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Abstract

The retinal pigment epithelium (RPE) forms a monolayer of cells at the blood:retina interface that plays important roles for photoreceptor renewal and function and is central to retinal health. RPE pigment is provided by melanin-containing melanosomes which offer protection against light and oxidative stress. Melanosome migration into the apical processes of the RPE following light onset is thought to contribute to preventing retinal degeneration with age, though the mechanism is not yet clear. Melanosomes are transported along microtubules to the apical surface where they are transferred to actin filaments within the apical processes. Melanosomes are lysosome-related organelles derived from endosomes and endosome transport along microtubules is heavily influenced by the endoplasmic reticulum (ER) through ER:endosome contact sites. Here we describe extensive connection between the ER and melanosomes in the RPE. We further show, in skin melanocytes, that the ER forms contact sites with all stages of melanosome maturation, but ER contact is reduced as melanosomes mature. Finally, we identify tripartite contact sites between the ER, melanosomes and mitochondria in both RPE tissue and cellular models, suggesting that the ER may influence melanosome biogenesis, maturation and interaction with mitochondria.

Keywords

RPE melanosomes endoplasmic reticulum contact sites electron microscopy

Introduction

The pigment melanin provides photoprotection to the skin and eyes by absorbing and scattering ultraviolet radiation and protecting against oxidative stress. Melanin is produced and stored within melanosomes, membrane-bound organelles found in skin epidermal melanocytes and in the retinal pigment epithelium (RPE) cells of the eye. Defined as lysosome-related organelles, melanosomes originate from endosomes but undergo a series of maturation stages (I to IV), involving the progressive deposition of melanin pigment (Seiji et al., 1963). Defects in melanosome biogenesis/function is associated with several human diseases including melanomas and cause syndromic forms of albinism (e.g., Hermansky-Pudlak Syndrome) and loss of vision (Coutant et al., 2024).

The RPE forms a monolayer of cells at the blood-retina interface at the back of the eye. Melanogenesis occurs prenatally in the RPE, which is densely packed with mature melanosomes from birth, providing cells with their characteristic pigmentation and offering protection against harmful backscattered light and damaging reactive oxygen species (ROS) (Wang et al., 2006). Melanosome distribution in the RPE is regulated, at least in part, by the light cycle. Light onset triggers apical migration of melanosomes along microtubules and their transfer to the actin filaments of the apical processes that extend between photoreceptor outer segments (Futter et al., 2004).

In contrast to the RPE, in skin and hair, melanosome biogenesis occurs continuously in melanocyte cells, predominantly in a central perinuclear region of melanocytes. Mature melanosomes traffic towards the cell periphery and along dendrites that extend through the epidermis to keratinocytes. Subsequent exocytosis from melanocyte dendrites releases the melanocore for phagocytosis into keratinocytes (Bento-Lopes et al., 2023). Melanin-containing phagosomes ultimately fuse with the lysosome to form “melanokerasomes”, weakly acidic terminal organelles that were recently shown to cluster around the nucleus forming melanokerasome:nuclear contact sites to provide a photoprotective perinuclear “cap” that protects DNA from UV damage (Neto et al., 2024).

Although melanosome distribution and its regulation differ between melanocytes and the RPE, basic mechanisms that drive melanosome movement are common to both cell types. Melanosomes are transported along microtubules to the periphery of the cell where they interact with the actin cytoskeleton via tripartite protein complexes consisting of Rab27A, it's effector and an actin motor protein (Myosin-7a in the RPE and Myosin-Va in melanocytes) (Barral and Seabra, 2004). In non-polarised cells, including melanocytes, the microtubule-organizing centre (MTOC), in which the minus ends of microtubules are anchored, is in a perinuclear position. In polarised RPE however, the MTOC is apically positioned, with microtubules extending vertically between minus ends in the apical region and plus ends facing the basal surface (Hazim and Williams, 2022). Transport along microtubules can be bidirectional, with anterograde transport towards plus ends (ie, at the periphery in melanocytes/basal surface in RPE) mediated by kinesin motor proteins, while retrograde transport towards the minus end (at the cell centre in melanocytes or apical side in RPE) is mediated by dynein motor proteins (Barral and Seabra, 2004). Thus dynein-dependent transport delivers mature melanosomes to the actin-rich apical domain of RPE cells but favours perinuclear positioning in melanocytes (Jiang et al., 2020).

Similarly, distribution of endosomes, from which melanosomes are derived, is also dependent on dynein and kinesin but endosome interactions with microtubule motor proteins is strongly influenced by the ER through ER:endosome membrane contact sites (MCS). In complementary, and likely coordinated processes, MCS in non-polarised cells promote kinesin-loading for plus-end directed endosome movement along microtubules to the plasma membrane (Raiborg et al., 2015) and prevent dynein interaction with endosomal RILP to reduce minus-end directed movement towards the peri-nuclear compartment (Rocha et al., 2009). ER:endosome MCSs thereby couple the regulation of both plus and minus-end-directed transport and interestingly ER contact with melanosomes has been identified in pigmented keratinocytes (Hurbain et al., 2018).

Melanosomes have also been shown to form contact sites with another organelle in melanocytes, where physical bridging with mitochondria has been implicated in melanosome biogenesis. These mitochondria:melanosome MCS are tethered by Mitofusin-2, are enhanced by the ocular albinism type 1 (OA1) protein that can stimulate melanogenesis and are increased in the perinuclear region where melanosomes are generated (Daniele et al., 2014). Melanosome biogenesis is heavily influenced by mitochondria, which likely supply ATP to meet the energy needs of melanogenesis, as well as buffering cytosolic Ca²⁺ since both the mitochondrial calcium uniporter (MCU) that mediates Ca²⁺ influx into mitochondria (Tanwar et al., 2024) and the mitochondrial voltage-dependent anion channel, VDAC1 (Wang et al., 2022) were recently found to be important in the regulation of melanogenesis.

Here we identify MCS between melanosomes and the ER both in RPE tissue and cellular models, from multiple species. Characterisation of ER:melanosome MCS reveals that the proportion of melanosomes forming an ER contact site is conserved across species and that the ER contacts all stages of melanosome maturation in melanocytes. Our data further suggests the existence of tripartite connections between the ER, melanosomes and mitochondria.

Results

Melanosomes Contact the ER and Mitochondria in Mouse, Porcine and Human RPE

To determine if melanosomes in the RPE form ER contact sites, we imaged mouse retina by electron microscopy (EM). Electron-dense pigmented melanosomes were readily visible both in central areas and apical processes of the RPE from mouse eyes two hours after light onset (Figure 1A), consistent with the previously described Rab27a-dependent increased apical positioning of melanosomes in response to light (Futter et al., 2004). Just as melanosome:mitochondria MCS have been identified in melanocytes (Daniele et al., 2014), we also found mitochondria tightly associated with melanosomes in mouse RPE (Figure 1A; mitochondria at MCS false-coloured green). Both the subpopulation of mitochondria in contact with the basal infoldings of the RPE (Neto et al., 2024) (Figure 1A, arrows), and mitochondria in central/apical regions (Figure 1A) appear to form contact sites with melanosomes.

Figure 1.

ER:melanosome MCS in RPE from adult mouse tissue. Ultrathin sections through the retina of mouse eyes were imaged by transmission electron microscopy. A) Highly pigmented, electron dense melanosomes form extensive MCS with ER, both in the apical (upper box) and central (lower box) areas of the RPE and with mitochondria in central and basal areas (mitochondria at MCS false-coloured green). Arrows, mitochondria in contact with basal infoldings; Br, Bruch’s membrane; BI, basal infoldings; AP, apical processes. Scale bar, 1 μm. Higher magnification images of apical (B) and central C) areas are shown with the ER false-coloured lilac in the lower panels. Scale bar, 250 nm.

Higher magnification imaging revealed that in addition to mitochondrial contact, melanosomes also form abundant connections with the ER, in both apical (Figure 1B) and central (Figure 1C) regions of mouse RPE. To determine if this RPE contact site population is unique to mouse tissue, we next examined RPE from human and porcine tissue. ER:melanosome MCS were readily visible in RPE from both species (supplemental Figure S1) indicating that formation of ER:melanosome MCS is conserved across different species and likely play important functional roles.

Having identified ER:melanosome MCS in mouse, porcine and human RPE tissue, we next investigated their formation in cellular RPE models in culture. Primary porcine RPE have been widely used to successfully generate pigmented polarized epithelia (Lakkaraju et al., 2020) and we found abundant ER:melanosome MCS visible by EM in primary porcine RPE cells (supplemental Figure S2A), comparable to the extent found in in vivo models (51% melanosomes had an ER contact in primary porcine RPE compared with 54% in RPE from mouse tissue).

The human ARPE19 cell line is a widely used RPE model that can be differentiated into RPE-like cells (Carr et al., 2018). Following six weeks of differentiation in X-Vivo medium, wild-type ARPE-19 cells formed a highly pigmented monolayer and analysis by EM revealed that ER:melanosome MCS also form in differentiated ARPE-19 cells (supplemental Figure S2B) to an even greater extent, with 74% melanosomes forming an ER contact.

ER:Melanosome MCS Form During Foetal Development in Mouse RPE

Biogenesis and maturation of melanosomes in the RPE occurs during foetal development in mice (Lopes et al., 2007). We therefore examined melanosome MCS in pre-existing sections from E14 foetal mouse eyes (not yet exposed to light). Melanosome synthesis is thought to be completed before birth in mouse RPE, with pigment granules retained throughout life (Wasmeier et al., 2008) and our data indicate that melanosome biogenesis and maturation has occurred by E14 during foetal development in mice (Figure 2). Using electron tomography, we were able to identify discreet ER contact sites with mature melanosomes in E14 mouse RPE, even though there appeared to be less ER content compared with postnatal RPE (Figure 2A-C). The ER leading to the melanosome contact was often decorated with ribosomes (false-coloured blue in Figure 2D), indicative of rough ER, though the ribosomes appeared to be excluded from the MCS (Figure 2B), as has been reported at other ER contact sites (Phillips and Voeltz, 2015).

Figure 2.

ER:melanosome MCS in E14 mouse RPE. A) Electron tomograph of RPE from E14 mouse tissue. Scale bar, 100 nm. B) Higher magnification tomographic slices of the orange boxed region in A showing the melanosome limiting membrane surrounding the melanocore forming an ER contact site (red arrows). Scale bar, 50 nm. C) A reconstruction of the region shown in B with the melanosomes false-coloured orange. D) Higher magnification of the purple boxed region in A showing the ER (false-coloured purple) decorated with ribosomes (false-coloured blue) which appear excluded from the site of contact with the melanosome limiting membrane (red arrows in B).

ER:Melanosome MCS Form with all Stages of Melanosome Maturation in Human Melanocytes

Melanosomes are derived from early endosomes with a four-step maturation process. Irregular amyloid fibrils start to form within endosomes in stage I melanosomes and by stage II the fibrils are fully formed and are organized into arrays of parallel sheets. Delivery of melanogenic enzymes results in progressive melanin deposition, causing the fibres to become thicker and darker in stage III and completely obscured by pigment in stage IV melanosomes. Whereas melanosome biogenesis and maturation is largely complete before birth in RPE cells, in skin melanocytes, melanosome synthesis occurs continuously throughout life (Wasmeier et al., 2008). Thus, melanocytes provide the opportunity to examine all four maturation stages and to determine the extent of ER contact with early as well as mature melanosomes. We therefore imaged different stages of melanosome maturation in the human melanoma MNT-1 cell line. In addition to forming abundant MCS with mature (stage IV) melanosomes as seen in RPE cells, the ER also made connections with melanosomes at earlier stages (I-III) of maturation (Figure 3A). The number of melanosomes with an ER contact was consistently high for all maturation stages (92–98%), and comparable to that of late endosome/lysosomes (95–99% Friedman et al., 2013). However, in contrast to the correlation between endosome maturation and extent of ER contact (Friedman et al., 2013), we found increased ER contact with early stage melanosomes compared with more mature organelles (Figure 3B), suggesting an inverse correlation between melanosome maturation and extent of ER contact.

Figure 3.

The ER forms MCS with all melanosome maturation stages. MNT-1 melanocytes were fixed and prepared for EM. A) Electron micrographs show ER (false-coloured lilac on the lower panels) interacting with melanosomes at all four stages of maturation. Scale bar, 250 nm. B) Quantitation of the average number of ER contacts/melanosome at each stage of maturation from 2 independent experiments with statistical significance determined by one-way ANOVA, Brown-Forsythe and Welch multiple comparison.

Tripartite MCS Between ER, Melanosomes and Mitochondria

In some areas of mitochondria:melanosome association, the ER appears to run intermittently between the two organelles, while in other areas, ER:melanosome contact is directly adjacent to mitochondria:melanosome MCS (Figure 4), suggesting the existence of tripartite contact sites between the three organelles. These three-way ER:melanosome:mitochondria contacts were present in both mouse tissue RPE (Figure 4A) and primary porcine RPE cells (Figure 4B), but were less frequent in mouse RPE, where 7% melanosomes were in a tripartite MCS compared with 13% in primary porcine RPE. This could reflect structural differences between in vitro and in vivo RPE models since a significant proportion of melanosomes migrate into the apical processes that interdigitate between photoreceptor outer segments following light onset, in vivo. In contrast, apical processes are much less pronounced in vitro and do not contain melanosomes. Distribution of mitochondria, like melanosomes, is also influenced by light. The majority of mitochondria localise to baso-lateral RPE, where a proportion are anchored to RPE basal infoldings through mitochondria:plasma membrane MCS (Neto et al., 2024), while light onset stimulates migration of a different mitochondrial subset into central RPE towards the apical surface, but not into apical processes (Neto et al., 2024). The mouse eyes were fixed two hours after light onset but it is likely that the number of tripartite MCS in mouse RPE might be higher without light-stimulated transport of melanosomes into the apical processes. Together, these data suggest that approximately 10% of melanosomes form multi-organelle platforms with the ER and mitochondria for non-vesicular communication and exchange.

Figure 4.

Tripartite MCS between ER, melanosomes and mitochondria. Electron micrographs of RPE showing ER:melanosome:mitochondria MCS (arrows). ER, false-coloured lilac; melanosomes, Mela; mitochondria, Mito; scale bar, 250 nm. A) RPE from adult mouse tissue. B) Primary porcine RPE cells.

Discussion

We have identified extensive ER contact sites with melanosomes that are conserved across species and in both RPE tissue and cells in culture. The frequency and extent of these MCS suggest important physiological roles which are as yet unknown. Melanosome traffic into the apical processes of the RPE is important in preventing retinal degeneration (Gibbs et al., 2004), but although ER contact has been shown to regulate the positioning of endosomes (Wenzel et al., 2022), from which melanosomes are derived, the influence of the ER over melanosome distribution has not yet been explored. Whereas in non-polarised melanocyte cells the microtubule-organizing centre (MTOC) is typically peri-nuclear, the MTOC is in the apical region of polarised RPE cells. Thus the minus-end directed motor protein dynein moves melanosomes away from dendrites, towards the cell body of melanocytes, while in the RPE, dynein-mediated transport traffics melanosomes in the opposite direction, to the apical surface where they are retained by myosin-7a/RAB27A/MYRIP-dependent transfer to actin filaments (Jiang et al., 2020). Endosomal engagement of dynein for minus-end directed transport along microtubules is negatively regulated by ER contact. The endosomal sterol-binding protein ORP1L forms a complex with the small GTPase Rab7 and the Rab7 effector RILP. In the absence of ER contact, the p150Glued subunit of the dynein/dynactin microtubule motor complex directly binds RILP, mediating minus-end-directed transport. However, interaction of ER-localised VAP with ORP1L at ER:endosome MCS results in removal of p150Glued and dynein, preventing dynein-dependent minus-end directed transport (Rocha et al., 2009). Our data has revealed that the majority (over 50%) of mature melanosomes form an ER contact in mouse RPE two hours after light onset, when maximal melanosome positioning in apical processes is expected (Futter et al., 2004). This suggests a potential role for ER contact sites in returning melanosomes from apical processes to the cell body and in restricting further dynein-dependent transport to the apical domain of the RPE. If MCS orchestrate melanosome positioning, the light-dependent migration of melanosomes into apical processes would suggest that light exposure may have some influence over contact site formation.

Whereas melanosome biogenesis and maturation are mostly prenatal in the RPE, in melanocytes it is continuous throughout life with more mature melanosomes being trafficked to the cell periphery prior to transfer to keratinocytes (Barral and Seabra, 2004). Interestingly ER contact with early melanosomes was more extensive than with more mature organelles in melanocytes, suggesting a possible role for the ER in melanosome biogenesis/maturation, likely through lipid and/or Ca²⁺ flux as has been described at ER:endosome MCS (Wenzel et al., 2022). After six weeks of differentiation in X-Vivo medium, ARPE19 cells continued to gain pigment, suggesting that melanosome maturation was incomplete, which may explain the greater extent of ER contact in these cells (74% melanosomes formed an ER contact compared with 54% in mouse RPE) since we found that ER contact is increased with less mature organelles.

The tripartite ER:melanosome:mitochondria MCS may also point to a role for the ER in melanosome biogenesis/maturation as has been shown for mitochondria. Melanosome biogenesis is subject to regulation by microphthalmia-associated transcription factor (MITF), a key transcription factor for expression of melanogenic enzymes (Kawakami and Fisher, 2017) and increased cytosolic Ca²⁺ on loss of mitochondrial VDAC1 induced downstream MITF activation and melanosome biogenesis (Wang et al., 2022). Lysosome MCS with ER and mitochondria have both been implicated in contributing to Ca²⁺ flux (Atakpa et al., 2018; Bretou et al., 2024; Kilpatrick et al., 2013; Peng et al., 2020) and melanosome Ca²⁺ import from mitochondria has been implicated in melanosome biogenesis (Zhang et al., 2019). Thus, these tripartite contacts may act as Ca²⁺ signalling hubs to regulate melanosome biogenesis. An additional role for melanosomes in quenching mitochondrial ROS has also been proposed (Daniele et al., 2014), (Seagle et al., 2005); the ER may help to coordinate melanosome:mitochondria contact through tripartite MCS to facilitate ROS scavenging by melanosomes. Identifying the molecular composition of melanosome MCS tethering complexes will be key to elucidating their function in the RPE.

Methods

RPE tissue: E14 foetal (Burgoyne et al., 2022) and adult mouse tissue (Neto et al., 2024) were isolated as previously described. E14 mice were isolated from sacrificed pregnant females and adult mice were sacrificed 2 h after light onset. All eyes were extracted and immediately plunged into EM fixative for 1 h prior to dissection. Human eye tissue from a donor with no known eye disease was approved for research and received from the Moran Eye Institute, Salt Lake City, UT, USA, courtesy of Dr GS Hageman. Porcine eyes were collected in the morning after light onset, kept on ice during transport and fixed for electron microscopy in the afternoon.

Cell culture: Primary porcine RPE cells were isolated from porcine eyes as previously described (Falcão et al., 2021) and seeded into 6 well plates. Once fully confluent, cells were passaged onto Corning transwell cell culture inserts. Cells were cultured in DMEM Glutamax supplemented with 1% FBS/PS. ARPE19 cells were passaged onto transwells and cultured in X-Vivo medium (Lonza Bioscience) for 6 weeks until they formed a pigmented cobblestone monolayer. MNT-1 cells (ATCC, CRL-3450) were cultured in MEM (no glutamine) supplemented with 10% AIM V Medium, 20% FBS, 2 mmol/L L-glutamine, 10 mmol/L HEPES and 1% PS. MNT-1 cells were passaged onto glass coverslips and cultured for 48–96 h before being fixed and processed for electron microscopy.

Electron microscopy (EM): The cornea and lens were removed from all eyes prior to subsequent incubation in EM fixative (2% gluteraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer) for 1 h (mouse tissue), overnight (porcine tissue) or >24 h, until further processing (human tissue). Cells in culture were fixed in EM fixative for 30 min. Following fixation, all samples were prepared for EM as previously described (Neto et al., 2024). For electron tomography, 150 nm sections were cut and ±60° tilt images collected over two perpendicular axes on a JEOL 1400+ TEM. A dual-axis tomogram was subsequently generated using IMOD (University of Colorado, Boulder) (Kremer et al., 1996) and 3D rendered images were generated in Chimera (UCSF).

Membrane contact site (MCS) analysis: MCS were imaged and analysed in random ultrathin sections, which we have previously shown to reflect MCS extent by serial sectioning to image entire organelles (Eden et al., 2016). For quantitation from EM images, MCS were defined as regions where the membranes of apposing organelles were less than 40 nm apart; greater than 100 melanosomes were analysed/condition or experiment and MCS extent expressed as the percent melanosomes with one or more ER contact, or as the number of ER contacts/melanosome. ER:melanosome MCSs were quantified in MNT-1 cells by EM from two independent experiments, with greater than 100 melanosomes analysed per experiment. The quantification represents the number of ER contacts with melanosomes at maturation stages I-IV, with statistical significance determined by one-way ANOVA, Brown-Forsythe and Welch multiple comparisons.

Supplemental Material

sj-pptx-1-ctc-10.1177_25152564251340949 - Supplemental material for Identification of ER:Melanosome Membrane Contact Sites in the Retinal Pigment Epithelium

Supplemental material, sj-pptx-1-ctc-10.1177_25152564251340949 for Identification of ER:Melanosome Membrane Contact Sites in the Retinal Pigment Epithelium by Burgoyne T., Doncheva D. and Eden E.R in Contact

Supplemental Material

sj-pptx-2-ctc-10.1177_25152564251340949 - Supplemental material for Identification of ER:Melanosome Membrane Contact Sites in the Retinal Pigment Epithelium

Supplemental material, sj-pptx-2-ctc-10.1177_25152564251340949 for Identification of ER:Melanosome Membrane Contact Sites in the Retinal Pigment Epithelium by Burgoyne T., Doncheva D. and Eden E.R in Contact

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Moorfields Eye Charity, Medical Research Council, (grant number GR001506, MR/V013882/1).

ORCID iDs

Burgoyne T.

Eden E.R

Supplemental Material

Supplemental material for this article is available online.

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