N-Ethylmaleimide–Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium

Albinism is a congenital disorder that affects roughly 1 in 17,000 people worldwide1–3 and is characterized by a complete or partial deficiency of melanin. Ocular defects associated with albinism include nystagmus, foveal hypoplasia, low acuity, and photophobia.4,5 Hypopigmentation can occur in the eyes and skin (oculocutaneous albinism [OCA]), eyes only (ocular albinism [OA]), or can be accompanied by more severe phenotypes in the relatively rare syndromic forms of albinism, such as Hermansky-Pudlak and Chediak-Higashi syndromes. In all known forms of albinism, hypopigmentation results from mutations in genes encoding proteins involved in melanin synthesis (melanogenesis)4,6 or in melanosome transport (e.g., Refs. 7, 8). In the eye, albinism affects the retinal pigment epithelium (RPE), a layer of pigmented cells closely apposed to the photoreceptors at the back of the retina. Proper pigmentation of the RPE is critical for normal retinal development.9 Melanosomes are specialized lysosome-related organelles that contain pheomelanin and eumelanin pigments. Melanosomes in the RPE aid the visual system by absorbing stray photons to prevent light scatter and focus light onto the photoreceptors.10 Melanogenesis involves the trafficking of melanogenic enzymes to forming melanosomes, facilitating pigment synthesis and deposition, a process that occurs through several well-defined stages.11–14 In mammals, stage I premelanosomes, still a part of the endocytic pathway, undergo membrane invaginations to form intralumenal vesicles and begin to develop a proteinaceous fibril meshwork. This meshwork becomes fully formed in stage II melanosomes. Melanin precursors and enzymes are trafficked to stage II melanosomes, and melanin aggregates on the surface of the meshwork, generating a stage III melanosome with characteristic pigmented striations. Melanin aggregation continues until the organelle is completely filled in and becomes a mature stage IV melanosome. This process is well conserved amongst vertebrates, the only notable difference being that, in fish, melanin aggregates on the surfaces of intralumenal vesicles instead of on the striated meshwork.15 Melanogenesis depends on proper vesicular targeting and membrane fusion. Indeed, mutations in the genes encoding a number of components of the endocytic pathway result in albinism.6,16 Despite the number of albinism causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has yet been identified, suggesting that our understanding of the proteins involved in melanogenesis may be incomplete. Forward genetic screens in animal model systems can be very useful in this regard by utilizing a high-throughout and unbiased screening approach to identify mutations in a process of interest. Highlighting this utility, our laboratory previously reported a forward genetic screen that identified ∼20 mutations with defects in eye development.17 Of this collection, two mutants presented with oculocutaneous albinism, and in this study, we report the cloning and characterization of these mutations and identify them as mutant alleles affecting the N-ethylmaleimide–sensitive factor (nsfb) gene. N-ethylmaleimide–sensitive factor (Nsf) is a critical regulator of membrane fusion; NSF was initially identified in yeast as an enzyme required for protein transport between Golgi stacks18,19 and has since been identified as a AAA superfamily (ATPase associated with various cellular activities) protein that consists of three domains: two ATPase domains (D1 and D2) and an N-terminal domain (NSF-N).20–22 The NSF protomers form a hexamer, and this constitutes part of the soluble NSF attachment protein (SNAP) complex, which mediates vesicular fusion by disassembling SNARE (SNAP receptor) complexes. SNARE complexes form when SNARE proteins on different membranes interact and provide the requisite energy for membrane fusion by forming four-helix bundles and bringing the membranes into close apposition.23 As a part of the SNAP complex, NSF couples hydrolysis of ATP with the disassembly of SNARE complexes following membrane fusion, freeing individual SNARE proteins to participate in future vesicular fusion events.22,24,25 Zebrafish possess two nsf genes: nsfa and nsfb.26 nsfa is required for myelination of axons,27 the secretion of trophic factors during the maturation of hair cell synapses,28 and neuropeptide-dependent visually mediated background adaptation.26 In contrast, very little is known about nsfb. Here we demonstrate that nsfb mutants display hypopigmentation of the RPE, resulting from a defect in melanogenesis.