TPC2 controls pigmentation by regulating melanosome pH and size

TPC2 controls pigmentation by regulating melanosome pH and size

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...

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Published in:Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 20; pp. 5622 - 5627
Main Authors: Ambrosio, Andrea L., Boyle, Judith A., Aradi, Al E., Christian, Keith A., Di Pietro, Santiago M.
Format: Journal Article
Language:English
Published: United States National Academy of Sciences 17-05-2016
Subjects:
Biological Sciences
Calcium - metabolism
Calcium Channels - analysis
Calcium Channels - physiology
Cell Size
Cellular biology
Electron microscopy
Fluorescence
Genetic research
Humans
Hydrogen-Ion Concentration
Melanins - analysis
Melanosomes - chemistry
Melanosomes - metabolism
Membranes
pH sensors
Pigmentation
Pigments
pigmentation
organelle pH
melanosome
membrane traffic
two-pore channel 2
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Summary: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 Ca2+ release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca2+ sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.
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Author contributions: A.L.A. and S.M.D. designed research; A.L.A., J.A.B., A.E.A., K.A.C., and S.M.D. performed research; A.L.A. and S.M.D. contributed new reagents/analytic tools; A.L.A. and S.M.D. analyzed data; and A.L.A. and S.M.D. wrote the paper.
Edited by Graça Raposo, Institut Curie-CNRS, Paris, France, and accepted by the Editorial Board April 5, 2016 (received for review January 4, 2016)
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1600108113