TPC2 controls pigmentation by regulating melanosome pH and size

Abstract

Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

Keywords: melanosome; membrane traffic; organelle pH; pigmentation; two-pore channel 2.

Fig. S1.

CRISPR design and genotyping for knockout and EmGFP tagging of the TPCN2 gene, coding for the TPC2 protein. (A) Schematic of the TPC2 protein topology. Red stars indicate the two amino acid changes caused by single nucleotide polymorphisms strongly associated with pigmentation variations in a genome-wide association study (13). (B) Schematic of the CRISPR/Cas9 design. (C, Left) Genotyping of TPC2-KO CRISPR clones. PCR using the indicated primers (OUT-Fwd-N and OUT-Rev-N) was carried out for mock transfected MNT-1 cells and MNT-1 homozygous clones (TPC2-KO1 and TPC2-KO2), obtained by transfection with the indicated sgRNAs (sgRNA1+2 and sgRNA1+3, respectively). (Right) Genotyping of a heterozygous TPC2-EmGFP CRISPR clone. PCR using the indicated primers (OUT-Fwd-C and OUT-Rev-C primers or EmGFP primers) was carried out for mock-transfected MNT-1 cells and MNT-1 cells transfected with the indicated sgRNAs (sgRNA4+5) and donor plasmid.

Fig. 1.

TPC2 localizes to melanosomes. (A) Schematic of the CRISPR/Cas 9 design. (B) Confocal fluorescence microscopy images of live MNT-1 cells coexpressing endogenous TPC2-EmGFP and exogenous TPC2-Cherry, tyrosinase-iRFP, or Cherry-Rab27a. (Scale bars, 5 µm.) (C) Confocal fluorescence microscopy images of fixed/permeabilized MNT-1 cells expressing endogenous TPC2-EmGFP and immunostained for endogenous TYRP1. (Scale bar, 5 µm.) (D, Upper) Immunogold electron micrograph of an MNT-1 cell labeled with an anti-TPC2 antibody (15,000×). (Scale bar, 500 nm.) (Lower) Higher-magnification view from the region indicated in the upper panel showing examples of pigmented melanosomes (M) and endosomes (E) labeled with the TPC2 antibody. (Scale bar, 250 nm.) (E) Postnuclear supernatant extracts of MNT-1 cells expressing TPC2-GFP were subjected to immunomagnetic isolation with anti-GFP and irrelevant antibodies. Immunoblotting revealed the presence of tyrosinase, PMEL17, and Rab32 in the anti-GFP sample. Blots are representative of three experiments.

Fig. S2.

MNT-1 cells expressing TPC2-EmGFP from the endogenous locus were transfected with the peroxisomal marker mRFP-SKL and imaged by confocal fluorescence microscopy to calculate the empirical background MOC. MOC = 0.12 ± 0.03 (n = 15 cells). (Scale bar, 5 µm.)

Fig. S3.

Thin-section immunogold electron microscopy analysis of MNT-1 cells overexpressing TPC2-GFP and labeled with an anti-GFP antibody. E, endosome; M, pigmented melanosome. Pigmented melanosomes: 57 ± 9% of label; endosomes/lysosomes: 36 ± 6% of label; nucleus: 2 ± 2% of label; mitochondria: 2 ± 1% of label; 982 gold particles, nine cells. (Scale bar, 500 nm.)

Fig. 2.

TPC2 expression level determines the melanin content in MNT-1 cells. (A) RNA was isolated from wild-type MNT-1 cells or two independent clones of CRISPR/Cas9 TPC2-KO homozygous cells and reverse-transcribed into cDNA. Relative amounts of the TPC2 mRNA were determined in triplicate by real-time PCR. (B) Melanin content was determined for the cells in A. (C) Melanin content was determined for mock-transfected wild-type MNT-1 cells, CRISPR/Cas9 TPC2-KO cells, and TPC2-GFP transfected TPC2-KO cells 5 d posttransfection (n = 2 for WT+Mock, n = 3 for TPC2-KO+Mock, and n = 9 for TPC2-KO+TPC2-GFP cells).

Fig. S4.

TPC2 expression level and melanin content are inversely correlated in MNT-1 cells and primary human melanocytes. (A) Melanin content was determined in MNT-1 cells (black bars) and primary human melanocytes (white bars) subjected to control siRNA and TPC2 siRNA. (B) Melanin content was determined in MNT-1 cells transfected with GFP and TPC2-GFP plasmids.

Fig. S5.

Design and validation of MELOPS, a genetically encoded melanosome localized pH sensor. (A) Cartoon illustrating MELOPS is built on the basis of the oculocutaneous albinism type 2 (OCA2) protein. The pH-sensitive fluorescent protein Nectarine was inserted in the loop connecting the first and second transmembrane domains, which is exposed to the organelle lumen. The OCA2 V443I mutation that renders the OCA2 channel inactive was also introduced. (B) MNT-1 cells were transfected with MELOPS and incubated in buffers with pH values ranging from 4.5 to 6.5 in the presence of ionophores. The graph indicates the fluorescence intensity of MELOPS is proportional to the pH of the buffers from pH 5.0 up to pH 6.5 (R² = 0.99; pH, 4.5: 32 cells; pH, 5.0: 30 cells; pH, 5.5: 34 cells; pH, 6.0: 36 cells; pH, 6.5: 29 cells). (C) Spinning-disk confocal fluorescence microscopy image of a cell incubated at pH 6.5, showing the high degree of colocalization between MELOPS and Tyrosinase-iRFP. (Scale bar, 5 µm.) (D) Spinning-disk confocal fluorescence microscopy image of live wild-type MNT-1 cells expressing MELOPS and tyrosinase-iRFP. The graph shown in B was used to interpolate the luminal melanosomal pH of MNT-1 cells (pH, 5.7 ± 0.6; 44 cells). (Scale bar, 5 µm.)

Fig. 3.

TPC2 regulates melanosome luminal pH. (A) Wild-type or CRISPR TPC2-KO MNT-1 cells expressing MELOPS and tyrosinase-iRFP were analyzed by confocal fluorescence microscopy. In a rescue experiment performed in parallel, TPC2-KO MNT-1 cells expressing MELOPS and TPC2-iRFP were also analyzed. (Scale bar, 5 µm.) (B) The average MELOPS fluorescence intensity was determined for all treatments (WT+Tyr = 30 cells; TPC2-KO+Tyr = 25 cells; TPC2-KO+TPC2 = 21 cells).

Fig. S6.

TPC2 regulates melanosomal pH in primary human melanocytes. (A and B) The average fluorescence intensity of MELOPS was determined for primary human melanocytes subjected to either control siRNA (n = 38 cells) or TPC2 siRNA knockdown (n = 44 cells). (C and D) The average fluorescence intensity of MELOPS was determined for primary human melanocytes overexpressing LAMP2-GFP as a control (n = 47 cells) or TPC2-GFP (n = 50 cells).

Fig. S7.

TPC2 inhibitors alkalize the melanosomal lumen and increase melanin content in MNT-1 cells. (A) The average fluorescence intensity of MELOPS was determined for MNT-1 cells overexpressing LAMP2-GFP, as a control (n = 31 cells), or for TPC2-GFP, and treated for 1 h with vehicle (DMSO; n = 28 cells) or the TPC2 inhibitors Ned19 (100 µM; n = 36 cells) and tetrandrine (2 µM; n = 36 cells). (B) Melanin content was determined for MNT-1 cells treated with DMSO or Ned19 (100 µM) for 6 d.

Fig. 4.

TPC2 regulates melanosome size. (A) Wild-type or CRISPR TPC2-KO MNT-1 cells expressing Cherry-Rab27a were analyzed by confocal fluorescence microscopy. Insets are magnifications of the boxed regions. (Scale bar, 5 µm.) (B) The average melanosome size was measured for wild-type and TPC2-KO cells (n = 37 and 41 cells, respectively). (C) Electron micrographs of wild-type (Left) or CRISPR TPC2-KO (Right) MNT-1 cells (18,500×). Roman numerals indicate melanosome maturation stage. (Scale bar, 500 nm.) (D) The average stage II–IV melanosome size was measured for wild-type and TPC2-KO cells (wild-type: seven cells, 464 melanosomes; TPC2-KO: nine cells, 567 melanosomes).

Fig. S8.

Larger thin-section electron micrographs of the wild-type (Upper) and CRISPR TPC2-KO (Lower) MNT-1 cells shown in Fig. 4 (18,500×). (Scale bars, 500 nm.)

Fig. 5.

TPC2 regulates Ca²⁺ release from melanosomes. (A) Wild-type or CRISPR TPC2-KO MNT-1 cells expressing tyrosinase-GCaMP6 were analyzed by confocal fluorescence microscopy. (Scale bar, 5 µm.) (B) The tyrosinase-GCaMP6 fluorescence intensity was determined for wild-type and TPC2-KO cells (n = 37 and 41 cells, respectively).

Fig. S9.

TPC2 regulates Ca²⁺ release from melanosomes. (A) Spinning-disk confocal fluorescence microscopy images of live wild-type and TPC2-KO MNT-1 cells expressing tyrosinase-GCaMP6-Cherry. (Scale bars, 5 µm.) (B) The green/red fluorescence intensity ratio was calculated for wild-type and TPC2-KO MNT-1 cells expressing tyrosinase-GCaMP6-Cherry (n = 17 and 19 cells, respectively).