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No / The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanomacell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.

Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

Während bei Prokaryonten, Pflanzen und Pilzen der Transport von Zuckern über Membranen mit Hilfe von Disaccharid-Transportern erfolgt, ist bei Tieren, vor allem Säugern, nach der allgemein anerkannten Vorstellung nur der Transport von Monosacchariden bekannt. Die vorliegende Dissertation, in der ScrT, ein bislang unerforschtes Protein aus der Fruchtfliege Drosophila melanogaster untersucht und charakterisiert wurde, beinhaltet Daten, die diese Vorstellung in Frage stellen.Die Expression von ScrT in Hefestämmen ermöglichte das Wachstum auf einem Minimalmedium, das als einzige C-Quelle Saccharose enthält, und Tests mit radioaktiv (14C) markierter Saccharose belegten, dass Hefestämme, die das untersuchte Protein heterolog exprimieren, Transportaktivität für dieses Disaccharid aufweisen.Die mittels Immunhistochemie ermittelte in vivo-Lokalisation von ScrT führte zu unterschiedlichen Resultaten. Zum einen waren in den adulten Fruchtfliegen deutliche Signale im Mitteldarm zu erkennen. Zum anderen wurden vesikuläre Strukturen in Follikelzellen der Ovarien gefärbt. Diese offenbar Melanin enthaltenden Strukturen fanden sich auch im Chorion der abgelegten Embryonen. Somit scheint es sich bei diesen Vesikeln um Melanosomen oder funktionell analoge Organellen zu handeln, in deren Membran das untersuchte Protein lokalisiert ist und die sekretorisch bei der Bildung des Chorions an dieses abgegeben werden. Die Lokalisierung von ScrT in den Follikelzellen wurde mittels in situ-Hybridisierung bestätigt.Den vorliegenden Daten zufolge dürfte ScrT unter anderem den Eintritt von Saccharose in Melanosomen regulieren, wo das chemisch relativ inerte Disaccharid als kompatibler Osmolyt fungieren und das osmotische Gleichgewicht während der Polymerisation von Melanin ausbalancieren könnte. Im Einklang mit dieser Hypothese steht auch, dass Mutationen in MATP, dem menschlichen Ortholog von ScrT, zu einer bislang wenig erforschten Form des Albinismus (OCA4) führen.

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Melanosome biogenesis is a highly regulated endosomal maturation process wherein structural fibers harbouring immature melanosomes acquires its biosynthetic proteins through the secretory pathway and finally matures into a functional organelle. These processes were shown to be dependent on several cytosolic protein complexes such as AP (adaptor protein)-1, AP-3, BLOC (biogenesis of lysosome-related organelles complex)-1, -2 and -3; in addition to kinesin motor KIF13A and Rab GTPases 7, 32 or 38. Mutations in the subunits of these complexes or Rab38 result into defective melanosome maturation leading to occulocutaneous albinism, a clinical phenotype commonly observed in Hermansky-Pudlak syndrome (HPS). Moreover, molecular function of these complexes in regulating the biogenesis of melanosome is partially known. The delivery of cargo to maturing melanosomal membranes requires fusion machinery that includes Rab GTPases, tethering factors and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. However, the SNAREs involved in the transport of cargo to melanosomes is poorly understood. In this study entitled as “understanding the SNARE dynamics during melanosome biogenesis” we focus on functional role of endosomal Qa-SNARE protein, Syntaxin 13 (formally called STX12, herein referred to as STX13) in the organelle biogenesis and its transport in and out of melanosome. Moreover, these studies show that STX13-mediated cargo transport require a melanosomal membrane localized R-SNARE VAMP7 and these SNAREs are interdependent on each other in regulating their steady state distribution. In addition, this study illustrated the possible mechanism of SNARE recycling which occurs indirectly through AP-3 complex. Thus, these studies underscore the STX13‟s role in cargo transport to maturating melanosomes and its trafficking routes to and from the melanosomes. Chapter-I describes the literature review on melanosome biogenesis; Chapter-II lists the experimental procedures used in this study and Chapter-III to V focuses on results and discussion, segregated into three sections. Chapter-III: Screening and identification of endosomal SNAREs involved in the trafficking of melanosomal proteins. Our preliminary RNAi screen for SNAREs involved in melanosome biogenesis revealed STX13 as one of the Qa-SNARE affecting pigmentation and cargo transport. STX13, a recycling endosomal SNARE has been reported to interact with pallidin, a subunit of BLOC-1; however the functional role of this interaction in pigment formation is unknown. In addition, previous studies from our lab have shown that STX13 colocalize with endosomal Rab11 and partially with EEA1- or Rab5-positive organelles in melanocytes. Together, these observations insinuated us to characterize the functional role of STX13 in melanosome biogenesis. Upon STX13 inactivation, wild type mouse melanocytes showed hypopigmentation due to mistargeting of cargo such as TYRP1 and TYR to lysosomes. Knockdown of STX13 dramatically decrease the population of immature and mature melanosomes. Moreover, STX13 associate with the melanosome cargo on endosomal tubular structures. In addition, deletion of regulatory domain in STX13 increases the cargo transport to melanosomes due to its increased SNARE activity. This is possibly due to loss in intracellular regulation of SNARE occur through multiple factors such as SM (Sec1p/Munc18) proteins. Together this data suggests that STX13 mediates cargo transport to melanosomes from recycling endosomes. Chapter-IV: Functional characterization of the SNAREs involved in melanosomal maturation. Several in vitro studies have shown that a set of four SNAREs such as Qa, Qb, Qc (or Qbc) and R control the membrane fusion event duing the cargo transport. Additionally, this process is further regulated by SM proteins in in vivo. Electron microscopic studies in melanocytes have shown that melanosomal proteins were delivered to the melanosomal membrane through recycling endosomal tubular domains. Moreover, our RNAi screen show that STX13 possibly acts as Qa-SNARE in mediating the fusion events between melanosomal membranes and the endosomal tubular or vesicular intermediates. However, the role of other SNAREs for this membrane transport is unknown. It has been shown that the expression of VAMP family SNAREs such as VAMP3, VAMP7 and VAMP8 increased with melanogenesis upon differentiation of melanoma cells. VAMPs belong to the class of R-SNAREs, in which VAMP7 is known to interact with VARP (abbreviation) and AP-3 (mediates the trafficking of TYR) separately, and these molecules are known to regulate the cargo transport to melanosomes. However, the precise role of VAMP7 in pigment granule maturation is unknown. Therefore, we set out to characterize the functional role of VAMP7 in melanosome biogenesis. VAMP7 has been shown to localizes to multiple sub-cellular compartments and regulate the several transport steps in other cell types. Our study found that GFP-epitope tagged either human or rat VAMP7 localize to melanosomes at steady state in wild type mouse melanocytes. Knockdown of VAMP7 causes hypopigmentation of melanocytes and misroutes the cargo to lysosomes. Further, the inactivation of VAMP7 in melanocytes phenocopies the STX13 depletion, suggesting both the SNAREs are required for the melanosome biogenesis. In addition, knockdown of STX13 target the VAMP7 to lysosomes; while inactivation of VAMP7 affect the localization of STX13 to recycling tubular structures. Subsequently, the dominant active mutants of STX13 were not able to rescue the pigmentation or cargo transport defects in VAMP7 knockdown melanocytes. Together, the data suggests that STX13 functions from recycling endosomes and VAMP7 on melanosome membrane for the transport of cargo to melanosomes Chapter-V: Understanding the mechanism of STX13 recycling during melanosome biogenesis. At steady state, SNAREs are localized to the membranes of specific organelles where they mediate or regulate the membrane fusion. During this process, three or two Q-SNAREs on one membrane (in a trans-SNARE complex, possibly formed by Qa, Qb, Qc or Qbc) interact with a R-SNARE on another member to form a SNAREpin complex. Post-fusion, SNAREs are disassembled by SNAP and NSF proteins and then recycled back to the original compartment for next round of fusion. Here, we address the mechanism of post-fusion recycling of STX13 from melanosomes to endosomes. Previous studies have shown that STX13 mislocalize to melanosomes in AP-3-deficient melanocytes, suggesting a role for AP-3 in recycling the SNARE from melanosomes. Bioinformatic analysis of the N-terminal region of STX13 revealed the presence of two canonical adaptor binding motifs 3YGP6L and KETNE80L81L, resembling the tyrosine-based (YXXø) and dileucine-based motif [DE]XXXL[LI], recognized by several adaptor proteins. Point mutagenesis of these motifs in STX13 had no effect on their steady state distribution indicating that STX13 possibly uses non-canonical residues for its recycling. Further, deletion of the N-terminal region (either 1-129 or 14-129 aa) in STX13 redistributes the SNARE to melanosomes. Moreover, the activity and the trafficking of recycling defective STX13 mutants are dependent on another HPS complex, BLOC-2 and the SNARE, VAMP7. Absence of 1-129 region in STX13 or mutations in the subunits of AP-3 perturbs the steady state localization of STX13 suggesting an indirect role for AP-3 in recycling of STX13 to endosome via non canonical motifs present in its 1-129 aa region.

Nosso trabalho apresenta um modelo de simulação de transtornos de pigmentação humana. Nosso modelo é formado por um conjunto de equações diferenciais que definem um sistema reação-difusão. Nosso sistema simula algumas características do sistema pigmentar humano. Alterações nesse sistema podem levar a desequilíbrios na distribuição de melanina na pele resultando em artefatos conhecidos como lesões de pigmentação. Nosso modelo tem como objetivo reproduzir essas alterações e assim sintetizar lesões de pigmentação humanas. Nosso sistema reação-difusão foi elaborado tomando como base dados biológicos a respeito da pele humana, do sistema pigmentar e do ciclo de vida dos melanócitos, que são as principais células envolvidas nesse tipo de transtorno. A simulação desse tipo de transtorno apresenta diversas aplicações em dermatologia como, por exemplo, suporte para o treinamento de dermatologistas e auxílio no diagnóstico de transtornos de pigmentação. No entanto, nosso trabalho se concentra em aplicações relacionadas com computação gráfica. Assim, nós também apresentamos um método para transferir os resultados do nosso modelo de simulação para texturas e imagens de pele humana. Nesse contexto, o nosso modelo contribui para a geração de texturas de pele mais realistas e consequentemente para a geração de modelos de serem humanos mais realistas. Além disso, nós também comparamos os resultados da nossa simulação com lesões de pigmentações reais objetivando avaliar a qualidade das lesões geradas pelo nosso modelo. Para realizar essa comparação nós extraímos métricas das lesões sintetizadas e das lesões reais e comparamos os valores dessas métricas. Com base nessa comparação, nós observamos que as lesões sintetizadas apresentam as mesmas características das lesões reais. Ainda, para efeito de comparações visuais, nós também apresentamos imagens de lesões reais lado a lado com imagens sintetizadas e podemos observar que o método utilizado para produzir imagens de lesões a partir do resultado do nosso modelo de simulação produz resultados que são indistinguíveis das imagens reais. / Our work presents a simulation model of human pigmentation disorders. Our model is formed by a set of differential equations that defines a reaction-diffusion system. Our system simulates some features of the human pigmentary system. Changes in this system can lead to imbalances in the distribution of melanin in the skin resulting in artifacts known as pigmented lesions. Our model aims to reproduce these changes and consequently synthesize human pigmented lesions. Our reaction-diffusion system was developed based on biological data regarding human skin, pigmentary system and melanocytes life cycle. The melanocytes are the main cells involved in this type of human skin disorders. The simulation of such disorders has many applications in dermatology, for example, to assist dermatologists in diagnosis and training related to pigmentation disorders. However, our study focuses on applications related to computer graphics. Thus, we also present a method to transfer the results of our simulation model for textures and images of human skin. In this context, our model contributes to the generation of more realistic skin textures and consequently for the generation of more realistic human models. Moreover, we also compared the results of our simulation with real pigmented lesions to evaluate the quality of the lesions generated by our model. To perform this comparison we measured some features of real and synthesized pigmented lesions and we compared the results of these measurements. Based on this comparison, we observed that synthesized lesions exhibit the same characteristics of real lesions. Still, for the purpose of visual comparisons, we also present images of real lesions along with images of synthesized lesions. In this visual comparison, we can note that the method used to produce lesions images from the results of our simulation generates images that are indistinguishable from real images.