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Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation

  • Author Footnotes
    5 These authors contributed equally to this work.
    Silvia Benito-Martínez
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    Affiliations
    Structure and Membrane Compartments, Institut Curie, Paris Sciences & Lettres Research University, Centre National de la Recherche Scientifique, Paris, France
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    Yueyao Zhu
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    Affiliations
    Department of Biology Graduate Program, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA

    Department of Pathology and Laboratory Medicine and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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    Riddhi Atul Jani
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    Structure and Membrane Compartments, Institut Curie, Paris Sciences & Lettres Research University, Centre National de la Recherche Scientifique, Paris, France
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    Dawn C. Harper
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    Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA
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    Michael S. Marks
    Correspondence
    Correspondence: Michael S. Marks, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania 19104.
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    6 These authors contributed equally to this work.
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    Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA

    Department of Pathology and Laboratory Medicine and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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    Cédric Delevoye
    Correspondence
    Cédric Delevoye, Structure and Membrane Compartments, Institut Curie, Paris Sciences & Lettres Research University, Centre National de la Recherche Scientifique, UMR144, Paris, France.
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    Structure and Membrane Compartments, Institut Curie, Paris Sciences & Lettres Research University, Centre National de la Recherche Scientifique, Paris, France
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    6 These authors contributed equally to this work.
      Pigmentation of the skin and hair represents the result of melanin biosynthesis within melanosomes of epidermal melanocytes, followed by the transfer of mature melanin granules to adjacent keratinocytes within the basal layer of the epidermis. Natural variation in these processes produces the diversity of skin and hair color among human populations, and defects in these processes lead to diseases such as oculocutaneous albinism. While genetic regulators of pigmentation have been well studied in human and animal models, we are still learning much about the cell biological features that regulate melanogenesis, melanosome maturation, and melanosome motility in melanocytes, and have barely scratched the surface in our understanding of melanin transfer from melanocytes to keratinocytes. Herein, we describe cultured cell model systems and common assays that have been used by investigators to dissect these features and that will hopefully lead to additional advances in the future.

      Abbreviations:

      2D (2-dimensional), 3D (3-dimensional), CLEM (correlative light to EM), DOPA (L-3,4-dihydroxyphenylalanine), EM (electron microscopy), EPR (electron paramagnetic resonance spectrometry), ET (electron tomography), GS (Griscelli syndromes), HPLC (high performance liquid chromatography), HEM (human epidermal melanocyte), HEK (human epidermal keratinocyte), HPF (high-pressure freezing), HPS (Hermansky-Pudlak syndrome), IEM (immunolabeling EM), IFM (immunofluorescence microscopy), LRO (lysosome-related organelle), TYR (tyrosinase)

      Summary Points

      • Skin pigmentation provides protection against UV radiation and relies on melanin synthesis within melanosomes in melanocytes and subsequent melanin transfer to keratinocytes.
      • Two types of melanins exist; they absorb visible light and can be quantified from a variety of sources by a number of spectroscopy methods.
      • Cell culture models for melanocytes and melanoma cells allow for analyses of melanosome maturation and content by electron microscopy or light microscopy, and by biochemical, or immunofluorescence, or immunoelectron microscopy analyses for melanosome protein contents.
      • Melanin transfer from melanocytes or isolated melanosome cores to keratinocytes can be studied using cell culture models and a variety of microscopy techniques.

      Introduction

      One of the most prominent features of metazoans is their pattern of pigmentation. Variation in the type and amount of pigment produced yields differences in hair, skin, and eye color, an instantly recognizable feature that dictates social cues, impacts visual acuity, and protects against the harmful effects of UV radiation (
      • Jablonski N.G.
      • Chaplin G.
      The evolution of human skin coloration.
      ,
      • Pavan W.J.
      • Sturm R.A.
      The genetics of human skin and hair pigmentation.
      ). Deficiencies in pigment production result in various forms of albinism—with concomitant poor visual acuity and susceptibility to skin cancer—or patterned disorders such as vitiligo or piebaldism (
      • Pavan W.J.
      • Sturm R.A.
      The genetics of human skin and hair pigmentation.
      ). Understanding how pigments are made and distributed is thus an important and vibrant area of dermatological research.
      The major pigments in mammals, melanins, are synthesized by specialized pigment cells—melanocytes in the basal layer of the skin, the hair bulb, and the choroid of the eye, and pigmented epithelial cells of the retina, iris, and ciliary body of the eye. The black or brown eumelanins and red or yellow pheomelanins are both composed of polymerized products of sequential redox reactions in which tyrosine is the initial substrate. The process underlying melanin formation is referred to as melanogenesis (Figure 1a ) (
      • d’Ischia M.
      • Wakamatsu K.
      • Cicoira F.
      • Di Mauro E.
      • Garcia-Borron J.C.
      • Commo S.
      • et al.
      Melanins and melanogenesis: from pigment cells to human health and technological applications.
      ). Because many of the intermediates in melanogenesis are highly redox reactive, melanin synthesis in pigment cells is sequestered within specialized membrane-bound organelles called melanosomes (
      • Marks M.S.
      • Seabra M.C.
      The melanosome: membrane dynamics in black and white.
      ). Melanin synthesis in the eye is largely limited to prenatal and perinatal life, and ocular pigment cells maintain their melanosomes intracellularly throughout their lifetime (
      • Lopes V.S.
      • Wasmeier C.
      • Seabra M.C.
      • Futter C.E.
      Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis.
      ). In contrast, epidermal melanocytes synthesize melanins constitutively (although melanogenesis can be stimulated, e.g., by UV exposure) and transfer them to neighboring keratinocytes. Melanins in keratinocytes are retained within membrane-bound organelles that form a cap above the nucleus to protect keratinocyte DNA from irradiation (
      • Kobayashi N.
      • Nakagawa A.
      • Muramatsu T.
      • Yamashina Y.
      • Shirai T.
      • Hashimoto M.W.
      • et al.
      Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis.
      ). Thus, skin and hair pigment is synthesized in melanocytes but resides primarily in keratinocytes (
      • Wu X.
      • Hammer J.A.
      Melanosome transfer: it is best to give and receive.
      ). Natural variation in melanosome formation and maturation, melanogenesis, and melanin transfer to keratinocytes results in variation in skin, hair, and eye color and in visual acuity. Accordingly, pigmentary disorders arise from defects in these processes. For example, heritable disorders, such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome, associated with oculocutaneous albinism, are caused by the disrupted assembly of melanosomes and other cell type–specific lysosome-related organelles (LROs), whereas the Griscelli syndromes (GS) cause pigment dilution primarily in the skin and hair by interfering with the intracellular positioning of melanosomes and other LROs (
      • Bowman S.L.
      • Bi-Karchin J.
      • Le L.
      • Marks M.S.
      The road to lysosome-related organelles: insights into lysosome-related organelles from Hermansky-Pudlak syndrome and other rare diseases.
      ,
      • Delevoye C.
      • Marks M.S.
      • Raposo G.
      Lysosome-related organelles as functional adaptations of the endolysosomal system.
      ).
      Figure thumbnail gr1
      Figure 1Pigmentation analyses. (a) Melanogenesis pathway from
      • Ito S.
      • Wakamatsu K.
      Chemistry of mixed melanogenesis - pivotal roles of dopaquinone.
      , with permission from John Wiley and Sons. (b) Pigmentation analyzed in pellets of B16 melanoma cells treated for indicated times with inulavosin, a drug that targets TYR for degradation (
      • Fujita H.
      • Motokawa T.
      • Katagiri T.
      • Yokota S.
      • Yamamoto A.
      • Himeno M.
      • et al.
      Inulavosin, a melanogenesis inhibitor, leads to mistargeting of tyrosinase to lysosomes and accelerates its degradation.
      ). (c) Pigmentation visualized in mouse melan-Ink4a-/- cells expressing a control non-target shRNA or depleted of the transporter MFSD12 by shRNA. Bright field microscopy images are shown on the left (Bar = 10 μm); quantification of the percent area of each cell that is covered by melanin is shown on the right. From Crawford et al., (2017). (d) Use of spectroscopy or visual inspection to estimate the melanin content of MNT-1 cells treated with control siRNA or depleted of the small GTPase RAB6A/A’ by siRNA. The absorbance of the melanin content is normalized to the control sample. From
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      . shRNA, short hairpin RNA; siRNA, small inhibitory RNA; TYR, tyrosinase.
      This review describes cultured cell model systems in which to study melanogenesis in epidermal melanocytes (Supplementary Table S1) and assays for melanogenesis, melanosome formation and maturation, and melanin transfer to cultured keratinocytes. We focus on major approaches used by investigators in the field, their advantages and limitations, and alternative approaches. Owing to space limitations, we will not discuss different genetic systems used for whole animal pigmentation studies (e.g., zebrafish and mice), analyses of melanocyte-keratinocyte interactions in situ, or systems to study pigmentation in ocular pigment cells.

      Cell Culture Models of Melanogenesis

      Although some physiological controls on pigmentation require the intact architecture of the skin and hair, melanosome biogenesis and melanin synthesis in epidermal melanocytes in situ are largely maintained in cultured melanocytes and model cell lines. Hence, melanocytes or melanogenic melanoma cells provide outstanding and easily manipulated model systems with which to study these processes. A number of different models are frequently used, each with its own advantages and disadvantages (Supplementary Table S1); thus, validation of data using different models is recommended. With any of these models, vigilance is needed to ensure that cells maintain proper differentiation and optimal growth (Box 1).
      Assessing the health of cultured melanocytes
      The overall cellular health of melanocytes in culture and their melanosome content and morphology can be rapidly assessed by standard bright field light microscopy with an inverted compound microscope. Cells can be visualized in their growth medium and/or platform, yielding low contrast visualization of highly pigmented granules (likely stage IV melanosomes). Cell stress or prolonged culture may lead to any of several features that change the pigmentation pattern in a cohort of the cells, including (i) dedifferentiation, leading to reduced or lost pigment granules; (ii) pigment aggregation, revealed as seemingly enlarged melanosomes; (iii) change in the subcellular distribution of melanosomes (e.g., clustering in the perinuclear area or accumulation at the periphery); and (iv) elongation and thinning of cell architecture and an apparent increase in dendrite formation. These features may skew the results of more detailed analyses of melanosome biogenesis, transfer, or signaling. The accumulation of melanocytes in culture with these features should be taken as a sign to thaw a new vial of cells. Note that cell confluency impacts the pigmentation status of all cultured melanocytes and needs to be controlled for in experiments comparing pigmentation across different cultured cell sources. Most of the melanocyte models described can be efficiently transduced by infection with recombinant retroviruses or lentiviruses for exogenous gene expression, short hairpin RNA knockdown, or CRISPR/Cas9 mutagenesis, and most of the cell lines can be transfected as well.

      Analyzing Melanin

      Melanins are synthesized within melanosomes through a series of chemical reactions that are initiated by the enzyme, tyrosinase (TYR), which catalyzes the hydroxylation of tyrosine to form L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to DOPAquinone (Figure 1a) (
      • d’Ischia M.
      • Wakamatsu K.
      • Cicoira F.
      • Di Mauro E.
      • Garcia-Borron J.C.
      • Commo S.
      • et al.
      Melanins and melanogenesis: from pigment cells to human health and technological applications.
      ). At near neutral pH and oxidizing conditions, DOPAquinone is oxidized through several intermediates to indole subunits that polymerize to form eumelanins (Figure 1a). Within melanosomes, eumelanins polymerize onto a sheet-like matrix consisting of proteolytic fragments of the amyloid protein, PMEL (also known as Pmel17, Silver, gp100, and several other names) (
      • Watt B.
      • van Niel G.
      • Raposo G.
      • Marks M.S.
      PMEL: A pigment cell-specific model for functional amyloid formation.
      ). If cysteine is abundant, and the pH is slightly lower (
      • Wakamatsu K.
      • Nagao A.
      • Watanabe M.
      • Nakao K.
      • Ito S.
      Pheomelanogenesis is promoted at a weakly acidic pH.
      ), DOPAquinone is converted into cysteinylDOPA, which then undergoes a series of steps to form pheomelanins (
      • d’Ischia M.
      • Wakamatsu K.
      • Cicoira F.
      • Di Mauro E.
      • Garcia-Borron J.C.
      • Commo S.
      • et al.
      Melanins and melanogenesis: from pigment cells to human health and technological applications.
      ). Because melanosomes progress from highly acidic to near neutral during maturation (
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      ), melanin deposits in melanosomes of darkly pigmented melanocytes may have a pheomelanin core surrounded by a eumelanin cortex (
      • d’Ischia M.
      • Wakamatsu K.
      • Cicoira F.
      • Di Mauro E.
      • Garcia-Borron J.C.
      • Commo S.
      • et al.
      Melanins and melanogenesis: from pigment cells to human health and technological applications.
      ). Both classes of melanin polymers are highly stable and insoluble in aqueous buffers; methods of chemical analyses of melanins must take this into account.

      Melanin visualization in cell pellets

      A simple method to assess pigmentation in cultured melanocytes or melanoma cells is to pellet the cells and visually compare the resulting color (Figure 1b). This non-quantitative approach—limited in presentation to a representative image—allows for a qualitative comparison between samples that can document changes in the degree or color of pigmentation.

      Melanin visualization by light microscopy

      Because melanin efficiently absorbs visible light, melanin within mature melanosomes can be visualized by bright field light microscopy. At high magnification (original magnification ×63 or ×100), melanosome size, distribution, total cellular content, and number per unit area can be analyzed qualitatively or quantitatively in live or fixed cells (Figure 1c) (
      • Crawford N.G.
      • Kelly D.E.
      • Hansen M.E.B.
      • Beltrame M.H.
      • Fan S.
      • Bowman S.L.
      • et al.
      Loci associated with skin pigmentation identified in African populations.
      ,
      • Wasmeier C.
      • Romao M.
      • Plowright L.
      • Bennett D.C.
      • Raposo G.
      • Seabra M.C.
      Rab38 an Rab32 control post-Golgi trafficking of melanogenic enzymes.
      ). Moreover, the localization of proteins of interest, detected by immunofluorescence or from fluorescent protein conjugates, can be compared with pigment granules by fluorescence or bright field microscopy (see section on Analyzing Melanosomes below). Light microscopy cannot detect lightly pigmented melanosomes or distinguish between eumelanin and pheomelanin. Note that differential interference contrast and phase contrast microscopy are not recommended to detect melanin because they lend contrast to other subcellular structures, which can then be mistaken for pigment granules.

      Melanin quantification by spectrophotometry (absorption spectroscopy)

      This procedure measures light absorption (wavelength between 400 and 500 nm) by melanin in detergent-free cell lysates using a spectrophotometer (
      • Hu D.N.
      Methodology for evaluation of melanin content and production of pigment cells in vitro.
      ) (Figure 1d). This approach can also be applied to quantify melanin either in skin or 3-dimensional (3D) skin equivalents using a modified lysate preparation (
      • Lo Cicero A.
      • Delevoye C.
      • Gilles-Marsens F.
      • Loew D.
      • Dingli F.
      • Guéré C.
      • et al.
      Exosomes released by keratinocytes modulate melanocyte pigmentation.
      ) or secreted into the culture supernatant and recovered by centrifugation. For a quantitative comparison of melanin content among pigment-containing samples, the values can be normalized to lysates or supernatant fractions from a non-pigmented cell (
      • Delevoye C.
      • Hurbain I.
      • Tenza D.
      • Sibarita J.B.
      • Uzan-Gafsou S.
      • Ohno H.
      • et al.
      AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.
      ). The total melanin content can be calculated by comparison with a standard curve generated with known concentrations of synthetic melanin. This method does not distinguish between pheomelanin and eumelanin because of their similar absorption spectra (
      • Ito S.
      • Fujita K.
      Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography.
      ).

      Melanin quantification by other spectroscopy methods

      Melanin does not fluoresce intrinsically, but the fluorescence from melanin oxidized by hydrogen peroxide in strong alkaline solutions can be quantified by fluorescence spectroscopy. Although this method does not distinguish between different types of melanins, it correlates well with the melanin content (
      • Rosenthal M.H.
      • Kreider J.W.
      • Shiman R.
      Quantitative assay of melanin in melanoma cells in culture and in tumors.
      ) and is amenable to complex biological structures, such as zebrafish embryos or human hair (
      • Fernandes B.
      • Matamá T.
      • Guimarães D.
      • Gomes A.
      • Cavaco-Paulo A.
      Fluorescent quantification of melanin.
      ). In contrast, electron paramagnetic resonance (EPR) spectroscopy discriminates between eumelanins and pheomelanins (
      • Sealy R.C.
      • Hyde J.S.
      • Felix C.C.
      • Menon I.A.
      • Prota G.
      Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy.
      ). EPR spectroscopy detects different electron spin signatures from stable free semiquinone-type radicals within the different melanin types, which accurately correlate with pigment concentration (
      • Godechal Q.
      • Ghanem G.E.
      • Cook M.G.
      • Gallez B.
      Electron paramagnetic resonance spectrometry and imaging in melanomas: comparison between pigmented and nonpigmented human malignant melanomas.
      ). EPR analyses can measure eumelanin and pheomelanin ratios even from biological samples such as histological sections (
      • Sealy R.C.
      • Hyde J.S.
      • Felix C.C.
      • Menon I.A.
      • Prota G.
      Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy.
      ) but require specialized expertise.

      High performance liquid chromatography

      The best quantitative method to analyze eumelanin and pheomelanin is high performance liquid chromatography (HPLC). Based on the detection of melanin degradation products (
      • Ito S.
      • Fujita K.
      Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography.
      ), HPLC does not require melanin isolation from tissues or cells and can be applied to essentially any biological sample. Although highly quantitative, HPLC requires costly equipment and very special expertise.

      Analyzing Melanosomes

      Melanosomes within melanocytes mature through four distinct stages defined by their ultrastructure (Figure 2a) (
      • Seiji M.
      • Fitzpatrick T.B.
      • Simpson R.T.
      • Birbeck M.S.C.
      Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes.
      ). The early stages lack pigment but harbor irregular amyloid fibrils (stage I) that accumulate and assemble into regularly spaced sheets (stage II). The progressive accumulation of melanin on the sheets marks stage III and ultimately stage IV in which the underlying sheets are masked (Figure 2a). The morphological progression correlates with protein content (
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      ), with early stages marked by the amyloid protein PMEL on the fibrils and/or sheets and later stages enriched in melanogenic enzymes (TYR and the TYR-related proteins TYRP1 and dopachrome tautomerase) and transporters (e.g., OCA2, ATP7A, TPC2) (reviewed by
      • Sitaram A.
      • Marks M.S.
      Mechanisms of protein delivery to melanosomes in pigment cells.
      ). These contents derive by transport from the Golgi and endosomes. Thus, it is possible to identify distinct melanosome stages by their morphology using conventional electron microscopy (EM) analyses or by their protein content using biochemical approaches, fluorescence microscopy, or immunolabeling EM (IEM).
      Figure thumbnail gr2
      Figure 2Electron microscopy analyses of melanosome ultrastructure. (a) Ultrastructure of stage I-IV melanosomes. Top, a schematic of melanosome biogenesis as presented in the text. Bottom, thin section electron microscopy analysis of MNT-1 cells showing examples of each melanosome stage. Stage I has a planar clathrin coat (black arrow), intraluminal vesicles (*) and membranous tubules (white arrow). Stage II is characterized by intraluminal amyloid sheets (arrow) upon which electron dense melanin deposits in stage III (arrow), until filling the lumen of the stage IV melanosome. Bar = 200 nm. Adapted from
      • Ripoll L.
      • Figon F.
      • Delevoye C.
      Mécanismes inter- et intracellulaires contrôlant la pigmentation des mélanocytes de la peau.
      . (b, c) MNT-1 cells fixed chemically (b) or immobilized by high-pressure freezing (c) were processed for conventional electron microscopy and imaged by transmission electron microscopy. Pigmented melanosome (arrows) are detected. Note the rounder shape of melanosomes in c owing to better ultrastructural preservation. Bar = 500 nm. From
      • Delevoye C.
      • Heiligenstein X.
      • Ripoll L.
      • Gilles-Marsens F.
      • Dennis M.K.
      • Linares R.A.
      • et al.
      BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes.
      (b) and
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      (c).

      Ultrastructural characterization of melanosomes by EM

      Melanosome maturation is spatiotemporally regulated (
      • Bowman S.L.
      • Bi-Karchin J.
      • Le L.
      • Marks M.S.
      The road to lysosome-related organelles: insights into lysosome-related organelles from Hermansky-Pudlak syndrome and other rare diseases.
      ,
      • Delevoye C.
      • Marks M.S.
      • Raposo G.
      Lysosome-related organelles as functional adaptations of the endolysosomal system.
      ), and defects in the expression or delivery of melanogenic proteins can alter melanosome morphology (
      • Montoliu L.
      • Grønskov K.
      • Wei A.H.
      • Martínez-García M.
      • Fernández A.
      • Arveiler B.
      • et al.
      Increasing the complexity: new genes and new types of albinism.
      ). These changes, as well as melanosome apposition to other organelles (e.g., mitochondria or endosomes [
      • Daniele T.
      • Hurbain I.
      • Vago R.
      • Casari G.
      • Raposo G.
      • Tacchetti C.
      • et al.
      Mitochondria and melanosomes establish physical contacts modulated by Mfn2 and involved in organelle biogenesis.
      ,
      • Delevoye C.
      • Hurbain I.
      • Tenza D.
      • Sibarita J.B.
      • Uzan-Gafsou S.
      • Ohno H.
      • et al.
      AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.
      ]) or remodeling of their limiting membranes (
      • Delevoye C.
      • Heiligenstein X.
      • Ripoll L.
      • Gilles-Marsens F.
      • Dennis M.K.
      • Linares R.A.
      • et al.
      BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ) are best detected by conventional transmission EM (TEM). Although it requires specialized equipment and expertise (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ), TEM provides the resolution to accurately and quantitatively assess melanosome stages (Figure 2b, c). Samples for conventional TEM are often chemically fixed with aldehydes (Figure 2b), which can introduce ultrastructural artifacts by altering the melanosome size and shape and by disrupting tubular membranes (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ). To better preserve melanosomes and related structures close to their native state, melanocytes can instead be immobilized using high-pressure freezing (HPF) followed by freeze substitution (Figure 2c) (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ). HPF preservation reveals otherwise unappreciated details, such as membrane remodeling at the melanosome membrane (
      • Delevoye C.
      • Hurbain I.
      • Tenza D.
      • Sibarita J.B.
      • Uzan-Gafsou S.
      • Ohno H.
      • et al.
      AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ) or individual intraluminal PMEL fibrils (
      • Hurbain I.
      • Geerts W.J.C.
      • Boudier T.
      • Marco S.
      • Verkleij A.J.
      • Marks M.S.
      • et al.
      Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets.
      ). HPF requires complex instrumentation that may not be available to all EM users.
      While conventional TEM provides a 2-dimensional (2D) ultrastructural view of the melanocyte interior, electron tomography (ET) allows for the reconstruction of a 3D model of melanosomes and associated structures (
      • Delevoye C.
      • Hurbain I.
      • Tenza D.
      • Sibarita J.B.
      • Uzan-Gafsou S.
      • Ohno H.
      • et al.
      AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.
      ,
      • Hurbain I.
      • Geerts W.J.C.
      • Boudier T.
      • Marco S.
      • Verkleij A.J.
      • Marks M.S.
      • et al.
      Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets.
      ). A specific area of a thick EM section (250–350 nm) is imaged at incremental angles, and the images are aligned using embedded gold particles as fiduciary marks. ET is preferentially combined with HPF and compatible with IEM (
      • Hess M.W.
      • Vogel G.F.
      • Yordanov T.E.
      • Witting B.
      • Gutleben K.
      • Ebner H.L.
      • et al.
      Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.
      ). An alternative and rapidly advancing (but lower resolution) 3D approach is focused ion beam scanning EM, in which a sample is repeatedly imaged on a scanning electron microscope after sequentially removing thin surface layers with a focused ion beam (
      • Titze B.
      • Genoud C.
      Volume scanning electron microscopy for imaging biological ultrastructure.
      ). 3D-EM approaches are costly, time-consuming, and require very specialized expertise.

      Melanosome analyses based on protein content

      Melanosome stages can be assessed by their protein content using antibodies or expressed fusion proteins in biochemical or imaging analyses. Analyses of the distribution of these proteins in melanocytes in which melanosome biogenesis is disrupted, either by natural mutations or by experimental manipulation, can reveal the mechanisms by which these proteins are delivered to melanosomes.

      Immunofluorescence microscopy of fixed cells

      Immunofluorescence microscopy (IFM) uses antibodies to reveal the steady-state subcellular distribution of proteins to melanosomes or other organelles in fixed melanocytes. An altered cellular distribution of a protein in melanocytes from disease states, such as HPS and GS, relative to normal melanocytes can enlighten the trafficking pathways by which that protein is delivered to melanosomes. The degree of overlap of a given protein with a pigment detected by bright field microscopy can define whether or not that protein localizes to mature (mainly stage IV) melanosomes, and localization to other compartments can be identified by overlap with markers detected by IFM. Frequently used melanosome markers include PMEL (stage II melanosomes) and the melanogenic enzymes TYR and TYRP1 (stage III and IV melanosomes), but others can be used for specific situations (Supplementary Table S2). An example showing TYRP1 overlap with pigment granules but not with PMEL or the lysosomal marker LAMP2 is shown in Figure 3a. Although this technique is subject to many potential pitfalls (Box 2), it is relatively simple to do and many protocols are available (eg, the protocol by
      • Donaldson J.G.
      Immunofluorescence staining.
      ).
      Figure thumbnail gr3
      Figure 3Analyses of melanosome content. (a) IFM and bright field analysis of immortalized mouse melan-Ink4a melanocytes labeled with antibodies to the mature melanosomal enzyme TYRP1 (green, with TA99 antibody) and either the lysosomal membrane protein LAMP2 (top, with GL2A7 antibody) or the early stage melanosomal protein PMEL (bottom, with HMB45 antibody). Melanin detected by bright field microscopy is pseudocolored red in the left and right panels. Insets show the original magnification ×5 of the boxed regions, emphasizing the "donut"-like structure of TYRP1 surrounding melanin. Bar = 10 μm. (b) IEM on MNT-1 cells using antibodies to PMEL (HMB50) and TYRP1 (TA99) and Protein A conjugated to 5, 10, or 15 nm gold particles. PMEL labeling (small gold particles) is associated mainly with unpigmented stage II melanosomes, and TYRP1 (large gold particles) labels melanin-containing stage IV melanosomes. Bar = 100 nm. Modified from
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      . (c) Lysates of melanosome-enriched fractions isolated from MNT-1 cells treated with siRNA control (siCtrl) or siRNA to Myosin VI or WASH1 were analyzed by immunoblotting for TYRP1 (with H-90 antibody) and the melanosome-associated SNARE VAMP7 (top). Data collected from several experiments were quantified and expressed as protein expression level normalized to the siRNA control (bottom). From
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      . (d) Melanosome-enriched fraction from normal human epidermal melanocytes (NHEM) or MNT-1 cells expressing GFP (left) or GFP-RAB6 (right) were deposited on EM grids and subjected to IEM using antibodies directed against RAB6 or GFP and 10 or 15 nm gold particles coupled to Protein A. Pigmented melanosomes labeled by gold particles (arrows) are shown. Bar = 500 nm. From
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      . EM, electron microscopy; GFP, green fluorescent protein; IEM, immunolabeling electron microscopy; IFM, immunofluorescence microscopy; siRNA, small inhibitory RNA.
      Potential pitfalls to avoid in pigment cells analyzed by immunofluorescence microscopy
      (i) Immunofluorescence microscopy measures steady state localization and represents the entire distribution of the epitope recognized by the antibody. Thus, there is no such thing as 100% localization to a given compartment. For example, melanosomal proteins traverse the biosynthetic and endosomal pathways (
      • Bowman S.L.
      • Bi-Karchin J.
      • Le L.
      • Marks M.S.
      The road to lysosome-related organelles: insights into lysosome-related organelles from Hermansky-Pudlak syndrome and other rare diseases.
      ,
      • Delevoye C.
      • Marks M.S.
      • Raposo G.
      Lysosome-related organelles as functional adaptations of the endolysosomal system.
      ), and thus, a fraction of these proteins are always in such compartments en route. Additionally, a protein may be subjected to posttranslational modifications or protein: protein interactions that prevent the detection of its total pool by a given antibody. For example, different antibodies to various regions of PMEL yield distinct labeling patterns in melanocytes (Supplementary Table S2) (
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      ). (ii) The expression level of a transgene product (e.g., TYR, TYRP1) (
      • Calvo P.A.
      • Frank D.W.
      • Bieler B.M.
      • Berson J.F.
      • Marks M.S.
      A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery.
      ,
      • Setty S.R.G.
      • Tenza D.
      • Truschel S.T.
      • Chou E.
      • Sviderskaya E.V.
      • Theos A.C.
      • et al.
      BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles.
      ) can alter its localization and detection. (iii) Melanosomes are ∼ 300–500 nm in length, close to the resolution limit of light microscopy. The resolution of the microscope objective and pixel size of the camera must be considered. The fluorescence imaging of melanosomes can be improved by image deconvolution of sequential z-plane images from a conventional fluorescence microscopy or by a super-resolution technique, such as structural illumination microscopy (
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ). Fluorescence extinction approaches to super-resolution such as photoactivation localization microscopy or stochastic optical reconstruction microscopy cannot be used with pigmented cells because melanin emits substantial fluorescence and heats up using these techniques.

      Live cell imaging

      The imaging of live pigment cells allows for the visualization of melanosome movement or the detection of membrane dynamics during melanosome biogenesis. Cells can be visualized with standard light (for melanin granules) or fluorescent light (for expressed fluorescent protein conjugates). Analyses can be continuous for a short time period to detect rapid movements or membrane dynamics during cargo transport or at intervals over long time frames to detect slow processes such as melanin transfer.

      Bright field analysis by wide field microscopy

      The intracellular dynamics of mature melanosomes can be monitored by live bright field imaging to study the mechanisms regulating melanosome motility (
      • Wu X.
      • Bowers B.
      • Rao K.
      • Wei Q.
      • Hammer 3rd, J.A.
      Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo.
      ). While imaging can be done without fear of phototoxicity, it is limited to the analysis of highly pigmented melanosomes.

      Fluorescence microscopy

      Fluorescent protein conjugates to various melanosomal proteins, expressed in melanocytes by transfection or recombinant virus transduction, have been used to visualize melanosome motility and membrane dynamics to and from melanosomes. The coexpression of fluorescent markers of other compartments, the concomitant visualization of pigment granules by bright field microscopy, or the identification of melanosomes by their characteristic "donut" structure (in which fluorescence in the melanosome interior is quenched by melanin) can reveal dynamic and transient interactions between melanosomes and endosomal transport intermediates (
      • Delevoye C.
      • Hurbain I.
      • Tenza D.
      • Sibarita J.B.
      • Uzan-Gafsou S.
      • Ohno H.
      • et al.
      AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.
      ,
      • Dennis M.K.
      • Mantegazza A.R.
      • Snir O.L.
      • Tenza D.
      • Acosta-Ruiz A.
      • Délevoye C.
      • et al.
      BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.
      ), lysosomes (
      • Bissig C.
      • Croisé P.
      • Heiligenstein X.
      • Hurbain I.
      • Lenk G.M.
      • Kaufman E.
      • et al.
      The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.
      ), or Golgi-derived vesicles (
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      ). Such interactions are best captured by spinning disk confocal microscopy. Alternatively, to visualize the dynamics of melanosomes associated with the melanocyte cortical cytoskeleton and their transfer to keratinocytes (
      • Bruder J.M.
      • Pfeiffer Z.A.
      • Ciriello J.M.
      • Horrigan D.M.
      • Wicks N.L.
      • Flaherty B.
      • et al.
      Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.
      ), total internal reflection fluorescence microscopy is ideal.

      Quantitative imaging analysis

      Proper interpretation of IFM and live cell imaging requires quantitative analysis, such as the degree to which a test protein is spatially associated with or localized to pigment granules (for an excellent review, see
      • Dunn K.W.
      • Kamocka M.M.
      • McDonald J.H.
      A practical guide to evaluating colocalization in biological microscopy.
      ). However, keep in mind that the diffraction limit for light microscopy (∼200–250 nm) and the congregation of melanosomes and other organelles in the perinuclear region of melanocytes will make some organelles erroneously appear to overlap; to address the latter, we often limit our analyses to the melanocyte periphery where organelles are more spatially separated. Colocalization can be quantified using either of two predominant theoretical approaches. Pixel-intensity-based correlation analysis (e.g., Pearson’s correlation or Mander’s overlap coefficients) measure how the signal intensity for two proteins correlate at each pixel. It is not an absolute measure of the degree of overlap, and accuracy requires similar maximal intensities for both proteins. Alternatively, object-based overlap considers the size and shape of the object, provides a more multidimensional analysis of colocalization, and correlates better with IEM analyses (
      • Dennis M.K.
      • Mantegazza A.R.
      • Snir O.L.
      • Tenza D.
      • Acosta-Ruiz A.
      • Délevoye C.
      • et al.
      BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.
      ,
      • Setty S.R.G.
      • Tenza D.
      • Truschel S.T.
      • Chou E.
      • Sviderskaya E.V.
      • Theos A.C.
      • et al.
      BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles.
      ) but requires subjective thresholding to define objects. Another measure is the distance between or coincidence of the centers of two test objects (
      • Lachmanovich E.
      • Shvartsman D.E.
      • Malka Y.
      • Botvin C.
      • Henis Y.I.
      • Weiss A.M.
      Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.
      ). This requires a centroid to be defined for each object, which may be challenging for non-isotropic structures like melanosomes (e.g., TYRP1 is detected in uneven rings around pigment granules, and PMEL does not uniformly label the melanosome lumen) (Figure 3a) and/or for examining melanosome interactions with tubulo-vesicular structures (
      • Dennis M.K.
      • Mantegazza A.R.
      • Snir O.L.
      • Tenza D.
      • Acosta-Ruiz A.
      • Délevoye C.
      • et al.
      BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.
      ). Box 3 presents additional approaches for quantifying melanosome imaging.
      Specific live cell quantitative approaches for melanosome biology
      There are many ways to quantify melanosome motility (
      • Hume A.N.
      • Wilson M.S.
      • Ushakov D.S.
      • Ferenczi M.A.
      • Seabra M.C.
      Semi-automated analysis of organelle movement and membrane content: understanding Rab-motor complex transport function.
      ,
      • Oberhofer A.
      • Spieler P.
      • Rosenfeld Y.
      • Stepp W.L.
      • Cleetus A.
      • Hume A.N.
      • et al.
      Myosin Va's adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro.
      ,
      • Palmisano I.
      • Bagnato P.
      • Palmigiano A.
      • Innamorati G.
      • Rotondo G.
      • Altimare D.
      • et al.
      The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells.
      ,
      • Rogers S.L.
      Gelfand VI. Myosin cooperates with microtubule motors during organelle transport in melanophores.
      ,
      • Wu X.
      • Bowers B.
      • Rao K.
      • Wei Q.
      • Hammer 3rd, J.A.
      Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo.
      ) and distribution to the perinuclear or peripheral regions (
      • Caviston J.P.
      • Zajac A.L.
      • Tokito M.
      • Holzbaur E.L.
      Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes.
      ,
      • Reilein A.R.
      • Tint I.S.
      • Peunova N.I.
      • Enikolopov G.N.
      • Gelfand V.I.
      Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).
      ). We have quantified the dynamics of melanosome-bound tubular transport intermediates during melanosome biogenesis using ImageJ (NIH, Bethesda, MD) and freeware called Icy (http://icy.bioimageanalysis.org/) (
      • Delevoye C.
      • Heiligenstein X.
      • Ripoll L.
      • Gilles-Marsens F.
      • Dennis M.K.
      • Linares R.A.
      • et al.
      BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes.
      ,
      • Dennis M.K.
      • Delevoye C.
      • Acosta-Ruiz A.
      • Hurbain I.
      • Romao M.
      • Hesketh G.G.
      • et al.
      BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.
      ,
      • Dennis M.K.
      • Mantegazza A.R.
      • Snir O.L.
      • Tenza D.
      • Acosta-Ruiz A.
      • Délevoye C.
      • et al.
      BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ). These and other behaviors may require custom-designed analyses. Note, a simple method to control for correct measurement using any quantification approach is to repeat the quantification on the same set of images but after rotating one of the image channels by 30–180° (
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ).

      Immunolabeling electron microscopy

      Based on antibody detection in thin sections using electron dense gold particles and on a different sample preparation than for conventional EM (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ), IEM is the best approach to assign an ultrastructural localization to an endogenous or expressed epitope-tagged protein. IEM in melanocytic cells or human skin biopsies has been invaluable to define protein distributions to distinct melanosome stages (Figure 3b) (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ,
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      ) or to pigment granules in keratinocytes (
      • Hurbain I.
      • Romao M.
      • Sextius P.
      • Bourreau E.
      • Marchal C.
      • Bernerd F.
      • et al.
      Melanosome distribution in keratinocytes in different skin types: melanosome clusters are not degradative organelles.
      ). An alternative state-of-the-art technique is correlative light to EM (CLEM) (
      • Hurbain I.
      • Romao M.
      • Bergam P.
      • Heiligenstein X.
      • Raposo G.
      Analyzing lysosome-related organelles by electron microscopy.
      ), in which an organelle of interest is first identified by fluorescence microscopy and then analyzed by TEM, thus associating a fluorescent spot to an ultrastructure. CLEM can be coupled to HPF (
      • Delevoye C.
      • Heiligenstein X.
      • Ripoll L.
      • Gilles-Marsens F.
      • Dennis M.K.
      • Linares R.A.
      • et al.
      BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes.
      ) and/or associated with 3D-ET (
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ) and has revealed unappreciated membrane trafficking steps during melanosome biogenesis and maturation that are targeted in HPS (
      • Delevoye C.
      • Heiligenstein X.
      • Ripoll L.
      • Gilles-Marsens F.
      • Dennis M.K.
      • Linares R.A.
      • et al.
      BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes.
      ).

      Biochemical characterization of melanosomes by subcellular fractionation

      Melanosomes can also be identified biochemically by subcellular fractionation. Detergent-free melanocyte homogenates are fractionated by centrifugation on a sucrose density gradient; pigmented melanosomes are denser than other membranous organelles, and thus migrate further (
      • Watabe H.
      • Kushimoto T.
      • Valencia J.C.
      • Hearing V.J.
      Isolation of melanosomes.
      ). Protein content in melanosome fractions from different samples can then be compared by immunoblotting (Figure 3c) or by proteomics analyses (
      • Chi A.
      • Valencia J.C.
      • Hu Z.Z.
      • Watabe H.
      • Yamaguchi H.
      • Mangini N.J.
      • et al.
      Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.
      ). Although such subcellular fractions are enriched for melanosomes, they are contaminated with other organelles, and thus caution should be exercised in interpreting their contents. Organelle contamination should be assessed by immunoblotting isolated fractions with antibodies to components of these organelles and may be complemented by ultrastructural inspection of the melanosome-enriched fraction by conventional TEM or IEM (Figure 3d) (
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      ).

      Analyzing Melanin Transfer and/or Uptake

      Melanin transfer to keratinocytes requires the peripheral positioning and immobilization of pigmented melanosomes near the melanocyte plasma membrane. Proposed transfer modes include (i) melanosome exchange by the fusion of melanocyte and keratinocyte plasma membranes or phagocytosis by keratinocytes either of (ii) melanosome-containing melanocyte dendrites, (iii) melanosome-containing membrane fragments shed from melanocytes, or (iv) the luminal content of melanosomes (melanocores) exocytosed by melanocytes (
      • Wu X.
      • Hammer J.A.
      Melanosome transfer: it is best to give and receive.
      ). Regardless of the model, genetic defects in melanosome positioning (
      • Bowman S.L.
      • Bi-Karchin J.
      • Le L.
      • Marks M.S.
      The road to lysosome-related organelles: insights into lysosome-related organelles from Hermansky-Pudlak syndrome and other rare diseases.
      ) or maturation (
      • Bultema J.J.
      • Boyle J.A.
      • Malenke P.B.
      • Martin F.E.
      • Dell'Angelica E.C.
      • Cheney R.E.
      • et al.
      Myosin vc interacts with rab32 and rab38 proteins and works in the biogenesis and secretion of melanosomes.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ) can affect melanin transfer.

      In vitro coculture system

      Most transfer assays described to date rely on a 2D in vitro coculture system of primary or immortalized melanocytes and keratinocytes (Supplementary Table S3). Because keratinocytes secrete factors that promote melanocyte pigmentation and dendricity and potentiate melanin exchange, donor-matched pairs of primary human epidermal melanocytes (HEMs) and human epidermal keratinocytes (HEKs) are optimal. The derivation of HEM and HEK from human pluripotent stem cells (
      • Nissan X.
      • Larribere L.
      • Saidani M.
      • Hurbain I.
      • Delevoye C.
      • Feteira J.
      • et al.
      Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.
      ) might provide an ‘infinite’ source of paired epidermal cells that may be used in the future to generate epidermal cells bearing patient mutations to study the pathophysiology of pigmentary disorders.

      Assays for monitoring melanin transfer

      IFM of fixed cells

      Pigment transfer in a 2D coculture model can be assessed by IFM based on the specific labeling of the keratinocytes and the transferred melanin. Keratinocytes can be detected by antibodies to components not expressed in melanocytes (e.g., cytokeratins [
      • Kasraee B.
      • Pataky M.
      • Nikolic D.S.
      • Carraux P.
      • Piguet V.
      • Salomon D.
      • et al.
      A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.
      ] or epidermal growth factor receptor [
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ]), whereas the transferred melanin and/or melanosomes can be detected by bright field microscopy for the pigment or by antibodies specific for melanosomal proteins (e.g., PMEL, TYR, and TYRP1). Note that the labeling for melanosomal membrane proteins (TYRP1 and to some degree, TYR) preferentially reflects the transfer of intact melanosomes and not of melanocores, whereas PMEL antibodies mainly detect lightly pigmented melanosomes and/or melanocores (Figure 4a, c, d, and e, arrowheads) because PMEL epitopes on the fibrils in the melanosome lumen are buried upon melanin deposition (
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      ). A comprehensive analysis would combine bright field imaging of the pigment with melanosome staining for PMEL. Transferred melanin or PMEL can be quantified relative to a negative control in which keratinocytes are grown without melanocytes (see
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      for further details).
      Figure thumbnail gr4
      Figure 4Analyses of melanosome transfer. (a) HEM and/or HEK coculture processed for IFM using antibodies specific to HEKs (epidermal growth factor receptor, green) or melanosomes (HMB45 anti-PMEL, red). The contours of HEKs were drawn manually using ImageJ (white lines) to detect transferred melanosomes and/or melanocores (HMB45-positive structures, arrowheads). Note that HEMs are heavily stained by HMB45 antibodies owing to the abundance of PMEL inside melanosomes, while transferred melanosomes and/or melanocores appear as small and dim fluorescent puncta or pigment granules in keratinocytes (arrowheads). Bar = 10 μm. From
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      . (b) Conditioned medium of MNT-1 culture deposited on top of a porous filter-containing column (3,000 kDa, left). After centrifugation, the medium passed through the filter (middle), but the black melanocore-enriched fraction was retained on top of the filter (right). (c-e) Melanocores purified from the conditioned medium of MNT-1 cells were fixed directly (c) or incubated with HEKs for (d) 10 minutes or (e) 8 hours before fixation, labeling for PMEL with HMB45, and analysis by IFM (left panels) and bright field microscopy (middle panels). Merged images are shown at right, and cell contours are drawn on the IFM images at left in (d) and (e). Arrowheads and arrows indicate melanin granules that do or do not overlap with HMB45 labeling, respectively. Note that structures in the cell periphery or exterior at 10 min accumulate into a perinuclear “cap” by 8 hours. Bar = 10 μm. HEM, human epidermal melanocyte; HEK, human epidermal keratinocyte; IFM, immunofluorescence microscopy; MCs, melanocores.

      Live cell imaging

      Live cell fluorescence imaging can be used to track the transfer of intact melanosomes to keratinocytes (identified in corresponding phase contrast images) from cocultured melanocytes expressing fluorescent protein fusions to melanosomal proteins (
      • Bruder J.M.
      • Pfeiffer Z.A.
      • Ciriello J.M.
      • Horrigan D.M.
      • Wicks N.L.
      • Flaherty B.
      • et al.
      Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.
      ).
      • Wu X.S.
      • Masedunskas A.
      • Weigert R.
      • Copeland N.G.
      • Jenkins N.A.
      • Hammer J.A.
      Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes.
      similarly used time-lapse imaging to visualize the shedding mode of melanin transfer by labeling the plasma membranes of melanocytes and keratinocytes with different fluorescent probes. These approaches do not detect melanocore exchange, which may be the primary mode of melanin transfer in human epidermis (
      • Tarafder A.K.
      • Bolasco G.
      • Correia M.S.
      • Pereira F.J.C.
      • Iannone L.
      • Hume A.N.
      • et al.
      Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.
      ), and are prone to UV-induced photodamage and transfer underestimates because of the non-synchronous manner of exchange at multiple planes of focus. To our knowledge, fluorescent labeling of melanin using chemical approaches has not been successful, but the recent design of fluorescent genetically encoded probes that could label melanosomes and melanocores (
      • Ishida M.
      • Marubashi S.
      • Fukuda M.
      M-INK, a novel tool for visualizing melanosomes and melanocores.
      ) might facilitate pigment transfer analyses.

      Alternative methods

      Melanosome transfer in 2D cocultures can be assessed by flow cytometry analysis of keratinocytes labeled for melanosome-associated markers (e.g., TYR, TYRP1 [
      • Hu Q.M.
      • Yi W.J.
      • Su M.Y.
      • Jiang S.
      • Xu S.Z.
      • Lei T.C.
      Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.
      ,
      • Lin H.C.
      • Shieh B.H.
      • Lu M.H.
      • Chen J.Y.
      • Chang L.T.
      • Chao C.F.
      A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
      ]) or by conventional EM to detect pigment in keratinocytes (
      • Kasraee B.
      • Pataky M.
      • Nikolic D.S.
      • Carraux P.
      • Piguet V.
      • Salomon D.
      • et al.
      A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.
      ). Several studies have exploited 3D culture systems, including the seeding of epidermal cells on either side of a porous filter (
      • Kasraee B.
      • Pataky M.
      • Nikolic D.S.
      • Carraux P.
      • Piguet V.
      • Salomon D.
      • et al.
      A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.
      ) or generating 3D-reconstructed epidermis for analyses by immunohistochemistry and/or EM (
      • Lo Cicero A.
      • Delevoye C.
      • Gilles-Marsens F.
      • Loew D.
      • Dingli F.
      • Guéré C.
      • et al.
      Exosomes released by keratinocytes modulate melanocyte pigmentation.
      ). While these systems may be more physiological, they are also more complicated to analyze.

      Monitoring the uptake of biochemically isolated melanocores

      In the human epidermis, a main mode of melanin transfer results from the fusion of melanosomes with the melanocyte plasma membrane, releasing their intraluminal melanin content, or melanocores, for subsequent phagocytosis by keratinocytes (
      • Correia M.S.
      • Moreiras H.
      • Pereira F.J.C.
      • Neto M.V.
      • Festas T.C.
      • Tarafder A.K.
      • et al.
      Melanin transferred to keratinocytes resides in nondegradative endocytic compartments.
      ,
      • Tarafder A.K.
      • Bolasco G.
      • Correia M.S.
      • Pereira F.J.C.
      • Iannone L.
      • Hume A.N.
      • et al.
      Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.
      ). Melanocores can thus be used in vitro as physiologically relevant pigment sources to study pigment transfer and keratinocyte pigmentation. Some melanoma cells (e.g., MNT-1) (Supplementary Table S1) constitutively secrete melanin into the conditioned medium, from which melanocores can be collected by differential centrifugation (Figure 4b) (
      • Correia M.S.
      • Moreiras H.
      • Pereira F.J.C.
      • Neto M.V.
      • Festas T.C.
      • Tarafder A.K.
      • et al.
      Melanin transferred to keratinocytes resides in nondegradative endocytic compartments.
      ) and quantified by absorption spectrophotometry (see above). Note that these preparations can be contaminated with other cellular materials; thus, the quality of the melanocore-enriched fraction should be properly characterized by immunoblotting and EM. Melanocore fractions can be stored frozen and/or incubated with keratinocytes and tracked over time (hours to days) by bright field microscopy (
      • Correia M.S.
      • Moreiras H.
      • Pereira F.J.C.
      • Neto M.V.
      • Festas T.C.
      • Tarafder A.K.
      • et al.
      Melanin transferred to keratinocytes resides in nondegradative endocytic compartments.
      ) and/or by immunolabeling for PMEL and fluorescence microscopy (Figure 4c–e).

      Final Remarks

      Pigmentation of the skin and hair is easily visible but has not yet revealed the biological basis underlying its full complexity, and many fundamental questions regarding the cell biology of pigmentation remain underexplored. The following general questions must be addressed using a multi-disciplinary approach, including some of the methods described herein.
      • Melanosome biogenesis: While much has been learned about melanosome biogenesis over the past two decades, several major questions remain unsolved. How are melanosomes within melanocytes segregated from the endolysosomal system, despite sharing trafficking pathways with endolysosomes? How and why are some ubiquitously expressed components within melanocytes, such as HPS proteins, specifically adapted for melanosome biogenesis?
      • Melanosome maturation: While steps in the delivery of melanogenic enzymes and transporters are becoming increasingly understood, less is known about the final events in melanosome maturation prior to transfer. For example, when and how do melanocytes sense that melanosome biogenesis is complete? How are the intracellular effectors of transfer (e.g., RAB27A and RAB11B) recruited to melanosomes within melanocytes, and are all pigmented melanosomes equally capable of peripheral capture and ultimate transfer to keratinocytes?
      • Melanosome transfer: While some progress has been made in unmasking mechanisms of melanosome transfer, basic questions remain. What is the primary mode by which melanosomes are transferred to keratinocytes in vivo? Do several modes coexist, and do environmental factors influence the mode used? Is there a keratinocyte receptor for melanin or melanosomes?
      • Melanin storage in keratinocytes: What is the nature of the melanin storage organelles within keratinocytes, and how do they differ in individuals of different phototypes? Is the nature of these organelles subject to regulation by environmental cues or signaling cascades?
      • Melanocyte-keratinocyte cross-talk: Given that skin pigmentation relies on the tight interplay between two cell types, it is imperative to better define how melanocytes and keratinocytes interact and communicate to orchestrate pigment transfer at the right place and time. How do they adapt their intracellular mechanisms in response to this dialogue? Is melanocyte-keratinocyte communication altered in pigmentary disorders?

      Data availability statement

      No datasets were generated or analyzed during the current study.

      Conflict of Interest

      Part of the work by CD, RAJ and SBM was funded by a research grant from L'Oréal Research and Development. DCH, MSM and YY state no conflicts of interest to declare.

      Multiple Choice Questions

      • 1.
        Which of these imaging methods allows one to distinguish ALL melanosome stages at the same time?
        • A.
          Immunofluorescence microscopy
        • B.
          Transmission electron microscopy
        • C.
          Live cell imaging
        • D.
          Bright field microscopy
      • 2.
        Which of the following methods allows a quantitative measurement of both eumelanin AND pheomelanin?
        • A.
          Electron paramagnetic resonance spectrometry
        • B.
          Absorption spectroscopy
        • C.
          High performance liquid chromatography
        • D.
          Light microscopy
        • E.
          Both A and C
      • 3.
        What is the primary substrate for melanin synthesis?
        • A.
          Tryptophan
        • B.
          Tyrosine
        • C.
          Lysine
        • D.
          Histidine
      • 4.
        What is the melanocore?
        • A.
          Another name for a fully pigmented (Stage IV) melanosome
        • B.
          The intralumenal melanin content of a melanosome devoid of a limiting membrane
        • C.
          The degradation product of the intralumenal melanin content of a melanosome
        • D.
          The core substrate of melanin synthesis
      • 5.
        Which of these imaging approaches allows one to visualize pigmented melanosomes in TYRP1-GFP expressing melanocytes?
        • A.
          Bright field microscopy
        • B.
          Live cell imaging
        • C.
          Immunoelectron microscopy
        • D.
          Stochastic optical reconstruction microscopy (STORM)
        • E.
          A, B, and C but not D
      Note: See the online version of this article for a detailed explanation of correct answers.

      Acknowledgments

      We acknowledge funding from the United States National Institutes of Health’s National Eye Institute ( 5R01 EY015625 ) and National Institute of Arthritis, Musculoskeletal and Skin Diseases ( 5R01 AR071382 and 5R01 AR048155 ); Fondation pour la Recherche Médicale (Equipe FRM : EQU201903007827 ); L’Oréal; Institut Curie ; Institut National de la Santé et de la Recherche Médicale (INSERM); and the Centre National de la Recherche Scientifique (CNRS), France. CD is supported by program grant ANR-17-CE11-0029-03 from the Agence Nationale de la Recherche (ANR ‘Myoactions’). SBM has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 666003.

      Author Contributions

      Conceptualization: MSM, CD; Funding Acquisition: MSM, CD; Investigation: SBM, YZ, RAJ; Methodology: SBM, YZ, RAJ, DCH, MSM, CD; Project Administration: MSM, CD; Supervision: MSM, CD; Visualization: SBM, YZ, RAJ, DCH, MSM, CD; Writing – Original Draft Preparation: SBM, YZ, RAJ, DCH, MSM, CD; Writing – Review and Editing: SBM, YZ, RAJ, DCH, MSM and CD

      Detailed Answers

      • 1.
        Which of these imaging methods allows one to distinguish ALL melanosome stages at the same time?
      • Answer: B. Transmission electron microscopy
      • Four melanosome stages coexist in pigment cells. They are defined based on their ultrastructural morphologies, which can only be detected by electron microscopy. Immunofluorescence microscopy or live cell imaging can highlight stage combinations (e.g., anti-PMEL for stages I and II, anti-TYRP1 for stages III and IV) but cannot distinguish between all of them.
      • 2.
        Which of the following methods allows a quantitative measurement of both eumelanin AND pheomelanin?
      • Answer: E. Both A and C
      • Eumelanin and pheomelanin are chemically distinct, but they cannot be distinguished by conventional spectroscopy owing to their very similar absorption spectra or by light microscopy owing to the partial detection of lightly pigmented melanosomes. In contrast, the electron paramagnetic resonance spectrometry signatures (free radicals) of eumelanin and pheomelanin are distinct, and high performance liquid chromatography can distinguish specific by-products of the degradation of these two different pigments.
      • 3.
        What is the primary substrate for melanin synthesis?
      • Answer: B. Tyrosine
      • The black and/or brown eumelanins and red and/or yellow pheomelanins are both composed of polymerized products of sequential redox reactions in which tyrosine is the initial substrate.
      • 4.
        What is the melanocore?
      • Answer: B. The intralumenal melanin content of a melanosome devoid of a limiting membrane
      • The term “melanocore” refers to the naked pigment devoid of the melanosome limiting membrane. It is primarily used to refer to the melanin form found in the extracellular space in human epidermis or pigment cell culture as a result of the exocytosis of melanosome contents by melanocytes.
      • 5.
        Which of these imaging approaches allows one to visualize pigmented melanosomes in TYRP1-GFP expressing melanocytes?
      • Answer: E. A, B, and C but not D
      • Light- or electron-absorbing melanin is visualized by bright field microscopy or electron microscopy. Melanin visualized in melanocytes by bright field microscopy reflects highly pigmented (likely stage IV) melanosomes. Stage III and/or IV melanosomes express TYRP1 on the limiting membrane. Endogenous TYRP1 can be detected by immunofluorescence microscopy or immunoelectron microscopy, and an expressed fusion protein of TYRP1 fused to the green fluorescent protein (TYRP1-GFP) can be detected by either live cell imaging or by immunofluorescence or immuno-electron microscopy with anti-GFP antibodies. Immuno-electron microscopy can unambiguously assign TYRP1-GFP localization to an ultrastructure, while combining bright field microscopy with fluorescence microscopy allows visualization of pigment granules surrounded by TYRP1-GFP. Finally, STORM (or the related photoactivation localization microscopy) cannot be used with pigmented cells because melanin emits substantial fluorescence and heats up during the acquisition.
      Supplementary Table S1Cell Culture Models of Epidermal Pigment Cells and Keratinocytes
      Cell TypeSource and/or BackgroundAdvantagesLimitationsNotes
      Mouse pigment cells
       Primary mouse melanocytesIsolated and cultured from the skins of newborn (0–2 day old) miceHundreds of mouse genetic pigmentation mutants availableMust be cultured on a feeder layer of keratinocytes or a keratinocyte cell lineMay require G418 to kill off fibroblasts—melanocytes are partially resistant
      Cultured cell morphology (dendritic) mimics the in vivo cellular morphologySlow growth rate and limited culture time
      Cells are generally flat and thus useful for microscopy approachesDifficult to obtain the large number of cells needed for biochemical analysis
       Immortalized mouse melanocyte cell lines“Wild-type” mice (most on C57BL/6J background)—spontaneously immortalizedBear features of primary melanocytes; flat and dendritic morphology, dense pigmentation (if derived from pigmented mice)Loss of p16Ink4a expression impacts studies of cell proliferation, senescence, and possibly signaling pathwaysSome cell lines require growth on feeder layers of irradiated keratinocytes or keratinocyte cells lines
      Ink4a-Arf-/- mice (most on C57BL/6J background)—prone to spontaneously immortalizeCan be stably cultured indefinitely (with care)Prone to de-differentiate over long culture periodsSee holdings of the Wellcome Trust Functional Genomics Cell Bank; www.sgul.ac.uk/depts/anatomy/pages/Dot/Cell%20bank%20holdings.htm
      Can be cultured in large volumes for biochemical analysisLines derived from mice that primarily synthesize pheomelanins do not consistently maintain their phenotype during culture
      Genetically defined
      Can be used to analyze the transfer of melanin to keratinocytes
       Mouse melanoma cell linesMany—mostly sublines of B16Grow well in cultureVarying degree of melanin contentSublines of classic B16 mouse melanoma are the most widely used
      Well-pigmented melanosomes that are morphologically intactSignaling pathways in melanoma might differ from those in primary or non-transformed melanocytes
      Human pigment cells
       Primary HEMsSkin of adult donors or foreskins of neonatal donorsHEMs retain many of the morphological and signaling properties of melanocytesSlow and limited growth potentialThe “gold standard” for many studies of melanogenesis, melanocyte biology, signaling, and melanosome transfer to keratinocytes
      HEMs generate pheomelanins and eumelanins as they do in situInability to control for genetic background differences and limited knowledge of donor sources
      Medium components can be costly and complex
       Human melanocyte cell linesHermes linesExperimental reproducibility appears to be highGeneration of immortalized cells is timely, costly, and complexSeveral available from the Wellcome Trust Functional Genomics Cell Bank; www.sgul.ac.uk/depts/anatomy/pages/Dot/Cell%20bank%20holdings.htm
      Melanocytes derived from iPSCs or embryonic stem cellsKnown genetic backgroundCare must be taken in the initial characterization to ensure normal pigmentation and signaling
      Virtually unlimited growthRelatively new systems; thus, limited data available in the literature
      May represent valuable model systems of pigmentary disorders (iPSC-derived)Medium components can be costly and complex
       Melanoma cell line (human)Many from human sourcesCan be cultured in large volumes for biochemical analysesMelanomas differ in their background, pigmentation status, mutations, and differentiation stateMNT-1 will be soon available from ATCC
      MNT-1 is a well-studied, highly pigmented lineMNT-1: transcriptome, melanosome content, and morphology closely resembles primary human melanocytesMNT-1: morphology is fusiform, and thus unlike normal melanocytes
      MNT-1: secretes melanin in form of melanocores, allowing for melanin transfer and/or uptake studies
      Keratinocytes
       Primary HEKsSkin of adult donors or foreskins of neonatal donorsHEKs retain many of the properties of keratinocytes in situInability to control for genetic background differences and limited knowledge of donor sourcesA cell detachment agent such as Accutase may be preferred to trypsin, which activates PAR-1 in HEK
      Useful for melanin transfer studiesLow number of passages in culture before differentiation and/or senescenceHEK differentiation can be delayed by growing at low confluence (<70%, to limit cell contacts) in low Ca2+ culture medium, and not more than five passages
       Human HaCaT cellsTransformed keratinocyte cell line from adult human skinGrow well in cultureImmortalized cells lack some properties of primary keratinocytesWhen irradiated or treated with mitomycin C, useful as feeder layers for human melanocytes
      Useful for melanin transfer studies
       Mouse XB2 cellsMouse skinGrow well in cultureImmortalized cells lack some properties of primary keratinocytesWhen irradiated or treated with mitomycin C, useful as feeder layers for mouse melanocytes
      Useful for melanin transfer studies
      Abbreviations: HEK, human epidermal keratinocyte; HEM, human epidermal melanocyte; iPSC, induced pluripotent stem cells.
      Supplementary Table S2Reagents To Detect Melanosome Proteins and Markers of Other Organelles
      TargetAntibody
      Indicated are antibody name and source.
      Specificity
      Indicated is the species from which the antigen is derived; cross-reactivity with other species is indicated when known.
      Intracellular localization
      Intracellular localization determined by high confidence IFM or IEM analyses.
      ApplicationsAvailability
      Sources are indicated as commercial or available from a specific laboratory; specific commercial sources are not listed. Laboratories are as follows. Di Pietro lab: Santiago di Pietro, Colorado State Univ., Fort Collins, CO, USA; Galli lab: Thierry Galli, Institut de Psychiatrie et Neurosciences de Paris, Paris, France; Hearing lab: contact Julio Valencia, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; Marigo lab: Valeria Marigo, University of Modena and Reggio Emilia, Modena, Italy; Marks lab: Michael Marks, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Schiaffino lab: M. Vittoria Schiaffino, IRCCS San Rafaelle Scientific Institute, Milan, Italy; Seabra lab, Miguel Seabra, Universidade NOVA de Lisboa, Lisboa, Portugal; Tani Lab: Yoshihiko Tani, Japanese Red Cross Osaka Blood Center, Osaka, Japan; Theos lab: Alexander Theos, Georgetown University, Washington, DC, USA
      References
      Non-exhaustive list of references to earliest descriptions and/or to documentation of use in particular applications.
      Melanosomal proteins
       PMEL (stage I and/or II melanosomes)HMB45 (mMAb)Proteolytic fragments of mature PMEL from multiple speciesPrimarily stage I and/or II melanosomesIFM, IEM, WBcommercial(
      • Adema G.J.
      • de Boer A.J.
      • Vogel A.M.
      • Loenen W.A.M.
      • Figdor C.G.
      Molecular characterization of the melanocyte lineage-specific antigen gp100.
      ,
      • Berson J.F.
      • Harper D.C.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.
      ,
      • Gown A.M.
      • Vogel A.M.
      • Hoak D.
      • Gough F.
      • McNutt M.A.
      Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes.
      ,
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      ,
      • Kapur R.P.
      • Bigler S.A.
      • Skelly M.
      • Gown A.M.
      Anti-melanoma monoclonal antibody HMB45 identifies an oncofetal glycoconjugate associated with immature melanosomes.
      ,
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      )
      NKI-bteb (mMAb)Human PMELPrimarily stage I and/or II melanosomes; detectable at the plasma membraneIFM, IEM, IP, FCcommercial(
      • Adema G.J.
      • de Boer A.J.
      • Vogel A.M.
      • Loenen W.A.M.
      • Figdor C.G.
      Molecular characterization of the melanocyte lineage-specific antigen gp100.
      ,
      • Vennegoor C.
      • Hageman P.
      • van Nouhuijs H.
      • Ruiter D.J.
      • Calafat J.
      • Ringens P.J.
      • et al.
      A monoclonal antibody specific for cells of the melanocyte lineage.
      )
      HMB50 (mMAb)Human PMELPrimarily stage I and/or II melanosomes; detectable at the plasma membraneIFM, IEM, IP, FCcommercial(
      • Adema G.J.
      • de Boer A.J.
      • Vogel A.M.
      • Loenen W.A.M.
      • Figdor C.G.
      Molecular characterization of the melanocyte lineage-specific antigen gp100.
      ,
      • Berson J.F.
      • Harper D.C.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.
      ,
      • Esclamado R.M.
      • Gown A.M.
      • Vogel A.M.
      Unique proteins defined by monoclonal antibodies specific for human melanoma. Some potential clinical applications.
      ,
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      ,
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      )
      αPep13h (rAb)Human PMEL (C-terminus); cross-reacts with other speciesER, Golgi, limiting membrane of stage I melanosomes; cleaved in stage I melanosomesIFM, IEM, IP, WBHearing lab, Marks lab(
      • Berson J.F.
      • Harper D.C.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.
      ,
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      ,
      • Kobayashi T.
      • Urabe K.
      • Orlow S.J.
      • Higashi K.
      • Imokawa G.
      • Kwon B.S.
      • et al.
      The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.
      ,
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      )
      αPMEL-N (rAb)Human PMEL (N-terminus)ER, Golgi, Stage I melanosomes; detectable at plasma membrane; cleaved in melanosomesIFM, IP, WB, FCMarks lab(
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      ,
      • Theos A.C.
      • Berson J.F.
      • Theos S.C.
      • Herman K.E.
      • Harper D.C.
      • Tenza D.
      • et al.
      Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17.
      )
      αPMEL-I (rAb)Human PMEL residues 326–344 (unglycosylated)ER only—ablated by O-glycosylation in ER or cis GolgiWB, IPMarks lab(
      • Harper D.C.
      • Theos A.C.
      • Herman K.E.
      • Tenza D.
      • Raposo G.
      • Marks M.S.
      Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes.
      )
      I51 (rAb)Human PMEL residues 206–220Primarily used for immunoblotting to detect PMEL amyloid fibril coreWBMarks lab (from R. Leonhardt)(
      • Hee J.S.
      • Mitchell S.M.
      • Liu X.
      • Leonhardt R.M.
      Melanosomal formation of PMEL core amyloid is driven by aromatic residues.
      ,
      • Leonhardt R.M.
      • Vigneron N.
      • Rahner C.
      • Van den Eynde B.J.
      • Cresswell P.
      Endoplasmic reticulum (ER)-export, subcellular distribution and fibril formation by PMEL17 requires an intact N-terminal domain junction.
      )
       TYRP1 (stage III/ IV melanosomes)TA99 / Mel5 (mMAb)Human and mouse TYRP1Primarily stage III/IV melanosomes; also Golgi and endosomesIFM, IEM, IP, FCcommercial(
      • Raposo G.
      • Tenza D.
      • Murphy D.M.
      • Berson J.F.
      • Marks M.S.
      Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
      ,
      • Thomson T.M.
      • Mattes M.J.
      • Roux L.
      • Old L.J.
      • Lloyd K.O.
      Pigmentation-associated glycoprotein of human melanomas and melanocytes: definition with a mouse monoclonal antibody.
      ,
      • Vijayasaradhi S.
      • Bouchard B.
      • Houghton A.N.
      The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product.
      )
      H-90 (rAb)Human and mouse TYRP1Primarily stage III/IV melanosomesIFM, WBcommercial(
      • Setty S.R.G.
      • Tenza D.
      • Truschel S.T.
      • Chou E.
      • Sviderskaya E.V.
      • Theos A.C.
      • et al.
      BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles.
      )
      αPep1 (rAb)HumanOnly useful for immunoblottingWBHearing lab(
      • Kobayashi T.
      • Urabe K.
      • Orlow S.J.
      • Higashi K.
      • Imokawa G.
      • Kwon B.S.
      • et al.
      The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.
      )
       TYR (stage III/ IV melanosomes)αPep7h (rAb)Human TYR C-terminus; cross-reacts with mousePrimarily stage III/IV melanosomes; also Golgi, ER and endosomesIFM, IP, WBHearing lab, Marks lab(
      • Calvo P.A.
      • Frank D.W.
      • Bieler B.M.
      • Berson J.F.
      • Marks M.S.
      A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery.
      ,
      • Dennis M.K.
      • Mantegazza A.R.
      • Snir O.L.
      • Tenza D.
      • Acosta-Ruiz A.
      • Délevoye C.
      • et al.
      BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.
      ,
      • Kobayashi T.
      • Urabe K.
      • Orlow S.J.
      • Higashi K.
      • Imokawa G.
      • Kwon B.S.
      • et al.
      The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.
      )
      rabbit anti-mouse TYR (rAb)Mouse TYR lumenal domainDetects primarily ER, Golgi and endosomes by IFM; by IEM detects primarily stage III/IV melanosomes and endosomesIEM, WBTheos lab(
      • Theos A.C.
      • Tenza D.
      • Martina J.A.
      • Hurbain I.
      • Peden A.A.
      • Sviderskaya E.V.
      • et al.
      Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.
      )
      T311 (mMAb)Human TYR lumenal domainDetects human TYR by IFM under certain fixation conditionsIFM, WBcommercial(
      • Bultema J.J.
      • Ambrosio A.L.
      • Burek C.L.
      • Di Pietro S.M.
      BLOC-2, AP-3, and AP-1 function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.
      ,
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      )
       DCT (TYRP2)αPep8 (rAb)Human DCT C-terminusMay only be useful for immunoblottingWBHearing lab(
      • Kobayashi T.
      • Urabe K.
      • Orlow S.J.
      • Higashi K.
      • Imokawa G.
      • Kwon B.S.
      • et al.
      The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.
      )
      TRP-2 C-9 (mMAb)Human DCT (lumenal domain)Utility for immunolocalization is not clearWB; maybe IFM, IPcommercial(
      • Bultema J.J.
      • Ambrosio A.L.
      • Burek C.L.
      • Di Pietro S.M.
      BLOC-2, AP-3, and AP-1 function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.
      ,
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      )
       OA1/ GPR143anti-OA1 (rAb)Human OA1 (C-terminus)Melanosomes (primarily stage I and/or II), late endosomes and lysosomesIFM, IEM, WB, IPMarigo lab, Schiaffino lab(
      • Giordano F.
      • Bonetti C.
      • Surace E.M.
      • Marigo V.
      • Raposo G.
      The Ocular Albinism type 1 (OA1) G-protein-coupled receptor functions with MART-1 at early stages of melanogenesis to control melanosome identity and composition.
      ,
      • Schiaffino M.V.
      • Baschirotto C.
      • Pellegrini G.
      • Montalti S.
      • Tacchetti C.
      • De Luca M.
      • et al.
      The ocular albinism type 1 gene product is a membrane glycoprotein localized to melanosomes.
      )
      anti-OA1 W7 (rAb)Human OA1 residues 287–401Melanosomes, late endosomes, lysosomesIFM, IEM, WBSchiaffino lab(
      • Schiaffino M.V.
      • d'Addio M.
      • Alloni A.
      • Baschirotto C.
      • Valetti C.
      • Cortese K.
      • et al.
      Ocular albinism: evidence for a defect in an intracellular signal transduction system.
      , 1996)
       MART-1EP1422Y /M2-7C10 (mMAb)Human MART-1Broad distribution, particularly TGN, stage I and/or II melanosomes, tubules and vesiclesIFM, IEM, WB, IP, FCcommercial(
      • de Mazière A.M.
      • Muehlethaler K.
      • van Donselaar E.
      • Salvi S.
      • Davoust J.
      • Cerottini J.C.
      • et al.
      The melanocytic protein melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.
      ,
      • Giordano F.
      • Bonetti C.
      • Surace E.M.
      • Marigo V.
      • Raposo G.
      The Ocular Albinism type 1 (OA1) G-protein-coupled receptor functions with MART-1 at early stages of melanogenesis to control melanosome identity and composition.
      ,
      • Kawakami Y.
      • Battles J.K.
      • Kobayashi T.
      • Ennis W.
      • Wang X.
      • Tupesis J.P.
      • et al.
      Production of recombinant MART-1 proteins and specific antiMART-1 polyclonal and monoclonal antibodies: use in the characterization of the human melanoma antigen MART-1.
      ,
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      )
      A103 (mMAb)Human MART-1Late endosome, lysosomes and early stage melanosomesIFM, WB, IP, FCcommercial(
      • Chen Y.T.
      • Stockert E.
      • Jungbluth A.
      • Tsang S.
      • Coplan K.A.
      • Scanlan M.J.
      • et al.
      Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.
      ,
      • de Mazière A.M.
      • Muehlethaler K.
      • van Donselaar E.
      • Salvi S.
      • Davoust J.
      • Cerottini J.C.
      • et al.
      The melanocytic protein melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.
      )
      Rb anti MART-1 (rAb)Human MART-1Late endosome, lysosomes and early stage melanosomesIFM, IEM, WB, IPcommercial(
      • de Mazière A.M.
      • Muehlethaler K.
      • van Donselaar E.
      • Salvi S.
      • Davoust J.
      • Cerottini J.C.
      • et al.
      The melanocytic protein melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.
      ,
      • Kawakami Y.
      • Battles J.K.
      • Kobayashi T.
      • Ennis W.
      • Wang X.
      • Tupesis J.P.
      • et al.
      Production of recombinant MART-1 proteins and specific antiMART-1 polyclonal and monoclonal antibodies: use in the characterization of the human melanoma antigen MART-1.
      )
       OCA2NOCA2 (rAb)Human OCA2 N-terminusPigmented melanosomes (stage III and/or IV)IFM, WB, IPMarks lab(
      • Sitaram A.
      • Piccirillo R.
      • Palmisano I.
      • Harper D.C.
      • Dell’Angelica E.C.
      • Schiaffino M.V.
      • et al.
      Localization to mature melanosomes by virtue of cytoplasmic dileucine motifs is required for human OCA2 function.
      )
       ABCB6ABCB6 61.5 (mMAb)Human ABCB6Stage I and/or II melanosomes, lysosomesIFM, IEM, WBcommercial(
      • Bergam P.
      • Reisecker J.M.
      • Rakvács Z.
      • Kucsma N.
      • Raposo G.
      • Szakacs G.
      • et al.
      ABCB6 resides in melanosomes and regulates early steps of melanogenesis required for PMEL amyloid matrix formation.
      )
      OSK43 (hMAb)Human ABCB6Stage I and/or II melanosomes, lysosomesIFM, IEM, WB, IP, FCTani lab(
      • Helias V.
      • Saison C.
      • Ballif B.A.
      • Peyrard T.
      • Takahashi J.
      • Takahashi H.
      • et al.
      ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis.
      )
       RAB32anti-Rab32 (rAb)Human RAB32Melanosomes, endosomesIFM, WBDi Pietro lab(
      • Bultema J.J.
      • Ambrosio A.L.
      • Burek C.L.
      • Di Pietro S.M.
      BLOC-2, AP-3, and AP-1 function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.
      )
       RAB38anti-Rab38 (rAb)Human RAB38Melanosomes, endosomesIFM, WBDi Pietro lab(
      • Bultema J.J.
      • Ambrosio A.L.
      • Burek C.L.
      • Di Pietro S.M.
      BLOC-2, AP-3, and AP-1 function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.
      )
       RAB27Aanti-RAB27A (rAb)Human RAB27AStage IV melanosomesWBcommercial
      anti-RAB27A (rAb)Human RAB27AStage IV melanosomesWB, IFM, IEMSeabra lab(
      • Barral D.C.
      • Ramalho J.S.
      • Anders R.
      • Hume A.N.
      • Knapton H.J.
      • Tolmachova T.
      • et al.
      Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome.
      )
      4B12 (mMab)Human RAB27AStage IV melanosomesWB, IFMSeabra lab(
      • Hume A.N.
      • Collinson L.M.
      • Rapak A.
      • Gomes A.Q.
      • Hopkins C.R.
      • Seabra M.C.
      Rab27a regulates the peripheral distribution of melanosomes in melanocytes.
      )
      Melanosome-associated proteins (not specific to melanosomes)
       VAMP7/ TI-VAMP158.2 (mMab)Human VAMP7/TI-VAMPStage III and/or IV melanosomes, endosomes, lysosomesWB, IFM, IEMGalli lab(
      • Danglot L.
      • Zylbersztejn K.
      • Petkovic M.
      • Gauberti M.
      • Meziane H.
      • Combe R.
      • et al.
      Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.
      ,
      • Dennis M.K.
      • Delevoye C.
      • Acosta-Ruiz A.
      • Hurbain I.
      • Romao M.
      • Hesketh G.G.
      • et al.
      BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      )
       CD63anti-CD63 (mMab)HumanAll melanosome stages, early and late endosomes, lysosomesWB, IFM, EM, IP, FCcommercial (many)(
      • Patwardhan A.
      • Bardin S.
      • Miserey-Lenkei S.
      • Larue L.
      • Goud B.
      • Raposo G.
      • et al.
      Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.
      ,
      • van Niel G.
      • Charrin S.
      • Simoes S.
      • Romao M.
      • Rochin L.
      • Saftig P.
      • et al.
      The tetraspanin CD63 regulates ESCRT-independent and dependent sorting at a common endosome.
      )
       GPNMBAF2550 (gAb)Human GPNMB lumenal domainVarious compartments, perhaps melanosomesIFM, WBcommercial(
      • Theos A.C.
      • Watt B.
      • Harper D.C.
      • Janczura K.J.
      • Theos S.C.
      • Herman K.E.
      • et al.
      The PKD domain distinguishes the trafficking and amyloidogenic properties of the pigment cell protein PMEL and its homologue GPNMB.
      )
      hNMB-C (rAb)Human GPNMB (C-terminus)Various compartments, perhaps melanosomesIFM, WB, IPMarks lab(
      • Theos A.C.
      • Watt B.
      • Harper D.C.
      • Janczura K.J.
      • Theos S.C.
      • Herman K.E.
      • et al.
      The PKD domain distinguishes the trafficking and amyloidogenic properties of the pigment cell protein PMEL and its homologue GPNMB.
      )
      Markers of other compartments in melanocytes
      This is meant as a minimal, non-exhaustive list of commercially available antibodies that we have used routinely in our own laboratories to detect markers of various compartments.
       LAMP1H4A3 (mMAb)Human LAMP1Late endosomes, lysosomes—some on melanosomes
      LAMP1 labeling on melanosomes varies with growth conditions and is often undetectable by IFM.
      IFM, IEM, IP, FCcommercial
      1D4B (ratMAb)Mouse LAMP1Late endosomes, lysosomes—some on melanosomes
      LAMP1 labeling on melanosomes varies with growth conditions and is often undetectable by IFM.
      IFM, IEM, IP, FCcommercial
      anti-LAMP1 (rAb)Many species LAMP1 (C-terminus)Late endosomes, lysosomes—some on melanosomes
      LAMP1 labeling on melanosomes varies with growth conditions and is often undetectable by IFM.
      IFM, IEM, WB, IPcommercial
       LAMP2H4B4 (mMAb)Human LAMP2Late endosomes, lysosomesIFM, IEM, IP, FCcommercial
      GL2A7 (ratMAb)Mouse LAMP2Late endosomes, lysosomesIFM, IEM, IP, FCcommercial
      anti-LAMP2 (rAb)Many species LAMP2 (C-terminus)Late endosomes, lysosomesIFM, IEM, WB, IPcommercial
       TGN46anti-TGN46 (sAb)Human TGN46Trans Golgi networkIFM, IEM, WBcommercial
       Calnexinanti-calnexin (rAb)Many species calnexinEndoplasmic reticulumIFM, WBcommercial
       KDEL10C3 anti-KDEL (mMAb)Many proteins from all species ending in KDEL (ER retention signal)Endoplasmic reticulumIFM, WBcommercial
       EEA1Clone 14/EEA1 (mMAb)Human EEA1Early sorting endosomesIFM, WBcommercial
      anti-EEA1 (gAb)EEA1, many speciesEarly sorting endosomesIFM, WBcommercial
       Transferrin Receptor/CD71B3/25 (mMAb)Human CD71Early sorting and recycling endosomesIFM, IP, FCcommercial
      Clone C2 (ratMAb)Mouse CD71Early sorting and recycling endosomesIFM, FCcommercial
       RAB5C8B1 (rMAb)Human RAB5, cross-reacts with mouseEarly sorting endosomesIFM, WBcommercial
       RAB11D4F5 (rMAb)Human RAB11, cross-reacts with mouseEarly recycling endosomesIFM, WBcommercial
       RAB7D95F2 (rMAb)Human RAB7, cross-reacts with mouseEarly and/or late endosomesIFM, WBcommercial
       RAB6(rAb)RAB6GolgiIFM, WBcommercial (many)
      Abbreviations: ER, endoplasmic reticulum; FC, flow cytometry; gAb, goat antibody; GPNMB, glycoprotein non-metastatic “b”; hMab, human monoclonal antibody; IEM, immunoelectron microscopy; IFM, immunofluorescence microscopy; IP, immunoprecipitation; mMAb, mouse monoclonal antibody; rAb, rabbit antibody; rMAb, rabbit monoclonal antibody; ratMAb, rat monoclonal antibody; sAb, sheep antibody; TYR, tyrosinase; WB, western blotting.
      1 Indicated are antibody name and source.
      2 Indicated is the species from which the antigen is derived; cross-reactivity with other species is indicated when known.
      3 Intracellular localization determined by high confidence IFM or IEM analyses.
      4 Sources are indicated as commercial or available from a specific laboratory; specific commercial sources are not listed. Laboratories are as follows. Di Pietro lab: Santiago di Pietro, Colorado State Univ., Fort Collins, CO, USA; Galli lab: Thierry Galli, Institut de Psychiatrie et Neurosciences de Paris, Paris, France; Hearing lab: contact Julio Valencia, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; Marigo lab: Valeria Marigo, University of Modena and Reggio Emilia, Modena, Italy; Marks lab: Michael Marks, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Schiaffino lab: M. Vittoria Schiaffino, IRCCS San Rafaelle Scientific Institute, Milan, Italy; Seabra lab, Miguel Seabra, Universidade NOVA de Lisboa, Lisboa, Portugal; Tani Lab: Yoshihiko Tani, Japanese Red Cross Osaka Blood Center, Osaka, Japan; Theos lab: Alexander Theos, Georgetown University, Washington, DC, USA
      5 Non-exhaustive list of references to earliest descriptions and/or to documentation of use in particular applications.
      6 This is meant as a minimal, non-exhaustive list of commercially available antibodies that we have used routinely in our own laboratories to detect markers of various compartments.
      7 LAMP1 labeling on melanosomes varies with growth conditions and is often undetectable by IFM.
      Supplementary Table S3In Vitro Culture Systems To Study Melanin Transfer (Listed References are Non-Exhaustive)
      Culture systemReferences
      In Vitro System
       Melanocores with XB2 keratinocytes cell lines(
      • Correia M.S.
      • Moreiras H.
      • Pereira F.J.C.
      • Neto M.V.
      • Festas T.C.
      • Tarafder A.K.
      • et al.
      Melanin transferred to keratinocytes resides in nondegradative endocytic compartments.
      )
      2-Dimensional Coculture Systems
       Primary melanocytes with keratinocytes from human skin(
      • Ando H.
      • Niki Y.
      • Yoshida M.
      • Ito M.
      • Akiyama K.
      • Kim J.H.
      • et al.
      Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes.
      ,
      • Bruder J.M.
      • Pfeiffer Z.A.
      • Ciriello J.M.
      • Horrigan D.M.
      • Wicks N.L.
      • Flaherty B.
      • et al.
      Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.
      ,
      • Cardinali G.
      • Ceccarelli S.
      • Kovacs D.
      • Aspite N.
      • Lotti L.V.
      • Torrisi M.R.
      • et al.
      Keratinocyte growth factor promotes melanosome transfer to keratinocytes.
      ,
      • Ripoll L.
      • Heiligenstein X.
      • Hurbain I.
      • Domingues L.
      • Figon F.
      • Petersen K.J.
      • et al.
      Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.
      ,
      • Singh S.K.
      • Kurfurst R.
      • Nizard C.
      • Schnebert S.
      • Perrier E.
      • Tobin D.J.
      Melanin transfer in human skin cells is mediated by filopodia--a model for homotypic and heterotypic lysosome-related organelle transfer.
      )
       Primary melanocytes with keratinocytes from mouse skin(
      • Wu X.S.
      • Masedunskas A.
      • Weigert R.
      • Copeland N.G.
      • Jenkins N.A.
      • Hammer J.A.
      Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes.
      )
       B16 mouse melanoma cells with primary human keratinocytes(
      • Bruder J.M.
      • Pfeiffer Z.A.
      • Ciriello J.M.
      • Horrigan D.M.
      • Wicks N.L.
      • Flaherty B.
      • et al.
      Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.
      )
       B16 mouse melanoma cells with HaCat immortalized human keratinocytes(
      • Choi E.J.
      • Kang Y.G.
      • Kim J.
      • Hwang J.K.
      Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.
      ,
      • Jung H.
      • Chung H.
      • Chang S.E.
      • Kang D.H.
      • Oh E.S.
      FK506 regulates pigmentation by maturing the melanosome and facilitating their transfer to keratinocytes.
      )
       Mouse melan-a melanocytes with murine SP-1 epidermal keratinocytes(
      • Virador V.M.
      • Muller J.
      • Wu X.
      • Abdel-Malek Z.A.
      • Yu Z.X.
      • Ferrans V.J.
      • et al.
      Influence of alpha-melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes.
      )
       Mouse melan-ink4a melanocytes with XB2 keratinocyte cell lines(
      • Tarafder A.K.
      • Bolasco G.
      • Correia M.S.
      • Pereira F.J.C.
      • Iannone L.
      • Hume A.N.
      • et al.
      Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.
      )
       Melanocytes derived from human embryonic stem cells with normal human keratinocytes(
      • Nissan X.
      • Larribere L.
      • Saidani M.
      • Hurbain I.
      • Delevoye C.
      • Feteira J.
      • et al.
      Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.
      )
       Melanocytes and keratinocytes derived from human induced pluripotent stem cell(
      • Gledhill K.
      • Guo Z.
      • Umegaki-Arao N.
      • Higgins C.A.
      • Itoh M.
      • Christiano A.M.
      Melanin transfer in human 3D skin equivalents generated exclusively from induced pluripotent stem cells.
      )
      3-Dimensional Coculture Systems
       Epidermal cells on either side of a porous filter(
      • Kasraee B.
      • Pataky M.
      • Nikolic D.S.
      • Carraux P.
      • Piguet V.
      • Salomon D.
      • et al.
      A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.
      )
       3-Dimensional-human pigmented reconstructed epidermis(
      • Gledhill K.
      • Guo Z.
      • Umegaki-Arao N.
      • Higgins C.A.
      • Itoh M.
      • Christiano A.M.
      Melanin transfer in human 3D skin equivalents generated exclusively from induced pluripotent stem cells.
      ,
      • Lo Cicero A.
      • Delevoye C.
      • Gilles-Marsens F.
      • Loew D.
      • Dingli F.
      • Guéré C.
      • et al.
      Exosomes released by keratinocytes modulate melanocyte pigmentation.
      ,
      • Nissan X.
      • Larribere L.
      • Saidani M.
      • Hurbain I.
      • Delevoye C.
      • Feteira J.
      • et al.
      Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.
      )

      Supplementary Material

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