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Published in final edited form as: J Invest Dermatol. 2020 Feb;140(2):257–268.e8. doi: 10.1016/j.jid.2019.12.002

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

Silvia Benito-Martínez 1,*, Yueyao Zhu 2,3,4,*, Riddhi Atul Jani 1,*, Dawn C Harper 3,*, Michael S Marks 3,4,#, Cédric Delevoye 1,#
PMCID: PMC6986772  NIHMSID: NIHMS1546485  PMID: 31980058

Abstract

Pigmentation of the skin and hair represents the result of melanin biosynthesis within melanosomes of epidermal melanocytes, followed by 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.

INTRODUCTION

One of the most prominent features of metazoans is their pattern of pigmentation. Variation in the type and amount of produced pigment 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 ultraviolet (UV) radiation (Jablonski and Chaplin, 2000, Pavan and Sturm, 2019). 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 and Sturm, 2019). 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/brown eumelanins and red/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 1 and (d’Ischia et al., 2015)]. 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 and Seabra, 2001). 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 et al., 2007). By 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 et al., 1998). Thus, skin and hair pigment is synthesized in melanocytes but resides primarily in keratinocytes (Wu and Hammer, 2014). Natural variation in melanosome formation/maturation, melanogenesis, and melanin transfer to keratinocytes result 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 (OCA), are caused by disruption of melanosome assembly 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 et al., 2019, Delevoye et al., 2019).

Figure 1. Pigmentation analyses.

Figure 1.

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a. Melanogenesis pathway from ref. (Ito and Wakamatsu, 2008), 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 Tyrosinase for degradation. From (Fujita et al., 2009). 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 (Scale 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 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 et al., 2017).

This review describes cultured cell model systems in which to study melanogenesis in epidermal melanocytes (Supplemental Table 1) and assays for melanogenesis, melanosome formation/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. Due 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 (Supplemental Table 1); 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).

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/ 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: (1) dedifferentiation, leading to reduced or lost pigment granules; (2) pigment aggregation, revealed as seemingly enlarged melanosomes; (3) change in the subcellular distribution of melanosomes (e.g. clustering in the perinuclear area or accumulation at the periphery); and (4) elongation and thinning of cell architecture and an apparent increase in dendrite formation. These features may skew results of more detailed analyses of melanosome biogenesis, transfer, or signaling. 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 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, shRNA 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 hydroxylation of tyrosine to form L-DOPA and oxidation of L-DOPA to DOPAquinone (Figure 1a; (d’Ischia et al., 2015)). 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 [a.k.a. Pmel17, Silver, gp100, and several other names; (Watt et al., 2013)]. If cysteine is abundant and pH is slightly lower (Wakamatsu et al., 2017), DOPAquinone is converted into cysteinyldopa, which then undergoes a series of steps to form pheomelanins (d’Ischia et al., 2015). Because melanosomes progress from highly acidic to near neutral during maturation (Raposo et al., 2001), melanin deposits in melanosomes of darkly pigmented melanocytes may have a pheomelanin core surrounded by a eumelanin cortex (d’Ischia et al., 2015). 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 (63x or 100x), melanosome size, distribution, total cellular content, and number per unit area can be analyzed qualitatively or quantitatively in live or fixed cells (Crawford et al., 2017, Wasmeier et al., 2006). Moreover, the localization of proteins of interest, detected by immunofluorescence or from fluorescent protein conjugates, can be compared to pigment granules by fluorescence/ bright field microscopy (see Figure 3a below). Light microscopy cannot detect lightly pigmented melanosomes or distinguish between eumelanin/ 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 be mistaken for pigment granules.

Figure 3. Analyses of melanosome content.

Figure 3.

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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 a 5X magnification of the boxed regions, emphasizing the “donut”-like structure of TYRP1 surrounding melanin. Scale bar: 10 μm. b. IEM on MNT-1 cells using antibodies to PMEL (a.k.a Pmel 17; HMB50) and TYRP1 (a.k.a TRP1; 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. Scale bar: 100 nm. Modified from (Raposo et al., 2001). 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 a protein expression level normalized to siRNA control (bottom). From (Ripoll et al., 2018b). d. Melanosome-enriched fraction from normal HEM (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. Scale bar: 500 nm. From (Patwardhan et al., 2017).

Melanin quantification by spectrophotometry (absorption spectroscopy)

This procedure measures light absorption (wavelength between 400–500 nm) by melanin in detergent-free cell lysates using a spectrophotometer (Hu, 2008) (Figure 1c). This approach can also be applied to quantify melanin either in skin or 3D skin equivalents using a modified lysate preparation (Lo Cicero et al., 2015) or secreted into the culture supernatant and recovered by centrifugation. For a quantitative comparison of melanin content among pigment-containing samples, values can be normalized to lysates or supernatant fractions from a non-pigmented cell (Delevoye et al., 2009). Total melanin content can be calculated by comparison to 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 and Fujita, 1985).

Melanin quantification by other spectroscopy methods

Melanin does not fluoresce intrinsically, but 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 melanin content (Rosenthal et al., 1973) and is amenable to complex biological structures such as zebrafish embryos or human hair (Fernandes et al., 2016). By contrast, electron paramagnetic resonance spectrometry (EPR) discriminates between eumelanins and pheomelanins (Sealy et al., 1982). EPR detects different electron spin signatures from stable free semiquinone-type radicals within the different melanin types, which accurately correlate with pigment concentration (Godechal et al., 2013). EPR analyses can measure eumelanin/ pheomelanin ratios even from biological samples such as histological sections (Sealy et al., 1982), 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 and Fujita, 1985), 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 et al., 1963). 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 et al., 2001), with early stages marked by the amyloid protein PMEL on the fibrils/ sheets and later stages enriched in melanogenic enzymes [TYR and the TYR-related proteins TYRP1 and dopachrome tautomerase (DCT)] and transporters [e.g. OCA2, ATP7A, TPC2; reviewed in (Sitaram and Marks, 2012)]. These contents derive by transport from the Golgi and endosomes. It is thus possible to identify distinct melanosome stages by their morphology using conventional EM analyses or by their protein content using biochemical approaches, fluorescence microscopy, or immunolabeling EM (IEM).

Figure 2. Electron microscopy analyses of melanosome ultrastructure.

Figure 2.

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a. Ultrastructure of stage I-IV melanosomes. Top, a schematic of melanosome biogenesis as presented in the text. Bottom, thin section EM analysis of MNT-1 cells showing examples of each melanosome stage. Stage I has a planar clathrin coat (black arrow), ILVs (*) 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. Scale bar: 200 nm. Adapted from (Ripoll et al., 2018a). b. MNT-1 cells fixed chemically (left) or immobilized by HPF (right) were processed for conventional EM and imaged by TEM. Pigmented melanosome (arrows) are detected. Note the rounder shape of melanosomes due to better ultrastructural preservation (right). Scale bar: 500 nm. From (Delevoye et al., 2016) (left) and (Ripoll et al., 2018b) (right).

Ultrastructural characterization of melanosomes by electron microscopy

Melanosome maturation is spatiotemporally regulated (Bowman et al., 2019, Delevoye et al., 2019) and defects in the expression or delivery of melanogenic proteins can alter melanosome morphology (Montoliu et al., 2014). These changes, as well as melanosome apposition to other organelles [e.g. mitochondria or endosomes (Daniele et al., 2014, Delevoye et al., 2009)] or remodeling of their limiting membranes (Delevoye et al., 2016, Ripoll et al., 2018b), are best detected by conventional transmission EM (TEM). Although it requires specialized equipment and expertise (Hurbain et al., 2017), TEM provides the resolution to accurately and quantitatively assess melanosome stages (Figure 2b). Samples for conventional TEM are often chemically fixed with aldehydes, which can introduce ultrastructural artifacts by altering melanosome size and shape and by disrupting tubular membranes (Hurbain et al., 2017). 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 2b) (Hurbain et al., 2017). HPF preservation reveals otherwise unappreciated details, such as membrane remodeling at the melanosome membrane (Delevoye et al., 2009, Ripoll et al., 2018b) or individual intraluminal PMEL fibrils (Hurbain et al., 2008). HPF requires complex instrumentation that may not be available to all EM users.

While conventional TEM provides a 2-dimensional ultrastructural view of the melanocyte interior, electron tomography (ET) allows for reconstruction of a 3D model of melanosomes and associated structures (Delevoye et al., 2009, Hurbain et al., 2008). A specific area of a thick EM section (250–350 nm) is imaged at incremental angles, and images are aligned using embedded gold particles as fiduciary marks. ET is preferentially combined with HPF and compatible with IEM (Hess et al., 2018). 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 and Genoud, 2016). 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 mechanisms by which these proteins are delivered to melanosomes.

Immunofluorescence microscopy (IFM) of fixed cells

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 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/IV melanosomes), but others can be used for specific situations (Supplemental Table 2). An example showing TYRP1 overlap with pigment granules, but not with PMEL or the lysosomal marker LAMP2, is shown in Figure 3a. This technique is relatively simple to do and many protocols are available [e.g. (Donaldson, 2001)], although it is also subject to many potential pitfalls (Box 2).

Box 2. Potential pitfalls to avoid in pigment cells analyzed by IFM.

1-IFM 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 et al., 2019, Delevoye et al., 2019), and thus a fraction of these proteins are always in such compartments en route. Additionally, a protein may be subjected to post-translational modifications or protein: protein interactions that prevent detection of its total pool by a given antibody. For example, different antibodies to various regions of PMEL yield distinct labeling patterns in melanocytes [Supplemental Table 2 and (Harper et al., 2008)]. 2-The expression level of a transgene product [e.g. TYR, TYRP1; (Calvo et al., 1999, Setty et al., 2007)] can alter its localization and detection. 3-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. 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 et al., 2018b). Fluorescence extinction approaches to super-resolution such as photoactivation localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) cannot be used with pigmented cells because melanin emits substantial fluorescence and heats up using these techniques.

Live-cell imaging

Imaging of live pigment cells allows for the visualization of melanosome movement or 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 mechanisms regulating melanosome motility (Wu et al., 1998). While imaging can be done without fear of phototoxicity, it is limited to 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. Co-expression of fluorescent markers of other compartments, concomitant visualization of pigment granules by bright field microscopy, or 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 et al., 2009, Dennis et al., 2015), lysosomes (Bissig et al., 2019) or Golgi-derived vesicles (Patwardhan et al., 2017). Such interactions are best captured by spinning disk confocal microscopy. Alternatively, to visualize dynamics of melanosomes associated with the melanocyte cortical cytoskeleton and their transfer to keratinocytes (Bruder et al., 2012), 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 et al., 2011)]. 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 better correlates with IEM analyses (Dennis et al., 2015, Setty et al., 2007), but requires subjective thresholding to define objects. Another measure is the distance between or coincidence of the centers of two test objects (Lachmanovich et al., 2003); 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 et al., 2015). Box 3 presents additional approaches for quantifying melanosome imaging.

Box3: Specific live cell quantitative approaches for melanosome biology.

There are many ways to quantify melanosome motility (Hume et al., 2011, Oberhofer et al., 2017, Palmisano et al., 2008, Rogers and Gelfand, 1998, Wu et al., 1998) and distribution to the perinuclear or peripheral regions (Caviston et al., 2011, Reilein et al., 1998). We have quantified the dynamics of melanosome-bound tubular transport intermediates during melanosome biogenesis using ImageJ and freeware called Icy (http://icy.bioimageanalysis.org/) (Delevoye et al., 2016, Dennis et al., 2016, Dennis et al., 2015, Ripoll et al., 2018b). 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 et al., 2018b).

Immuno-EM (IEM)

Based on antibody detection in thin sections using electron dense gold particles and on a different sample preparation than for conventional EM (Hurbain et al., 2017), 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 distribution to distinct melanosome stages [Figure 3b; (Hurbain et al., 2017, Raposo et al., 2001)] or to pigment granules in keratinocytes (Hurbain et al., 2018). An alternative state-of-the-art technique is correlative light to EM (CLEM) (Hurbain et al., 2017), 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 et al., 2016) and/or associated with 3D-ET (Ripoll et al., 2018b), and has revealed unappreciated membrane trafficking steps during melanosome biogenesis and maturation that are targeted in HPS (Delevoye et al., 2016).

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 et al., 2005). Protein content in melanosome fractions from different samples can then be compared by immunoblotting (Figure 3c) or by proteomics analyses (Chi et al., 2006). 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 et al., 2017)].

ANALYZING MELANIN TRANSFER/ UPTAKE

Melanin transfer to keratinocytes requires the peripheral positioning and immobilization of pigmented melanosomes near the melanocyte plasma membrane. Proposed transfer modes include (1) melanosome exchange by fusion of melanocyte and keratinocyte plasma membranes, or phagocytosis by keratinocytes either of (2) melanosome-containing melanocyte dendrites, (3) melanosome-containing membrane fragments shed from melanocytes, or (4) luminal content of melanosomes (melanocores) exocytosed by melanocytes (Wu and Hammer, 2014). Regardless of the model, genetic defects in melanosome positioning (Bowman et al., 2019) or maturation (Bultema et al., 2014, Ripoll et al., 2018b) can affect melanin transfer.

In vitro co-culture system

Most transfer assays described to date rely on a 2D in vitro co-culture system of primary or immortalized melanocytes and keratinocytes (Supplemental Table 3). 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 et al., 2011) 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 co-culture 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 et al., 2011) or epidermal growth factor receptor (Ripoll et al., 2018b)], whereas the transferred melanin/ melanosomes can be detected by bright field microscopy for the pigment or by antibodies specific for melanosomal proteins (e.g. PMEL, TYR, TYRP1). Note that labeling for melanosomal membrane proteins (TYRP1 and to some degree, TYR) reflects preferentially the transfer of intact melanosomes and not of melanocores, whereas PMEL antibodies mainly detect lightly pigmented melanosomes/ melanocores (Figure 4a, c, d, e, arrowheads), because PMEL epitopes on the fibrils in the melanosome lumen are buried upon melanin deposition (Raposo et al., 2001). 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 et al., 2018b) for further details].

Figure 4. Analyses of melanosome transfer.

Figure 4.

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a. HEM/ HEK co-culture processed for IFM using antibodies specific to HEKs (EGFR, green) or melanosomes (HMB45 anti-PMEL, red). The contours of HEKs were drawn manually using ImageJ (white lines) to detect transferred melanosomes/ melanocores (HMB45-positive structures, arrowheads). Note that HEMs are heavily stained by HMB45 antibody due to the abundance of PMEL inside melanosomes, while transferred melanosomes/ melanocores appear as small and dim fluorescent puncta or pigment granules in keratinocytes (arrowheads). Scale bar: 10 μm. From (Ripoll et al., 2018b). 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 10 min (d) or 8 h (e) 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 h. Scale bar, 10 μm.

Live-cell imaging

Live cell fluorescence imaging can be used to track transfer of intact melanosomes to keratinocytes (identified in corresponding phase contrast images) from co-cultured melanocytes expressing fluorescent protein fusions to melanosomal proteins (Bruder et al., 2012). Wu and colleagues similarly used time lapse imaging to visualize the shedding mode of melanin transfer by labelling the plasma membranes of melanocytes and keratinocytes with different fluorescent probes (Wu et al., 2012). These approaches do not detect melanocore exchange, which may be the primary mode of melanin transfer in human epidermis (Tarafder et al., 2014), and are prone to UV-induced photodamage and transfer underestimates due to the non-synchronous manner of exchange at multiple planes of focus. To our knowledge, fluorescent labelling 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 et al., 2017) might facilitate pigment transfer analyses.

Alternative methods

Melanosome transfer in 2D co-cultures can be assessed by flow cytometry analysis of keratinocytes labeled for melanosome-associated markers [e.g. TYR, TYRP1 (Hu et al., 2017, Lin et al., 2008)] or by conventional EM to detect pigment in keratinocytes (Kasraee et al., 2011). Several studies have exploited 3D culture systems, including seeding of epidermal cells on either side of a porous filter (Kasraee et al., 2011), or generating 3D-reconstructed epidermis for analyses by immunohistochemistry and/ or EM (Lo Cicero et al., 2015). While these systems may be more physiological, they are also more complicated to analyze.

Monitoring uptake of biochemically isolated melanocores

In human epidermis, a main mode of melanin transfer results from fusion of melanosomes with the melanocyte plasma membrane, releasing their intra-luminal melanin content, or melanocores, for subsequent phagocytosis by keratinocytes (Correia et al., 2018, Tarafder et al., 2014). Melanocores can thus be used in vitro as a physiologically relevant pigment source to study pigment transfer and keratinocyte pigmentation. Some melanoma cells (e.g. MNT-1; Supplemental Table 1) constitutively secrete melanin into the conditioned medium, from which melanocores can be collected by differential centrifugation [Figure 4b and (Correia et al., 2018)] 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 et al., 2018) 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, like 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 co-exist, 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?

Supplementary Material

1

ACKNOWLEDGEMENTS

We acknowledge funding from the United States National Institutes of Health/ National Eye Institute (5R01 EY015625) and National Institute of Arthritis, Musculoskeletal and Skin Diseases (5R01 AR071382 and 5R01 AR048155), Agence Nationale de la Recherche (ANR ‘Myoactions’; ANR-17-CE11-0029-03 to C.D.), 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. SBM has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 666003.

Question 1

Which of these imaging methods allows one to distinguish ALL melanosome stages at the same time?

  1. Immunofluorescence microscopy

  2. Transmission electron microscopy

  3. Live cell imaging

  4. Bright field microscopy

Explanation

Four melanosome stages co-exist 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.

Question 2

Which of the following methods allows a quantitative measurement of both eumelanin AND pheomelanin?

  1. Electron paramagnetic resonance spectrometry

  2. Absorption spectroscopy

  3. High performance liquid chromatography

  4. Light microscopy

  5. Both a and c

Explanation

Eumelanin and pheomelanin are chemically distinct, but they cannot be distinguished by conventional spectroscopy due to their very similar absorption spectra or by light microscopy due to the partial detection of lightly pigmented melanosomes. In contrast, the EPR signatures (free radicals) of eumelanin and pheomelanin are distinct, and HPLC can distinguish specific by-products of the degradation of these two different pigments.

Question 3

What is the primary substrate for melanin synthesis?

  1. Tryptophan

  2. Tyrosine

  3. Lysine

  4. Histidine

Explanation

The black/ brown eumelanins and red/ yellow pheomelanins are both composed of polymerized products of sequential redox reactions in which tyrosine is the initial substrate.

Question 4

Is the melanocore:

  1. Another name for a fully pigmented (Stage IV) melanosome

  2. The intralumenal melanin content of a melanosome devoid of a limiting membrane

  3. The degradation product of the intralumenal melanin content of a melanosome

  4. The core substrate of melanin synthesis

Explanation

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.

Question 5

Which of these imaging approaches allows one to visualize pigmented melanosomes in TYRP1-GFP expressing melanocytes?

  1. Bright field microscopy

  2. Live cell imaging

  3. Immuno-electron microscopy

  4. Stochastic optical reconstruction microscopy (STORM)

  5. a, b, and c but not d

Explanation

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/ IV melanosomes express TYRP1 on the limiting membrane. Endogenous TYRP1 can be detected by immunofluorescence microscopy or immuno-electron microscopy, and an expressed fusion protein of TYRP1 fused to the green

Footnotes

CONFLICT OF INTEREST STATEMENT

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 have no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT

No datasets were generated or analyzed during the current study.

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REFERENCES

  1. Bissig C, Croisé P, Heiligenstein X, Hurbain I, Lenk GM, Kaufman E, et al. The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation. J Cell Sci 2019;132(5):jcs.229500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowman SL, Bi-Karchin J, Le L, Marks MS. The road to LROs: insights into lysosome-related organelles from Hermansky-Pudlak syndrome and other rare diseases. Traffic 2019;20(6):404–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruder JM, Pfeiffer ZA, Ciriello JM, Horrigan DM, Wicks NL, Flaherty B, et al. Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker. PLoS One 2012;7(8):e43465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bultema JJ, Boyle JA, Malenke PB, Martin FE, Dell’Angelica EC, Cheney RE, et al. Myosin vc interacts with rab32 and rab38 proteins and works in the biogenesis and secretion of melanosomes. J Biol Chem 2014;289(48):33513–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calvo PA, Frank DW, Bieler BM, Berson JF, Marks MS. A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery. J Biol Chem 1999;274(18):12780–9. [DOI] [PubMed] [Google Scholar]
  6. Caviston JP, Zajac AL, Tokito M, Holzbaur EL. Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes. Mol Biol Cell 2011;22(4):478–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chi A, Valencia JC, Hu ZZ, Watabe H, Yamaguchi H, Mangini NJ, et al. Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes. J Proteome Res 2006;5(11):3135–44. [DOI] [PubMed] [Google Scholar]
  8. Correia MS, Moreiras H, Pereira FJC, Neto MV, Festas TC, Tarafder AK, et al. Melanin transferred to keratinocytes resides in nondegradative endocytic compartments. J Invest Dermatol 2018;138(3):637–46. [DOI] [PubMed] [Google Scholar]
  9. Crawford NG, Kelly DE, Hansen MEB, Beltrame MH, Fan S, Bowman SL, et al. Loci associated with skin pigmentation identified in African populations. Science 2017;358(6365):eaan8433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. d’Ischia M, Wakamatsu K, Cicoira F, Di M, E., Garcia-Borron JC, Commo S, et al. Melanins and melanogenesis: from pigment cells to human health and technological applications. Pigment Cell Melanoma Res 2015;28(5):520–44. [DOI] [PubMed] [Google Scholar]
  11. 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. Curr Biol 2014;24(4):393–403. [DOI] [PubMed] [Google Scholar]
  12. Delevoye C, Heiligenstein X, Ripoll L, Gilles-Marsens F, Dennis MK, Linares RA, et al. BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes. Curr Biol 2016;26(1):1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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. J Cell Biol 2009;187(2):247–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Delevoye C, Marks MS, Raposo G. Lysosome-related organelles as functional adaptations of the endolysosomal system. Curr Opin Cell Biol 2019;59(4):147–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dennis MK, Delevoye C, Acosta-Ruiz A, Hurbain I, Romao M, Hesketh GG, et al. BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers. J Cell Biol 2016;214(3):293–308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dennis MK, Mantegazza AR, Snir OL, Tenza D, Acosta-Ruiz A, Délevoye C, et al. BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery. J Cell Biol 2015;209(4):563–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Donaldson JG. Immunofluorescence staining. Curr Protoc Cell Biol 2001;Chapter 4:Unit 4.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dunn KW, Kamocka MM, McDonald JH. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 2011;300(4):C723–C42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fernandes B, Matama T, Guimaraes D, Gomes A, Cavaco-Paulo A. Fluorescent quantification of melanin. Pigment Cell Melanoma Res 2016;29(6):707–12. [DOI] [PubMed] [Google Scholar]
  20. 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. J Invest Dermatol 2009;129(6):1489–99. [DOI] [PubMed] [Google Scholar]
  21. Godechal Q, Ghanem GE, Cook MG, Gallez B. Electron paramagnetic resonance spectrometry and imaging in melanomas: comparison between pigmented and nonpigmented human malignant melanomas. Mol Imaging 2013;12(4):218–23. [PubMed] [Google Scholar]
  22. Harper DC, Theos AC, Herman KE, Tenza D, Raposo G, Marks MS. Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of pmel17 that have been proteolytically processed in endosomes. J Biol Chem 2008;283(4):2307–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hess MW, Vogel GF, Yordanov TE, Witting B, Gutleben K, Ebner HL, et al. Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography. Traffic 2018;19(8):639–49. [DOI] [PubMed] [Google Scholar]
  24. Hu D-N. Methodology for evaluation of melanin content and production of pigment cells in vitro. Photochem Photobiol 2008;84(3):645–9. [DOI] [PubMed] [Google Scholar]
  25. Hu QM, Yi WJ, Su MY, Jiang S, Xu SZ, Lei TC. Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer. Cell Prolif 2017;50(6):e12372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hume AN, Wilson MS, Ushakov DS, Ferenczi MA, Seabra MC. Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function. Traffic 2011;12(12):1686–701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hurbain I, Geerts WJC, Boudier T, Marco S, Verkleij A, Marks MS, et al. Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci USA 2008;105(50):19726–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hurbain I, Romao M, Bergam P, Heiligenstein X, Raposo G. Analyzing lysosome-related organelles by electron microscopy. Methods Mol Biol 2017;1594:43–71. [DOI] [PubMed] [Google Scholar]
  29. 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. J Invest Dermatol 2018;138(3):647–56. [DOI] [PubMed] [Google Scholar]
  30. Ishida M, Marubashi S, Fukuda M. M-INK, a novel tool for visualizing melanosomes and melanocores. J Biochem 2017;161(4):323–6. [DOI] [PubMed] [Google Scholar]
  31. Ito S, Fujita K. Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal Biochem 1985;144(2):527–36. [DOI] [PubMed] [Google Scholar]
  32. Ito S, Wakamatsu K. Chemistry of mixed melanogenesis - pivotal roles of dopaquinone. Photochem Photobiol 2008;84(3):582–92. [DOI] [PubMed] [Google Scholar]
  33. Jablonski NG, Chaplin G. The evolution of human skin coloration. J Hum Evol 2000;39(1):57–106. [DOI] [PubMed] [Google Scholar]
  34. Kasraee B, Pataky M, Nikolic DS, Carraux P, Piguet V, Salomon D, et al. A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes. Exp Dermatol 2011;20(11):938–42. [DOI] [PubMed] [Google Scholar]
  35. Kobayashi N, Nakagawa A, Muramatsu T, Yamashina Y, Shirai T, Hashimoto MW, et al. Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis. J Invest Dermatol 1998;110(5):806–10. [DOI] [PubMed] [Google Scholar]
  36. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM. Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J Microsc 2003;212(Pt 2):122–31. [DOI] [PubMed] [Google Scholar]
  37. Lin HC, Shieh BH, Lu MH, Chen JY, Chang LT, Chao CF. A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro. Pigment Cell Melanoma Res 2008;21(5):559–64. [DOI] [PubMed] [Google Scholar]
  38. Lo Cicero A, Delevoye C, Gilles-Marsens F, Loew D, Dingli F, Guere C, et al. Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Commun 2015;6:7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lopes VS, Wasmeier C, Seabra MC, Futter CE. Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis. Mol Biol Cell 2007;18(10):3914–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Marks MS, Seabra MC. The melanosome: membrane dynamics in black and white. Nature Rev Mol Cell Biol 2001;2(10):738–48. [DOI] [PubMed] [Google Scholar]
  41. Montoliu L, Grønskov K, Wei AH, Martínez-García M, Fernández A, Arveiler B, et al. Increasing the complexity: new genes and new types of albinism. Pigment Cell Melanoma Res 2014;27(1):11–8. [DOI] [PubMed] [Google Scholar]
  42. 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. Proc Natl Acad Sci USA 2011;108(36):14861–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oberhofer A, Spieler P, Rosenfeld Y, Stepp WL, Cleetus A, Hume AN, et al. Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro. Proc Natl Acad Sci U S A 2017;114(24):E4714–E23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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. Hum Mol Genet 2008;17(22):3487–501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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. Nat Commun 2017;8:15835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pavan WJ, Sturm RA. The genetics of human skin and hair pigmentation. Annu Rev Genomics Hum Genet 2019;In press:doi: 10.1146/annurev-genom-083118-15230. [DOI] [PubMed] [Google Scholar]
  47. Raposo G, Tenza D, Murphy DM, Berson JF, Marks MS. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J Cell Biol 2001;152(4):809–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Reilein AR, Tint IS, Peunova NI, Enikolopov GN, Gelfand VI. Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A). J Cell Biol 1998;142(3):803–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ripoll L, Figon F, Delevoye C. Mécanismes inter- et intracellulaires contrôlant la pigmentation des mélanocytes de la peau In: Haftek M, editor. Biologie cutanée CoBIP: Fonctions normales et pathologiques de la barrière cutanée. Lyon, France: Matrix; 2018a. p. 4–28. [Google Scholar]
  50. Ripoll L, Heiligenstein X, Hurbain I, Domingues L, Figon F, Petersen KJ, et al. Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers. J Cell Biol 2018b;217(8):2709–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rogers SL, Gelfand VI. Myosin cooperates with microtubule motors during organelle transport in melanophores. Curr Biol 1998;8(3):161–4. [DOI] [PubMed] [Google Scholar]
  52. Rosenthal MH, Kreider JW, Shiman R. Quantitative assay of melanin in melanoma cells in culture and in tumors. Anal Biochem 1973;56(1):91–9. [DOI] [PubMed] [Google Scholar]
  53. Sealy RC, Hyde JS, Felix CC, Menon IA, Prota G. Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy. Science 1982;217(4559):545–7. [DOI] [PubMed] [Google Scholar]
  54. Seiji M, Fitzpatrick TM, Simpson RT, Birbeck MSC. Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature 1963;197:1082–4. [DOI] [PubMed] [Google Scholar]
  55. Setty SRG, Tenza D, Truschel ST, Chou E, Sviderskaya EV, Theos AC, et al. BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles. Mol Biol Cell 2007;18(3):768–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sitaram A, Marks MS. Mechanisms of protein delivery to melanosomes in pigment cells. Physiology 2012;27(2):85–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tarafder AK, Bolasco G, Correia MS, Pereira FJ, Iannone L, Hume AN, et al. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis. J Invest Dermatol 2014;134(4):1056–66. [DOI] [PubMed] [Google Scholar]
  58. Titze B, Genoud C. Volume scanning electron microscopy for imaging biological ultrastructure. Biol Cell 2016;108(11):307–23. [DOI] [PubMed] [Google Scholar]
  59. Wakamatsu K, Nagao A, Watanabe M, Nakao K, Ito S. Pheomelanogenesis is promoted at a weakly acidic pH. Pigment Cell Melanoma Res 2017;30(3):372–7. [DOI] [PubMed] [Google Scholar]
  60. Wasmeier C, Romao M, Plowright L, Bennett DC, Raposo G, Seabra MC. Rab38 an Rab32 control early post-Golgi trafficking of melanogenic enzymes. J Cell Biol 2006;175(2):271–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Watabe H, Kushimoto T, Valencia JC, Hearing VJ. Isolation of melanosomes. Curr Protoc Cell Biol 2005;Chapter 3:Unit 3.14. [DOI] [PubMed] [Google Scholar]
  62. Watt B, van Niel G, Raposo G, Marks MS. PMEL: A pigment cell-specific model for functional amyloid formation. Pigment Cell Melanoma Res 2013;26(3):300–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wu X, Bowers B, Rao K, Wei Q, Hammer JA 3rd. Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo. J Cell Biol 1998;143(7):1899–918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wu X, Hammer JA. Melanosome transfer: it is best to give and receive. Curr Opin Cell Biol 2014;29(4):1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wu XS, Masedunskas A, Weigert R, Copeland NG, Jenkins NA, Hammer JA. Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes. Proc Natl Acad Sci USA 2012;109(31):E2101–E9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

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