Extracellular vesicles released by keratinocytes regulate melanosome maturation, melanocyte dendricity, and pigment transfer

Abstract

Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids, and genetic material between cells and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthesize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes [(A. Lo Cicero et al., Nat. Commun. 6, 7506 (2015)]. However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here, we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites and mature melanosomes and promote their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulating pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors to cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.

Keywords: dendrites; melanocytes; pigment transfer; skin pigmentation; small extracellular vesicles.

Fig. 1.

Keratinocyte-derived sEVs contribute to the transfer of melanin pigment from melanocytes to keratinocytes. (A–C) HEM-D treated with HEK sEVs (sEVs) or PBS for 48 h were cocultured with HEK-D for 48 h. (A) IFM images of cocultures immunolabeled for HMB45 (premelanosome protein, PMEL, red) to label melanin pigment and EGFR (green) to label HEKs. HMB45 staining in HEKs corresponds to transferred melanin pigment. Boxed regions at higher magnification depict the transferred pigment (arrowheads). Bars: 10 µm. (B) Quantification of the percentage of HEKs with HMB45-positive structures in each condition. Values are the mean ± SEM of three independent experiments. ****P < 0.00001. (PBS, n = 238; sEVs, n = 252). (C) Quantification of the area covered by HMB45-positive structures per HEK normalized to the total HEK surface area in each condition. Data shown are the results of three independent experiments with the median indicated in red ****P < 0.0001. (PBS, n = 238; sEVs, n = 252).

Fig. 2.

Keratinocyte sEVs induce melanosome maturation, peripheral accumulation, and increased Myo Va at the tip of melanocyte dendrites. (A) Representative conventional TEM micrographs of the tip, middle, and base of HEM-D dendrites incubated for 48 h with PBS or HEK sEVs (sEVs). Insets show magnification of the boxed areas. I to IV depict different stages of maturation of melanosomes. Bars: 1 µm; Insets: 200 nm. (SI Appendix, Fig. S2 A and B). (B and C) Percentage of melanosomes at early and late stages of maturation (B) within the dendrites and (C) at the dendritic tip of HEM treated as in (A).(B) PBS: n = 291 melanosomes, 5 dendrites; sEVs: n = 262 melanosomes, 6 dendrites. (C) PBS: n = 117 melanosomes, 5 dendrites; sEVs: n = 113 melanosomes, 6 dendrites. Values are the mean ± SEM (****P < 0.00001). (D) IFM images of HEM-D treated as in A, immunolabeled for Myo Va (white). Red asterisks point to the punctate Myo Va staining throughout the cytoplasm. Bars: 50 µm; Insets: 10 µm. (E) IFM image of HEM-D treated with sEVs for 48 h, stained for F-actin (phalloidin, red), and immunolabeled for Myo Va (green). Note the colocalization of Myo Va and F-actin at the dendritic tips (white arrows; Inset). Bar: 50 µm; Inset: 10 µm. (F) Scatter dot plot of Myo Va intensity at the dendritic tip of HEMs incubated as in A. Only HEMs with three dendrites were considered. Data shown are the results of three independent experiments (a color per experiment), with the median indicated (n = 112 dendrites). ***P = 0.0002. (G) Scatter dot plot of Myo Va intensity in the whole HEMs (Myo Va total intensity) incubated as in A. The same HEMs as in F were considered. Data shown are the results of three independent experiments (a color per experiment), with the median indicated (PBS, n = 36 cells; sEVs, n=40 cells). n.s. P = 0.4735. (H) Scatter dot plot of the normalized Mean Deviation Product coefficient (nMDP) for colocalization of Myo Va with F-actin in HEMs incubated for 48 h with HEK sEVs which exhibited significant accumulation of MyoVa at dendritic tips compared to control (D). Comparison of nMDP values at the tip and average nMDP values at the middle and base of the dendrites. Data shown are the results of three independent experiments (a color per experiment), with the median indicated (n = 30 dendrites). ****P < 0.0001.

Fig. 3.

Keratinocyte sEVs alter melanocyte morphology and promote dendrite outgrowth. (A) IFM images of HEM-L treated for 24 h with DMSO or FSK, or for 48 h with PBS or HEK sEVs (sEVs), immunolabeled for α-tubulin (green), and stained for F-actin (phalloidin, red). The boxed regions mark the zoomed area. Bars: 25 µm. Extensions emanating from the cell soma, white arrows; protrusions, red arrows; dendrites, blue arrows. (B) Percentage of HEMs [treated as in (A)] harboring protrusions (DMSO, n = 186; FSK, n = 187; PBS and sEVs, n = 177). Values are the mean ± SEM of three independent experiments. Only significant P values are indicated. ***P = 5 × 10⁻⁴. (C and D) Percentage of HEMs [treated as in (A)] showing dendrites (C), or 0, 1, 2, or 3 dendrites (D) (DMSO, n = 186; FSK, n = 188; PBS, n = 178; sEVs, n = 183). Values are the mean ± SEM of three independent experiments. Only significant P values are indicated. (C) ****P = 5.5 × 10⁻⁵. (D) 0 dendrites ****P = 5.5 × 10⁻⁵. 2 dendrites, DMSO and FSK, ****P = 5.3 × 10⁻⁵; PBS and sEVs, **P = 0.008. 3 dendrites, **P = 0.0032. P values are calculated by comparing the number of dendrites in each category to the sum of the number of dendrites in the other categories. (E) Scatter dot plot of the AR of HEMs. Data shown are the result of three independent experiments (a color for each experiment) with the median indicated (DMSO, n = 186; FSK, n = 188; PBS, n = 177; sEVs, n = 183). Only significant P values are indicated. DMSO and FSK, ****P < 0.0001; PBS and sEVs ***P = 0.0004.

Fig. 4.

Melanocyte dendricity is mediated by Rac1-containing sEVs. (A) WB analysis of whole-cell lysate (L; 20 µg) and sEVs (sEVs; 10 µg) from siCtrl- and siRac1-transfected HEKs probed with CD63, GAPDH and Rac1 antibodies. Note that since the exposure is the same for the whole WB, the signal for CD63 and GAPDH is slightly saturated for lysates compared with sEVs. (B) Quantification of Rac1 mRNA expression normalized to GAPDH in whole-cell lysates and sEVs from siCtrl- or siRac1-transfected HEKs. Values are mean ± SEM of three independent experiments. Only significant P values are indicated. **P = 0.0022 for whole-cell lysates and sEVs. (C) IFM images of HEM-D incubated with PBS, sEVs from HEK transfected with siCtrl (siCtrl-sEVs), or sEVs from HEK transfected with siRac1 (siRac1-sEVs) immunolabeled for α-tubulin (green) and stained for F-actin (red) with fluorescently labeled phalloidin. The boxed regions mark the zoomed area. Blue arrows depict HEM dendrites. Bars, 50 µm. (D and E) Percentage of HEMs [treated as in (C)] with dendrites (D) or with 0, 1, or 2 to 3 dendrites (E) (PBS, n = 197; + siCtrl-sEVs, n = 206; + siRac1-sEVs, n = 204). Values are the mean ± SEM of three independent experiments. Only significant P values are indicated. (D) PBS and + siCtrl-sEVs ****P = 0; + siCtrl-sEVs and + siRac1-sEVs ****P =0.0001. (E) 0 dendrite: PBS and + siCtrl-sEVs ****P = 0; + siCtrl-sEVs and + siRac1-sEVs ***P = 0.00045. 2 to 3 dendrites: PBS and + siCtrl-sEVs *P = 0.0147; + siCtrl-sEVs and + siRac1-sEVs *P = 0.0191. P values are calculated by comparing the number of dendrites in each category to the sum of the number of dendrites in the other categories. (F) Scatter dot plot of the AR of HEMs [treated as in (C)]. Data shown are the result of three independent experiments (a color for each experiment) with the median indicated. (PBS, n = 197; + siCtrl-sEVs, n = 206; + siRac1-sEVs, n = 204). Only significant P values are indicated. ****P < 0.0001.