were established from primary cultures. The last two cell lines are null for p gene function. The pJ allele is characterized by an intragenic deletion (Oakey et al., 1996Oakey R.J. Keiper N.M. Ching A.S. Brilliant M.H. Molecular analysis of the cDNAs encoded by the pJ and pun alleles of the pink-eyed dilution locus.Mammalian Genome. 1996; 7: 315-316Crossref PubMed Scopus (19) Google Scholar) and pcp/p6H is a compound heterozygote of two deletion alleles and lacks all protein encoding sequences of the p gene (Gardner et al., 1992Gardner J.M. Nakatsu Y. Gondo Y. Lee S. Lyon M.F. King R.A. Brilliant M.H. The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes.Science. 1992; 257: 1121-1124Crossref PubMed Scopus (181) Google Scholar;Nakatsu et al., 1993Nakatsu Y. Tyndale R.F. DeLorey T.M. et al.A cluster of three GABAA receptor subunit genes is deleted in a neurological mutant of the mouse, p. locus.Nature. 1993; 364: 448-450Crossref PubMed Scopus (83) Google Scholar;Lehman et al., 1998Lehman A.L. Nakatsu Y. Ching A. et al.A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice.Proc Natl Acad Sci USA. 1998; 95: 9436-9441Crossref PubMed Scopus (86) Google Scholar). After establishing pure cultures of these melanocytes, electron microscopy and immunofluorescent studies were performed, comparing cells of the same passages (passages 3–6). Confluent flasks of melanocytes were treated with 0.05% trypsin and 0.02% EDTA in Ca2+,Mg2+-free PBS, pelleted, and washed twice in PBS. Cell pellets were fixed for at least 18 h at 4°C in 3% glutaraldehyde and 0.1 M phosphate buffer (pH 7.2). The fixed pellets were dissected to 1 mm3 pieces. Postfixation was in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h followed by en bloc staining for 30 min in 1% uranyl acetate in 50% ethanol. The tissues were then dehydrated using serial alcohol and acetone incubations and embedded in Spurr resin. A Sorvall MT-2B ultramicrotome was used to section the tissues to 80 nm (silver-gold). Sections were stained with uranyl acetate and lead citrate. Grids were viewed on a Philips 400 electron microscope at an accelerating voltage of 80 kV. To visualize tyrosinase activity in melanocytes, L-DOPA conversion to melanin was assayed in vitro. Trypsinized cell pellets were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h, and then washed three times in cacodylate buffer. These fixed pellets were dissected into 1 mm3 pieces and incubated in 0.1% L-DOPA in cacodylate buffer at 4°C overnight. The next day, cell pellets were incubated in fresh 0.1% L-DOPA cacodylate buffer at room temperature for 2 h, and then incubated a third time in 0.1% L-DOPA cacodylate buffer at 37°C for 2 h. Postfixation was done in 1% osmium tetroxide in 0.1 M cacodylate buffer, and all further steps of electron microscopy processing were performed as described above. Melanocytes (1 × 105) from pJ/pJ or pcp/p6H mice were plated on glass coverslips in six-well plates. Cells were allowed to grow for 48 h at 37°C, and then were incubated with 30 μM DAMP for 30 min at 37°C and washed with Ham's F10 medium. For monensin studies, monolayers were treated with 25 μM of monensin (Sigma Chemical, St. Louis, MO) for 5 min after DAMP treatment. To localize DAMP, cells were fixed at room temperature for 15 min in 3% (wt/vol) paraformaldehyde in buffer A (10 mM sodium phosphate, 150 mM sodium chloride, 2 mM magnesium chloride; pH 7.4), and then were washed once with 50 mM ammonium chloride and twice with buffer A. Each monolayer was permeabilized with 0.1% (vol/vol) Triton X-100 in buffer A for 5 min at -10°C. Coverslips were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 30 min. They were then covered with 100 μl of monoclonal mouse anti-dinitrophenol (DNP) IgG diluted 1:10 (Oxford Biomedical Research) and incubated at 37°C for 60 min. After three washes with buffer A, the cells were incubated with 100 μl fluorescein-5-isothiocyanate (FITC) conjugated goat antimouse IgG diluted 1:200 (Organon Teknika Corporation) at 37°C for 60 min. After three additional washes with buffer A, melanocytes were blocked again with 5% goat serum for 30 min and incubated with rabbit polyclonal antiserum PEP1 diluted 1:500 (a kind gift of Vince Hearing, NIH) generated against the carboxyl terminus of murine Tyrp1 (Jimenez et al., 1991Jimenez M. Tsukamoto K. Hearing V. Tyrosinase from two different loci are expanded by normal and by transformed melanocytes.J Biol Chem. 1991; 266: 1147-1156Abstract Full Text PDF PubMed Google Scholar) for 60 min. Coverslips were washed in buffer A, incubated with lissamine rhodamine-conjugated goat antirabbit IgG diluted 1:100 (Jackson ImmunoResearch Laboratories) for 60 min and washed with buffer A. These coverslips were then mounted in Vecta shield (Vector Laboratories, Burlingame, CA) and viewed under a Bio-Rad MRC-600 confocal laser scanning microscope (Bio-Rad, Richmond, CA) equipped with an argon krypton laser coupled to a Nikon Optiphot II fluorescence microscope and a 60× plan Apo oil objective. A standard k1/k2 filter set was used. Simultaneous two-channel recording was performed using excitation wavelengths of 488 and 568 nm. Images were processed and merged using a Voel view ultra 2.5 software (Vital Images, Airfield, IA). The numbers of vesicles that stained for Tyrp1, DAMP, or both were scored in four independent experiments. In each experiment, 25 independent fields (47 μm2) were counted from cells established from C57BL/6+/+ and the p mutants. The data from 100 (4 × 25) representative fields of approximately equivalent vesicular densities of peripheral cytoplasm were used to calculate the percentage of vesicles stained with Tyrp1 and DAMP in all three cell lines. C57BL/6 wild-type melanocytes were grown and treated with DAMP as detailed above, and pelleted in Ca2+,Mg2+-free PBS, pH 7.2. The cells were then fixed for 1 h in 1% glutaraldehyde in Ca2+,Mg2+-free PBS, pH 7.2, and washed for 5 min three times in Ca2+,Mg2+-free PBS. The remaining glutaraldehyde was quenched by incubation in 0.1 M NH4Cl in Ca2+,Mg2+-free PBS, pH 7.2, for 30 min, followed by two 5 min washes in Ca2+,Mg2+-free PBS, pH 7.2. The treated cell pellets were then dehydrated by serial alcohol dilution and embedded in LRWhite at 50°C, under vacuum. The cell pellets were cut in 80 nm sections onto 200 mesh formvar/carbon coated nickel grids. The grids were floated on a drop of blocking buffer (0.5 M NaCl, 0.1% Na-azide, 1% ovalbumin, in 0.01 M Tris-HCl, pH 7.2) for 30 min at room temperature. The grids were then floated on a drop of blocking buffer plus mouse monoclonal anti-DNP (Oxford Biomedical Research) at 1:10 dilution for 16 h at 4°C, in a moisture chamber. The grids were then washed with rinse buffer (0.15 M NaCl in 0.01 M Tris-HCl, pH 7.2) and floated on rinse buffer plus 0.02% polyethylene glycol-20, 0.1% Na-azide, plus Protein A gold, 10 nm (Amersham), at 1:20 for 1 h at room temperature and washed again with rinse buffer. Fixation of the antigen-antibody complex was with 2% glutaraldehyde in PBS for 10 min followed by two washes with ddH2O. The grids were then stained with 2% uranyl acetate (aqueous) for 10 min and lead citrate for 10 min and viewed on a Philips 400 electron microscope at 80 kV. Cultures of melanocytes were established from C57BL/6+/+, pJ/pJ, and pcp/p6H mice. In comparison with melanocytes established from wild-type C57BL/6+/+ mice, both p null mutant cell lines exhibited minimal amounts of visible pigmentation in their cell pellets or culture media. Electron microscopy demonstrated that melanocytes from C57BL/6+/+ (Figure 1a) contained numerous elliptical melanosomes that were highly melanized (stage III-IV). Melanocytes from p null mutants also contained numerous melanosomes, but these were poorly melanized (stage I-II) (Figure 1b, c); similar results were obtained byRosemblat et al., 1998Rosemblat S. Sviderskays E.V. Easty D.J. Wilson A.M. Kwon B.S. Bennett D.C. Orlow S.J. Melanosomal defects in melanocytes from mice lacking expression of the pink-eyed dilution (, p.) gene: correction by culture in the presence of excess tyrosine.Exp Cell Res. 1998; 239: 1-9Crossref PubMed Scopus (51) Google Scholar. In addition, we tested L-DOPA reactivity of the pcp/p6H melanocytes that we established in vitro. These melanocytes exhibited L-DOPA oxidase activity of tyrosinase that revealed the presence of this enzyme in the trans-Golgi network and in stage I-II melanosomes (Figure 1d). Normal melanosomes have been shown to be acidic organelles (Moellmann et al., 1988Moellmann G. Slominski A. Kuklinska E. Lerner A.B. Regulation of melanogenesis in melanocytes.Pigment Cell Res. 1988; 1: 79-87Crossref Scopus (58) Google Scholar;Bhatnagar et al., 1993Bhatnagar V. Anjaiah S. Puri N. Darshanam B.N.A. Ramaiah A. pH of melanosomes of B16 murine melanoma is acidic: its physiological importance in the regulation of melanin biosynthesis.Arch Biochem Biophys. 1993; 307: 183-192Crossref PubMed Scopus (95) Google Scholar). To determine whether melanosomes from p mutant melanocytes are acidic, we used DAMP and anti-DNP staining to visualize acidic organelles.Anderson et al., 1984Anderson R.G.W. Falck A.J.R. Goldstein J.L. Brown M.S. Visualization of acidic organelles in intact cells by electron microscopy.Proc Natl Acad Sci USA. 1984; 81: 4838-4842Crossref PubMed Scopus (178) Google Scholar have shown that DAMP penetrates the membranes of living cells, accumulates in acidic vesicles within the cell, and can be retained in these vesicles after aldehyde fixation. Melanocytes from C57BL/6+/+ and p mutant mice were treated with DAMP, fixed in paraformaldehyde, and processed for indirect immunofluorescence (Figure 2) using mouse monoclonal antibodies directed against dinitrophenol (anti-DNP) and visualized with goat antimouse antibodies coupled to FITC. The polyclonal antibody PEP1 directed against Tyrp1 was used to detect melanosomes and their precursors and visualized with goat antirabbit IgG conjugated with lissamine rhodamine. Tyrp1-staining vesicles were detected primarily in the perinuclear area but were also scattered throughout the cytoplasm in melanocytes from all three genotypes (Figure 2b, e, h). To ensure that our assay for DAMP accumulation and binding was specific for acidic vesicles, we included control experiments employing monensin treatment of melanocytes from both C57BL/6+/+ and p mutants. The carboxylic ionophore monensin is known to dissipate proton gradients by exchanging protons for potassium ions across membranes (Pressman, 1976Pressman B. Biological applications of ionophores.Ann Rev Biochem. 1976; 45: 501-530Crossref PubMed Scopus (1421) Google Scholar). Melanocytes from all genotypes were allowed to take up DAMP, and were then washed and exposed to monensin for 5 min. After monensin treatment, DAMP was no longer visualized within the cells (Figure 2j, k). These results confirm that in our assays the accumulation of DAMP and its retention in cell organelles is dependent on a pH gradient across the membrane of these organelles. To determine the number of vesicles that stained for Tyrp1, DAMP, or both, we counted a total of 100 fields of 47 μm2 (25 fields in each of four independent experiments) of peripheral cytoplasm. A total of 7846 vesicles were counted. The average number of stained vesicles per 47 μm2 field was 22.4 in C57BL/6+/+ 27.9 in pcp/p6H; and 28.1 in pJ/pJ. The three cell lines contained roughly the same number of Tyrp1-staining vesicles per single 47 μm2 field (C57BL/6+/+, 20.0; pcp/p6H, 18.4; pJ/pJ, 19.1). In C57BL/6+/+ melanocytes 94.2% of melanosomes defined by Tyrp1 staining were also acidic by DAMP staining (i.e., colocalization of Tyrp1 and DAMP, Figure 2c; Table 1). In melanocytes from pcp/p6H and pJ/pJ mice, however, only 7.4% and 8.4%, respectively, of the Tyrp1 staining vesicles exhibited colocalization with DAMP (Figure 2f, i; Table 1).Table IPercentage of organelles in wild-type and p mutant melanocytes that are melanosomes (defined by Tyrp1 staining), acidic vesicles (defined by DAMP staining), or bothMelanosomesSource experiment number+ Tyrp1 + DAMP (yellow fluorescence) number of vesicles;%+ Tyrp1 - DAMP (red fluorescence) number of vesicles;%Non-melanosomes – Tyrp1 + DAMP (green fluorescence) number of vesicles;%C57/BL6+/+ I.457; 82.3%29; 5.3%68; 12.4% II.483; 82.8%30; 5.1%70; 12.0% III.481; 84.5%30; 5.3%58; 10.2% IV.465; 86.6%27; 4.8%46; 8.6% total vesicles84.0 ± 1.7%5.1 ± 0.2%10.8 ± 1.5% melanosomes aTyrp1 staining vesicles only. The identity of the non-Tyrp1 staining acidic vesicles is unknown; among the possibilities are very early (non-Tyrp1 expressing) melanosomal precursors or other acidic endosomal-derived vesicles. In each of the four sets of experiments (I-IV), 25 independent fields of 47 μm2 of peripheral melanocyte cytoplasm were evaluated (see Materials and Methods). The data summarized for each cell line (in bold) represent the percentages of each vesicle type (mean values ± standard deviation) in all four experiments, i.e., 100 independent fields of peripheral cytoplasm of similar vesicular densities.94.2 ± 0.3%5.7 ± 0.3%pcp/p6H I.25; 3.5%403; 57.0%279; 39.4% II.48; 6.9%410; 59.2%235; 33.9% III.36: 5.0%465; 64.2%224; 30.8% IV.27; 4.0%423; 63.0%222; 33.0% total vesicles4.8 ± 1.3%60.8 ± 2.9%34.3 ± 3.2% melanosomes7.4 ± 1.9%92.6 ± 1.9%pJ/pJ I.30; 4.3%440; 63.4%224; 32.2% II.51; 7.2%428; 60.1%232; 32.6% III.47; 6.5%467; 64.6%208; 28.9% IV.33; 4.9%418; 61.6%227; 33.5% total vesicles5.7 ± 1.2%62.4 ± 1.7%31.8 ± 1.7% melanosomes8.4 ± 1.7%91.6 ± 1.7%a Tyrp1 staining vesicles only. The identity of the non-Tyrp1 staining acidic vesicles is unknown; among the possibilities are very early (non-Tyrp1 expressing) melanosomal precursors or other acidic endosomal-derived vesicles. In each of the four sets of experiments (I-IV), 25 independent fields of 47 μm2 of peripheral melanocyte cytoplasm were evaluated (see Materials and Methods). The data summarized for each cell line (in bold) represent the percentages of each vesicle type (mean values ± standard deviation) in all four experiments, i.e., 100 independent fields of peripheral cytoplasm of similar vesicular densities. Open table in a new tab To confirm that the acidic vesicles visualized by DAMP staining were indeed melanosomes, DAMP staining vesicles were revealed by electron microscopy using immuno-gold conjugated anti-DNP. As shown in Figure 3, structurally identifiable melanosomes in wild-type melanocytes were clearly stained in this assay. Early stage to mature melanosomes were stained, indicating that melanosomes are acidic even before melanin synthesis and deposition. We also found a class of organelles that were acidic (DAMP staining) but were not melanosomes (not Tyrp1 staining) in wild-type and mutant cell lines. These acidic nonmelanosomal vesicles accounted for about 11% of the vesicles detected in wild type and for about 33% of the vesicles detected in the two mutant cell lines (Table 1). In addition, the mutant cell lines appear to have a 25% increase in the total number of staining vesicles per field (Table 1). These extra vesicles seen in mutant cell lines were primarily nonmelanosomal acidic vesicles and were not further characterized in this study. We have established melanocyte cell lines that recapitulate the in vivo phenotypes of melanocytes from wild-type and p null mutant mice. Electron microscopy revealed mature and highly pigmented stage IV melanosomes in the wild-type cell line, whereas only immature stage I and stage II melanosomes were detected in the p null mutant cell lines, although the mutant melanosomes did possess tyrosinase activity using L-DOPA as substrate (Figure 1). Similar observations have been made in vivo (Orlow and Brilliant, 1999Orlow S.J. Brilliant M.H. The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.Exp Eye Res. 1999; 68: 147-154Crossref PubMed Scopus (56) Google Scholar) and in vitro (Potterf et al., 1998Potterf S.B. Furumura M. Sviderskaya E.V. Bennett D.C. Hearing V.J. Normal tyrosine transport and abnormal tyrosinase routing in pink-eyed dilution melanocytes.Exp Cell Res. 1998; 244: 319-326Crossref PubMed Scopus (53) Google Scholar;Rosemblat et al., 1998Rosemblat S. Sviderskays E.V. Eas
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