The Recycling Endosome Protein Rab17 Regulates Melanocytic Filopodia Formation and Melanosome Trafficking

Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072 QLD, Australia.
Traffic (Impact Factor: 4.35). 02/2011; 12(5):627-43. DOI: 10.1111/j.1600-0854.2011.01172.x
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Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.

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Available from: Mitsunori Fukuda, Oct 07, 2014
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    • "Rab27A, whose deficiency causes type 2 GS, was the first Rab protein discovered to be associated with pigmentation (Ménasché et al., 2000; Wilson et al., 2000), and it has been shown to regulate actin-dependent melanosome transport through formation of a tripartite protein complex with Slac2-a/melanophilin and myosin- Va (reviewed in Fukuda, 2005). Rab1A and Rab36 are involved in movements on microtubules through their roles in anterograde melanosome transport and retrograde melanosome transport, respectively (Ishida et al., 2012; Matsui et al., 2012), and Rab11B and Rab17 are involved in the melanosome transfer step from melanocytes to keratinocytes (Beaumont et al., 2011; Tarafder et al., 2014). Two closely related Rabs, Rab32 and Rab38, cooperatively regulate trafficking of melanogenic enzymes, e.g., tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in the melanosome biogenesis step (Wasmeier et al., 2006; Tamura et al., 2009; Tamura et al., 2011; Bultema et al., 2012). "
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    ABSTRACT: Varp (VPS9-ankyrin repeat protein) was originally identified as an activator of small GTPase Rab21 through its VPS9 domain, but it has subsequently been shown to function as a Rab32/38 effector through its first ANKR1 domain. Although these functions of Varp are important for melanogenesis, Varp contains a second ANKR2 domain, whose function remained completely unknown. Here we identified Rab40C, an atypical Rab containing a SOCS box that recruits a ubiquitin ligase complex, as a novel ANKR2-binding protein and investigated its involvement in melanogenic enzyme trafficking in melanocytes. The results showed that overexpression of Rab40C in melanocytes caused a dramatic reduction in melanogenic enzyme Tyrp1 signals by promoting proteasomal degradation of Varp in a SOCS-box-dependent manner and that knockdown of Rab40C in melanocytes caused an increase in the amount of Varp. Intriguingly, Rab40C knockdown also caused a dramatic reduction in Tyrp1 signals, the same as Varp overexpression did. These findings indicated that Rab40C is a previously unexpected regulator of Tyrp1 trafficking in melanocytes through controlling the proteasomal degradation of Varp. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 03/2015; 4(3). DOI:10.1242/bio.201411114 · 2.42 Impact Factor
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    • "It showed that multiple filopodia were associated with the pigment globules which consisted of multiple melanosomes tightly packaged into a single membrane structure. The observation that pigment globules appeared to aggregate and arise from the tips of the filopodia suggests that filopoida play an important role in shedding pigment globules [19] [20] [21]. AFM, which is gaining ever-increasing attention in biology, is one type of the scanning probe microscopy with high resolution at nanometer level. "
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    ABSTRACT: Background Skin pigmentation is accomplished by production of melanin in melanosome and by transfer of these organelles from melanocytes (MCs) to surrounding keratinocytes (KCs). However, the detailed mechanism is still unknown. Objective We aimed to investigate the morphological structure changes on human epidermal MCs and KCs, which were either mono-cultured or co-cultured, with or without the treatment of both α-Melanocyte-stimulating hormone (α-MSH) and prostaglandin E2 (PGE2), by atomic force microscopy (AFM) and to provide more direct proofs for process of melanosome transfer. Methods Human epidermal MCs and KCs were isolated and co-cultured with 1:10 ratio in a defined Keratinocyte-serum free medium (K-SFM). After exposure with 100. nM α-MSH or 20. μM PGE2 for 3 days, cells were fixed with 0.5% glutaraldehyde and AFM images of scanning observation were captured by contacting and tapping model under normal atmospheric pressure and temperature. ResultsIt showed that human epidermal MCs in culture had secondary or tertiary branches. Except for globular granules structure on the surface of dendrites, some filopodia were protruded on the tips and lateral sides of the dendrites. The administration of α-MSH and PGE2 made not only the dendrites thinner and longer, but also the globular granules more intensive and denser. Many spheroid granules were shed from branches of dendrite and most of them adhered with dense filopodia. Compared with untreated group, the number of filopodia per cell, diameter of filopodia, and shedding spheroid granules per field all increased following α-MSH and PGE2 exposure (. P<. 0.05, n=. 3). However, many crest-like protrusions, which were distributed homogenously on the surface of mono-cultured KCs, were less changed after α-MSH and PGE2 exposure. In co-culture model, α-MSH and PGE2 increased the number of transferred melanosomes in KCs under laser confocal microscopic examination. Filopodia were observed only on the adhesion area of KCs and MCs in a coiled style by AFM examination. In addition, the number of filopodia per field, diameter of filopodia and shedding spheroid granules per field all increased after the administration of α-MSH and PGE2 (. P<. 0.05, n=. 3). Conclusions Our data suggest that shedding spheroid granules, filopodia delivery and KC phagocytosis are major mode of melanosome transfer between MCs and KCs. PGE2, as well as α-MSH, drives melanosome transfer by promoting filopodia delivery and numbers of shedding spheroid granules in MCs, but no direct morphological effects on KCs. These findings open a new path in our understanding of MCs-KCs communication regulating pigmentation.
    Journal of Dermatological Science 09/2014; 76(3). DOI:10.1016/j.jdermsci.2014.09.005 · 3.42 Impact Factor
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    • "RAB17 particularly, is phylogenetically very close to RAB5 genes (RAB5A, RAB5B and RAB5C) [31]. RAB17 expression [32] is regulated by MITF in mammalian cell lines (melanomas) and melanocytes with RAB17 knockdown have reduced filopodia formation, leading to impaired melanosome transfer. Then, RAB17 might make some contribution to the quail plumage colour but it is probably not responsible for lavender in quail because hybrids between lavender chicken and quail would have the RAB17 function rescued by the normal chicken RAB17, and should have shown a wild-type, undiluted, plumage colour, which they did not [14]. "
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    ABSTRACT: Background The lavender phenotype in quail is a dilution of both eumelanin and phaeomelanin in feathers that produces a blue-grey colour on a wild-type feather pattern background. It has been previously demonstrated by intergeneric hybridization that the lavender mutation in quail is homologous to the same phenotype in chicken, which is caused by a single base-pair change in exon 1 of MLPH. Results In this study, we have shown that a mutation of MLPH is also associated with feather colour dilution in quail, but that the mutational event is extremely different. In this species, the lavender phenotype is associated with a non-lethal complex mutation involving three consecutive overlapping chromosomal changes (two inversions and one deletion) that have consequences on the genomic organization of four genes (MLPH and the neighbouring PRLH, RAB17 and LRRFIP1). The deletion of PRLH has no effect on the level of circulating prolactin. Lavender birds have lighter body weight, lower body temperature and increased feed consumption and residual feed intake than wild-type plumage quail, indicating that this complex mutation is affecting the metabolism and the regulation of homeothermy. Conclusions An extensive overlapping chromosome rearrangement was associated with a non-pathological Mendelian trait and minor, non deleterious effects in the lavender Japanese quail which is a natural knockout for PRLH.
    BMC Genomics 08/2012; 13(1):442. DOI:10.1186/1471-2164-13-442 · 3.99 Impact Factor
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