title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Ambrosio
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Boyle
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870.
authors.forename: Al E
authors.initials: AE
authors.lastname: Aradi
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Christian
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870 santiago.dipietro@colostate.edu.
authors.forename: Santiago M
authors.initials: SM
authors.lastname: Di Pietro
mesh_terms: D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis / Q000502:physiology*; D048429:Cell Size; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008543:Melanins / Q000032:analysis; D020460:Melanosomes / Q000737:chemistry / Q000378:metabolism*; D010858:Pigmentation
chemical_list: D015220:Calcium Channels; D008543:Melanins; C530224:TPCN2 protein, human; D002118:Calcium
keywords: melanosome; membrane traffic; organelle pH; pigmentation; two-pore channel 2