PIKfyve regulates melanosome biogenesis.

Marc C Liggins,Priya Vasudeva,Junko Sasaki,Anand K Ganesan,Jessica L Flesher,Sohail Jahid,Takehiko Sasaki,Shunsuke Takasuga,Victoria Eby,Raymond E Boissy

doi:10.1371/journal.pgen.1007290

Abstract

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.

Highlights

Melanin, a pigment produced within uveal and epidermal melanocytes, absorbs UV radiation, protecting the eyes and skin from UV-induced DNA damage [1]
We used a well-established model for organelle biogenesis to understand how phosphoinositides regulate vesicle trafficking and melanogenesis
VAC14 and FIG4 mutants are characterized by early lethality and accumulation of vacuoles in the central nervous system (CNS) with accompanying coat color defects [37, 41, 42]

Summary

Methods

Ethics statementAll experiments involving mice conform to the NIH guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, approval number 2011–3020.Antibodies and primersAll antibodies used in experimental assays are listed in S3 Table. PIKfyve genotyping primers and PCR parameters are described in [44]. Other genotyping primers and PCR settings were taken from the mouse mutant resource website, (Jackson Laboratory)). Human MNT-1 cells were cultured in DMEM (Genesee Scientific) supplemented with 15% fetal bovine serum (Corning), AIM-V medium (Life Technologies), MEM vitamin solution (Invitrogen), and antibiotic-antimycotic (Life Technologies). MNT-1 cells were switched to DMEM lacking phenol red (Fisher Scientific) supplemented with 10% fetal bovine serum, L-glutamine (Invitrogen), and antibiotic-antimycotic. Harvested primary melanocytes were grown in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FBS, antibiotic-antimycotic, 200nM 12-O-tetradecanoylphorbol 13-acetate (TPA) (Abcam), and 200pM cholera toxin (Sigma-Aldrich) or RPMI-1640 supplemented with 5% FBS, 50ng/ml Stem cell Factor (SCF) (Protech International), 20nM Endothelin-3 (END3) (Sigma-Aldrich), 2.5ng/ml Fibroblast growth Factor (FGF) (Sigma-Aldrich), 100nM α-Melanocyte stimulating hormone (α-MSH) (Sigma-Aldrich), 1μM Phosphoethanolamine (Sigma-Aldrich), 10μM Ethanolamine (Sigma-Aldrich), and 1mg/ml Insulin (Sigma-Aldrich)

Results

Discussion

Conclusion