Tissue-specific geometry and chemistry of modern and fossilized melanosomes reveal internal anatomy of extinct vertebrates.

Recent discoveries of nonintegumentary melanosomes in extant and fossil amphibians offer potential insights into the physiological functions of melanin not directly related to color production, but the phylogenetic distribution and evolutionary history of these internal melanosomes has not been characterized systematically. Here, we present a holistic method to discriminate among melanized tissues by analyzing the anatomical distribution, morphology, and chemistry of melanosomes in various tissues in a phylogenetically broad sample of extant and fossil vertebrates. Our results show that internal melanosomes in all extant vertebrates analyzed have tissue-specific geometries and elemental signatures. Similar distinct populations of preserved melanosomes in phylogenetically diverse vertebrate fossils often map onto specific anatomical features. This approach also reveals the presence of various melanosome-rich internal tissues in fossils, providing a mechanism for the interpretation of the internal anatomy of ancient vertebrates. Collectively, these data indicate that vertebrate melanins share fundamental physiological roles in homeostasis via the scavenging and sequestering of metals and suggest that intimate links between melanin and metal metabolism in vertebrates have deep evolutionary origins.

Nanosecond Laser Treatment for Age-Related Macular Degeneration Does Not Induce Focal Vision Loss or New Vessel Growth in the Retina.

Purpose: Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. Methods: Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. Results: Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. Conclusions: In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.

Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules.

To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV-vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects - modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents.

Inulavosin, a melanogenesis inhibitor, leads to mistargeting of tyrosinase to lysosomes and accelerates its degradation.

The melanosome is a highly specialized organelle where melanin is synthesized. Tyrosinase and tyrosinase-related protein-1 (Tyrp1) are major melanosomal membrane proteins and key enzymes for melanin synthesis in melanocytes. Inulavosin, a melanogenesis inhibitor isolated from Inula nervosa (Compositae), reduced the melanin content without affecting either the enzymatic activities or the transcription of tyrosinase or Tyrp1 in B16 melanoma cells. To our knowledge, this inhibitor is previously unreported. Electron-microscopic analyses revealed that inulavosin impaired late-stage development of melanosomes (stages III and IV), in which melanin is heavily deposited. However, it did not alter the early stages of melanosomes (stages I and II), when filamentous structure is observed. Immunofluorescence analyses showed that tyrosinase, but not Tyrp1, was specifically eliminated from melanosomes in cells treated with inulavosin. Unexpectedly, inulavosin specifically accelerated the degradation of tyrosinase but not other melanosomal/lysosomal membrane proteins (Tyrp1, Pmel17, and LGP85). The degradation of tyrosinase induced by inulavosin associated with lysosomes but not the proteasome. Interestingly, lysosomal protease inhibitors restored the melanogenesis but not the targeting of tyrosinase to melanosomes in the cells treated with inulavosin. Instead, colocalization of tyrosinase with lysosome-associated membrane protein-1 at late endosomes/multivesicular bodies and lysosomes was accentuated. Taken together, inulavosin inhibits melanogenesis as a result of mistargeting of tyrosinase to lysosomes.

Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis.

The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine (l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.