Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced by melanocytes into signaling cues to promote their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we found that caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released by keratinocytes. Preventing caveolae formation in melanocytes results in increased production of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, our data establish that, in melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and intercellular communication, skin pigmentation and tissue homeostasis.

polarized caveolae as compared to cells grown in their own medium (Figures 1D and S1C).

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The proportion of melanocytes with polarized caveolae was similar between cells co-cultured 123 with keratinocytes ( Figure 1C) and cells incubated with conditioned medium (Figure 1D) (Figures 1E and S1D). The melanocyte-keratinocyte interface revealed 134 numerous plasma membrane-associated cup-shaped invaginations, with a diameter between 135 43 and 102 nm and an average size of 63.9 nm, that lacked an electron dense cytoplasmic coat 136 ( Figures 1E and S1D, arrowheads). Immunogold labelling on ultrathin cryosections of human 137 skin samples revealed that these invaginations were positive for Cav1 in melanocytes (Figure 138 S1E) and were thus identified as caveolae. To access caveolae 3D ultrastructure, thick-139 sectioned (300 nm) human skin samples were subjected to double-tilt electron tomography 140 ( Figures 1F and S1F). The reconstructed 3D model ( Figure 1F and Video 1) depicts an 141 epidermal area consisting of a transversal section of a melanocyte dendrite (plasma membrane 142 in green) containing pigmented melanosomes (red) and surrounded by a keratinocyte (plasma 143 membrane in blue, presenting keratin bundles on the cytosol). Caveolae (white) were observed 144 in the melanocyte as single or clustered structures known as rosettes (arrowhead and arrow, 145 respectively) that were connected to the cell surface (Richter et al., 2008;Stan, 2005).

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3D human reconstructed pigmented epidermis (3D-HRPE) composed of normal human 147 epidermal melanocytes (Mel) and keratinocytes (Ker) are used to study epidermis stratification 148 and pigmentation (Ali et al., 2015). The development of the synthetic tissue includes the initial 149 epidermis stratification at day 4, pigmentation at day 6 and formation of a fully stratified and 7 pigmented epidermis at day 12. To address the distribution and modulation of caveolae during 151 human epidermis formation at cell-cell interface, representative samples of each day were 152 chemically fixed, thin-sectioned and analyzed by conventional TEM (Figures 1G, H and S1G, 153 H). From day 4 to 12, the melanocyte-keratinocyte interface showed increased numbers of 154 caveolae per 10 µm-length of plasma membrane when compared to homologous keratinocyte-155 keratinocyte interface (Figures 1G and S1G). Although the number of caveolae was constant at 156 the melanocyte-keratinocyte interface (Figure 1G), differences in caveolae enrichment 157 appeared with time for each cell type (Figure 1H). At day 4, when the tissue stratified, caveolae 158 were 4-fold enriched in keratinocytes when compared to melanocytes. However, from day 4 to 159 6, when the tissue started to pigment, caveolae biogenesis showed a 5-fold increase in 160 melanocytes ( Figure 1H). As a control, we observed that the number of CCPs, identified by the 161 presence of a characteristic electron dense coat (Heuser, 1980), was similar at both interfaces   (Figures 1I and S1I). Cav1 protein levels were increased 6-fold 174 in melanocytes after 3 consecutive irradiations ( Figure 1I) while keratinocytes only slightly up-175 regulated Cav1 protein levels in comparison to non-exposed cells ( Figure S1I). Thus, UV-B   (Figures 2A and S2B). Cav1-depleted melanocytes increased the 196 intracellular cAMP dramatically by 7.5-fold upon stimulation while in control cells, the increase in 197 cAMP was only 3.5-fold (Figure 2A). The 2-fold gain in the cAMP production observed in the 198 absence of Cav1 suggests that Cav1 and/or caveolae inhibit tmACs activity in melanocytes.

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Several studies have reported that caveolae could regulate the activity of various signaling  CavTratin-treated melanocytes resulted in a 30% reduction of cAMP intracellular levels 205 (Figures 2B and S2C). These results strongly suggest that caveolin-1 reduces the activity of 206 tmACs and the production of cAMP in melanocytes through direct binding to the Cav1-CSD.

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Interestingly, Cav1-depleted melanocytes were more frequently deprived of physical contact by 284 keratinocytes during the total time of acquisition ( Figure 3E). In contrast, the frequency of

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The cell mechanical response to changes in shape is correlated with adjustments in the plasma

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To address the role of caveolae in melanin transfer, siCtrl-and siCav1-treated melanocytes 315 were co-cultured with keratinocytes for 3 days, after which the cells were analyzed by 316 immunofluorescence ( Figure 4A). Keratinocytes co-cultured with Cav1-depleted melanocytes 317 were less frequently positive for melanin ( Figure 4B) and, when positive, showed decreased 318 staining for the pigment ( Figure 4C). This result shows that caveolae are required for the 319 efficient transfer of melanin from melanocytes to keratinocytes in co-culture. showed decreased Cav1 protein expression levels ( Figure S4A). When co-cultured with 323 melanocytes treated with pre-miR-203a, melanin transfer had occurred in fewer keratinocytes

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( Figures 4A and B), which also showed a decrease content of melanin ( Figure 4C). The miR- pigmented epidermis when control melanocytes were added ( Figure S4C). In contrast, a non-335 homogenous pigmentation was observed in the epidermis reconstructed with siCav1-treated 336 melanocytes ( Figure S4C, arrow). The pigmentation defect was further characterized at the 337 ultrastructural level ( Figure 4D) and revealed that keratinocytes juxtaposed to Cav1-depleted 338 melanocytes contained less melanin than when adjacent to control cells ( Figure 4E). This data

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shows that caveolae is a novel player in melanin transfer from melanocytes to keratinocytes in 340 the human epidermis.    to the extracellular context, will enable to decipher its defects and associated consequences in 431 disease.

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Competing financial interests: The authors declare no competing financial interests.    to stratify, at day 6 it started to pigment and at day 9 to 12, the epidermis was fully stratified and 634 pigmented. All the incubation steps were performed at 37ºC in a 5% CO 2 incubator. The detailed protocol for the melanin transfer assay is described elsewhere (Ripoll et al., 2018).

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Melanocytes were transfected twice with the indicated siRNA or miRNAs at day 1 and day 3 for 671 a total of 5 days. At day 3, Melanocytes were co-cultured with keratinocytes for a total of 2 days.