Melanocytes are the neural crest–derived pigment-producing cells of the skin that possess dendrites. Yet little is known about how melanocyte dendrites receive and process information from neighboring cells. Here, using a co-culture system to interrogate the interaction between melanocyte dendrites and keratinocytes, we show that signals from neighboring keratinocytes trigger local compartmentalized Ca 2+ transients within the melanocyte dendrites. The localized dendritic Ca 2+ transients could be triggered by two keratinocyte-secreted factors, endothelin and acetylcholine, which acted via specific melanocyte receptors. Furthermore, compartmentalized Ca 2+ transients were also generated on discrete dendritic spine-like structures on the melanocytes. These spines were also present in intact human skin. Our findings provide insights into how melanocyte dendrites communicate with neighboring cells and offer a new model system for studying compartmentalized signaling in dendritic structures.

As the pigment-producing cells of the skin, melanocytes have melanosomes, specialized lysosome-related organelles. Melanin pigment is made within the lumen of melanosomes and transferred, via melanocyte dendrites, to neighboring keratinocytes, epithelial cells that comprise the bulk of the epidermis ( Marks and Seabra, 2001 ; Wu and Hammer, 2014 ). Keratinocytes are known regulators of melanocyte behavior, and much work has been done to understand how keratinocytes influence melanocyte cell proliferation and the production and transfer of pigment throughout the skin ( Gordon et al., 1989 ; Hirobe, 2014 ). Nonetheless, cell–cell communication between melanocytes and keratinocytes, at the single-cell level, is poorly understood.

In intact human skin, similar interactions were observed from thin transmission EM (TEM) sections. Processes from basal and suprabasal keratinocytes enveloped melanocyte dendrites in multiple layers of overlapping projections ( Fig. 2 D ), often physically isolating multiple dendrites within the same region ( Fig. 2 E ). Interestingly, some keratinocytes had pools of small vesicles (44–70 nm diameter) in the cytosol adjacent to the plasma membrane that was juxtaposed to the melanocyte dendrite ( Fig. 2 E , right panel). These pooled vesicles were distinct in their aggregated localization from other intracellular vesicles present throughout the cytosol of all cells and suggested that melanocyte dendrites might receive localized signaling from individual keratinocytes. To confirm that the keratinocyte processes observed in situ were indeed wrapping around melanocyte dendrites, focused ion beam scanning EM (FIB-SEM) was used to obtain serial sections of intact neonatal foreskin with a volume of 30 × 27 × 27 µm at 30 nm z resolution and an XY pixel size of 14–18 nm. 3D reconstruction of two keratinocyte processes showed that both processes interacted with and wrap around the melanocyte dendrite ( Fig. 2, F–H ).

To visualize melanocytes and keratinocytes in living cultures, the two cell types were cultured separately, and then a subset of each population was transduced with lentivirus encoding a fluorescent protein plasma membrane reporter: EGFPmem (melanocytes) or iRFPmem (keratinocytes). After 24 h, both cell types were combined to initiate the co-culture ( Fig. 1 B ). Mosaic labeling of each cell type allowed for real-time morphological observation of individual cells in the dense 3D culture. In regions with only a few labeled keratinocytes, we observed processes that extended from the cell surface of the keratinocytes, which contacted and wrapped adjacent melanocyte dendrites ( Fig. 2 A ). The keratinocyte processes and melanocyte dendrites made a stable interaction, which was maintained over the course of an hour even while each of the processes was moving ( Fig. 2, B and C ).

To determine the source of Ca 2+ responsible for the dendritic transients, we quantified the number of Ca 2+ transients before and after the removal of external CaCl 2 and/or addition of 1 µM thapsigargin, which releases Ca 2+ from internal stores ( Lytton et al., 1991 ). The percentage of melanocytes with Ca 2+ transients was reduced by either removal of external CaCl 2 or addition of thapsigargin to the imaging media (0.63 ± 0.04 and 0.30 ± 0.05 fold change from before treatment, respectively) compared with the control (0.88 ± 0.5 fold change from before treatment), with the combination of removal of CaCl 2 and addition of thapsigargin having the greatest effect (0.07 ± 0.4 fold change from before treatment; Fig. 5 A ). This was also true for the number of Ca 2+ transients per cell ( Fig. 5, B–E ), where removal of external CaCl 2 plus the addition of thapsigarin reduced the number of transients per cell to a level similar to that of mono-cultured melanocytes (Fig. S3 H and Fig. S4 D).

Analysis of dendritic Ca 2+ transients from 53 melanocytes grown with keratinocytes in co-culture media showed a range of detectable Ca 2+ spread from 8–42 µm with the majority (58%) of transients being 14–22 µm in length ( Fig. 3 H ). Individual dendrites had transients that either originated from a single point ( Fig. 4, A–C ; and Video 2) or from multiple distinct locations within a region several microns long of the dendrite ( Fig. 4, D and E ). In those dendrites that had multiple Ca 2+ transients over time, repetitive transients initiated from the same region of the dendrite ( Fig. 4 F ).

To determine if the dendritic transients of Ca 2+ were intrinsic to melanocytes or were dependent on the presence of keratinocytes, we altered the growth conditions and cell types present in cultures of melanocytes expressing GCaMP6f. Cultures were then imaged in modified DPBS without serum or other exogenous growth factors. The high percentage of melanocytes with Ca 2+ transients (64 ± 11%) was only observed when melanocytes were co-cultured with keratinocytes. Significantly, fewer melanocytes had Ca 2+ transients when the keratinocytes were replaced with HEK293T cells ( Fig. 3 F , 10 × 293T: 13 ± 6%) or melanocytes were grown in mono-culture ( Fig. 3 F , 10 × Mel and Mel alone: 16 ± 2% and 11 ± 4%, respectively), or when melanocytes were separated from keratinocytes by a semiporous membrane (Fig. S2 B, TW 10 × Ker: 9 ± 8% to 20 ± 10%, depending on growth conditions). This was irrespective of growth media formulation (Fig. S2, A and B). In co-cultures, the number of local dendritic transients detected during 2.5 min ranged from 0–70 per melanocyte. Of the melanocytes with local dendritic transients, 91% had 1–9 transients while 9% had >10 transients ( Fig. 3 G ). In comparison, of the few mono-cultured melanocytes that had dendritic transients, 90% had 1–4 transients and 10% had 5–12 transients ( Fig. 3 G , inset). Thus, close proximity to, and possibly direct contact with, keratinocytes was required for the high frequency of Ca 2+ transients.

Endothelins, a family of 21 amino acid peptides, play a critical role in melanocyte development, maturation, and homeostasis within the epidermis (Reid et al., 1996). Epidermal keratinocytes produce and secrete endothelin 1 (ET-1; Yohn et al., 1993), which can elicit increases of Ca2+ in the cell bodies of melanocytes (Kang et al., 1998). Addition of 10nM ET-1 to co-cultures produced recurring Ca2+ transients across the entire melanocyte, including dendrites and the cell body (Fig. 6, A–D; Fig. S3 A; and Video 3). The percentage of melanocytes with a response to ET-1 was dose-dependent (Fig. S3 B), and the number of transients per melanocyte also increased with the addition of 10 nM ET-1 (Fig. 6 B). ET-1–induced increase of Ca2+ in melanocytes co-cultured with keratinocytes was blocked by preincubation with a selective inhibitor of endothelin receptor B (ET B ), BQ788, but not an inhibitor of endothelin receptor A (ET A ), BQ123 (Fig. 6 B; Fig. S2, C and D; and Video 4), consistent with previous work that showed many patient-derived human melanocytes do not express ET A (Eberle et al., 1999). This was also true of melanocytes in mono-culture (Fig. S3, E–H).

The localized Ca2+ transients in melanocytes elicited by ET-1 (Fig. 6, C–E; and Video 3) resembled the spontaneous Ca2+ transients in co-cultures of melanocytes and keratinocytes without the addition of exogenous ET-1 (Fig. 4 and Video 1). To test if the spontaneous local Ca2+ transients in melanocytes was caused by keratinocyte secreted endothelin, we treated co-cultures with endothelin receptor antagonists. Co-cultures treated with the ET B antagonist BQ788 had a significant reduction in the percentage of melanocytes with Ca2+ transients, but no change was observed in Ca2+ transients in co-cultures treated with the ET A antagonist BQ123 (Fig. 7 A). To confirm that the effects were due to inhibition of the ET B on melanocytes and not the inhibition of ET B on keratinocytes, we used Dicer-substrate siRNA (DsiRNA) against EDNRA (the gene that encodes ET A ) or EDNRB (the gene that encodes ET B ) to reduce expression of either ET A or ET B in melanocytes before co-culture with keratinocytes. Consistent with the endothelin receptor antagonist data, knockdown of ET B but not ET A reduced the number of co-cultured melanocytes with Ca2+ transients (Fig. 7 B). We next knocked down EDN1, the ET-1 gene, in keratinocytes and found that co-cultures with keratinocytes expressing shRNA against EDN1 had significantly fewer melanocytes with Ca2+ transients (Fig. 7, C and D). Together, these data confirmed that keratinocyte-secreted endothelin acts on melanocyte ET B to produce local dendritic Ca2+ transients.