Michael S. Marks

We previously found that skin tone correlates with intronic polymorphisms influencing the expression of the Joubert Syndrome (JS) ciliopathy gene TMEM138 , suggesting that primary cilia might regulate pigment production in skin melanocytes. Herein, we show that many ciliary proteins regulate basal pigmentation in wild-type mouse and human melanocytes by facilitating primary cilium-dependent Melanocortin-1 receptor signaling, and downstream activation of the Microphthalmia transcription factor and melanogenic gene expression. Additionally, we identify an extra-ciliary moonlighting role for TMEM138 and other JS proteins in endolysosomal organization required for the biogenesis of melanosomes, the pigment-producing lysosome-related organelle in melanocytes. Our results provide new mechanistic insights into the physiological regulation of pigmentation and suggest that JS-specific pathology might be mediated by ciliary-independent mechanisms.

Summary: Photoprotective melanins in the skin are synthesised by epidermal melanocytes within specialised lysosome-related organelles called melanosomes. Melanosomes coexist with lysosomes; thus, melanocytes employ specific trafficking machineries to ensure correct cargo delivery to either the endolysosomal system or maturing melanosomes. Mutations in some of the protein complexes required for melanogenic cargo delivery, such as biogenesis of lysosome-related organelles complex 1 (BLOC-1), result in hypopigmentation due to mistrafficking of cargo to endolysosomes. We show that hypopigmented BLOC-1-deficient melanocytes retain melanogenic capacity that can be enhanced by treatment with cAMP elevating agents despite the mislocalisation of melanogenic proteins. The melanin formed in BLOC-1-deficient melanocytes is not generated in melanosomes but rather within late endosomes/lysosomes to which some cargoes mislocalise. Although these organelles generally are acidic, a cohort of late endosomes/lysosomes have a sufficiently neutral pH to facilitate melanogenesis, perhaps due to mislocalised melanosomal transporters and melanogenic enzymes. Modulation of the pH of late endosomes/lysosomes by genetic manipulation or via treatment with lysosomotropic agents significantly enhances the melanin content of BLOC-1-deficient melanocytes. Our data suggest that upregulation of mistargeted cargoes can facilitate reprogramming of a subset of endolysosomes to generate some functions of lysosome-related organelles.

Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for MFSD12, HMG20B, OCA2, MITF, LEF1, TRPS1, BLOC1S6 and CYB561A3 that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an OCA2 enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of MITF, LEF1 and TRPS1, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified CYB561A3 as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.

Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KIIα and PI4KIIβ) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIβ with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KIIα and PI4KIIβ function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation toward melanosomes by generating local pools of PtdIns4P. The data show that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are downstream of likely redundant functions in BLOC-1-dependent tubule initiation.

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.

SUMMARY: Color variation is a frequent evolutionary substrate for camouflage in small mammals but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus , exhibits a range of colors including the cinnamon morph which has a similar color to the brown bear, U. arctos , and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus ( P < 10 ⁻¹³ ). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 ( TYRP1 ) that impaired protein localization and decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos , but is not fixed across the range. HIGHLIGHTS: The cinnamon morph of American black bears and brown bears have different missense mutations in TYRP1 that account for their similar coloration TYRP1 variants in American black bears and brown bears are loss-of-function alleles associated with impaired protein localization to melanosomes In American black bears, the variant causing the cinnamon morph arose 9,360 years ago in the western lineage where it provides an adaptive advantage, and has spread northwards and eastwards by migration

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.

Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KIIα and PI4KIIβ) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of PI4KIIα or PI4KIIβ with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KIIα and PI4KIIβ function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation towards melanosomes by generating local pools of PtdIns4P. The data demonstrate that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are likely downstream of redundant functions in BLOC-1-dependent tubule initiation. SUMMARY Contents are delivered from early endosomal intermediates to maturing melanosomes through tubular transport carriers. Zhu et al show that two type II phosphatidylinositol kinases, PI4KIIα and PI4KIIβ, sequentially generate phosphatidylinositol-4-phosphate during tubule initiation and elongation for ultimate melanosome content delivery.

Synopsis: Melanins, the main pigments of the skin and hair in mammals, are synthesized within membrane-bound organelles of melanocytes called melanosomes. Melanosome structure and function are determined by a cohort of resident transmembrane proteins, many of which are expressed only in pigment cells and localize specifically to melanosomes. Defects in the genes that encode melanosome-specific proteins or components of the machinery required for their transport in and out of melanosomes underlie various forms of ocular or oculocutaneous albinism, characterized by hypopigmentation of the hair, skin, and eyes and by visual impairment. We review major components of melanosomes, including the enzymes that catalyze steps in melanin synthesis from tyrosine precursors, solute transporters that allow these enzymes to function, and structural proteins that underlie melanosome shape and melanin deposition. We then review the molecular mechanisms by which these components are biosynthetically delivered to newly forming melanosomes—many of which are shared by other cell types that generate cell type-specific lysosome-related organelles. We also highlight unanswered questions that need to be addressed by future investigation.