The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform

Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of Melanosome number, shape and movement in the zebrafish retinal pigment epithelium by OA1 and PMEL
    Journal of Cell Science, 2015
    Co-Authors: Thomas Burgoyne, M. N. O'connor, D F Cutler, Miguel C. Seabra, Clare E Futter
    Abstract:

    Analysis of Melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of Melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in Melanosome number, recapitulating a key feature of the mammalian disease where reduced Melanosome numbers precede macroMelanosome formation. We further show that PMEL, a key component of mammalian Melanosome biogenesis, is required for the generation of cylindrical Melanosomes in zebrafish, which in turn is required for Melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical Melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical Melanosomes enter the apical processes. Taken together, our findings indicate that Melanosome number and shape are independently regulated and that Melanosome shape controls a function in the RPE that depends on localisation in the apical processes.

  • rab27a and myova are the primary mlph interactors regulating Melanosome transport in melanocytes
    Journal of Cell Science, 2007
    Co-Authors: Alistair N. Hume, Dmitry S Ushakov, Abul K Tarafder, Michael A Ferenczi, Miguel C. Seabra
    Abstract:

    Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for Melanosome targeting and stability of Mlph. Mutants and siRNA that affect Mlph-MyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to Melanosomes, MyoVa but not EB1 interaction is required for transport of Melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on Melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows Melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and Melanosome transfer.

  • The ternary Rab27a-Myrip-Myosin VIIa complex regulates Melanosome motility in the retinal pigment epithelium
    TRAFFIC, 2007
    Co-Authors: Miguel C. Seabra
    Abstract:

    The retinal pigment epithelium (RPE) contains Melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE Melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE Melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to Melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in Melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile Melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of Melanosomes onto actin filaments, a process that ensures Melanosome movement towards the cell periphery.

  • a coiled coil domain of melanophilin is essential for myosin va recruitment and Melanosome transport in melanocytes
    Molecular Biology of the Cell, 2006
    Co-Authors: Alistair N. Hume, Jose S Ramalho, Elena V Sviderskaya, Abul K Tarafder, Miguel C. Seabra
    Abstract:

    Melanophilin (Mlph) regulates retention of Melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the Melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel Melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to Melanosomes correlated with rescue of Melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for Melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of Melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

  • the role of rab27a in the regulation of Melanosome distribution within retinal pigment epithelial cells
    Molecular Biology of the Cell, 2004
    Co-Authors: Clare E Futter, Jose S Ramalho, G B Jaissle, Mathias W Seeliger, Miguel C. Seabra
    Abstract:

    Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of Melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of Melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, Melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, Melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates Melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar Melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.

  • Rab7B/42 Is Functionally Involved in Protein Degradation on Melanosomes in Keratinocytes.
    Cell structure and function, 2020
    Co-Authors: Soujiro Marubashi, Mitsunori Fukuda
    Abstract:

    Keratinocytes uptake Melanosomes from melanocytes and retain them in the perinuclear region, where they form melanin caps. Although these processes are crucial to protecting nuclear DNA against ultraviolet injury, the molecular basis of Melanosome uptake and decomposition in keratinocytes is poorly understood. One of the major reasons for its being poorly understood is the lack of a specific marker protein that can be used to visualize or monitor Melanosomes (or Melanosome-containing compartments) that have been incorporated into keratinocytes. In this study, we performed a comprehensive localization screening for mammalian Rab family small GTPases (Rab1-45) and succeeded in identifying 11 Rabs that were enriched around Melanosomes that had been incorporated into keratinocytes. We also established a new assay by using a recently developed Melanosome probe (called M-INK) as a means of quantitatively assessing the degradation of proteins on incorporated Melanosomes in control and each of a series of Rab-knockdown keratinocytes. The results showed that knockdown or CRISPR/Cas9-mediated knockout of Rab7B (also identified as Rab42) in keratinocytes caused strong inhibition of protein degradation on Melanosomes. Our findings indicated that Rab7B/42 is recruited to Melanosome-containing compartments and that it promotes protein degradation on Melanosomes in keratinocytes.Key words: degradation, keratinocytes, melanocytes, Melanosome, Rab small GTPase.

  • SNARE dynamics during Melanosome maturation.
    Biochemical Society transactions, 2018
    Co-Authors: Norihiko Ohbayashi, Mitsunori Fukuda
    Abstract:

    Historically, studies on the maturation and intracellular transport of Melanosomes in melanocytes have greatly contributed to elucidating the general mechanisms of intracellular transport in many different types of mammalian cells. During Melanosome maturation, Melanosome cargoes including melanogenic enzymes (e.g. tyrosinase) are transported from endosomes to immature Melanosomes by membrane trafficking, which must require a membrane fusion process likely regulated by SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors]. In the present study, we review the literature concerning the expression and function of SNAREs (e.g. v-SNARE vesicle-associated membrane protein 7 and t-SNAREs syntaxin-3/13 and synaptosomal-associated protein-23) in melanocytes, especially in regard to the fusion process in which Melanosome cargoes are finally delivered to immature Melanosomes. We also describe the recent discovery of the SNARE recycling system on mature Melanosomes in melanocytes. Such SNARE dynamics, especially the SNARE recycling system, on Melanosomes will be useful in understanding as yet unidentified SNARE dynamics on other organelles.

  • M-INK, a novel tool for visualizing Melanosomes and melanocores
    Journal of biochemistry, 2017
    Co-Authors: Morié Ishida, Soujiro Marubashi, Mitsunori Fukuda
    Abstract:

    Melanosome transfer from melanocytes to surrounding keratinocytes is one of the crucial but less well-characterized steps in the process of skin pigmentation. Although several markers have generally been used to detect Melanosomes in melanocytes, no suitable markers for Melanosomes that have been transferred into keratinocytes have ever been reported. The melanocore-interacting Kif1c-tail (M-INK) probe we developed and reported here specifically recognizes melanocores and thus makes visualizing Melanosomes that have been incorporated into keratinocytes possible even in a fluorescent field. M-INK staining makes it possible to reconstruct 3D images of Melanosome-containing keratinocytes, and thereby precisely localize Melanosomes in keratinocytes.

  • Rab1A regulates anterograde Melanosome transport by recruiting kinesin-1 to Melanosomes through interaction with SKIP
    Scientific reports, 2015
    Co-Authors: Morié Ishida, Norihiko Ohbayashi, Mitsunori Fukuda
    Abstract:

    Rab1A regulates anterograde Melanosome transport by recruiting kinesin-1 to Melanosomes through interaction with SKIP

  • The GTPase-deficient Rab27A(Q78L) mutant inhibits Melanosome transport in melanocytes through trapping of Rab27A effector protein Slac2-a/melanophilin in their cytosol: development of a novel Melanosome-targetinG tag.
    The Journal of biological chemistry, 2014
    Co-Authors: Morié Ishida, Saki P. Arai, Norihiko Ohbayashi, Mitsunori Fukuda
    Abstract:

    The small GTPase Rab27A is a crucial regulator of actin-based Melanosome transport in melanocytes, and functionally defective Rab27A causes human Griscelli syndrome type 2, which is characterized by silvery hair. A GTPase-deficient, constitutively active Rab27A(Q78L) mutant has been shown to act as an inhibitor of Melanosome transport and to induce perinuclear aggregation of Melanosomes, but the molecular mechanism by which Rab27A(Q78L) inhibits Melanosome transport remained to be determined. In this study, we attempted to identify the primary cause of the perinuclear Melanosome aggregation induced by Rab27A(Q78L). The results showed that Rab27A(Q78L) is unable to localize on mature Melanosomes and that its inhibitory activity on Melanosome transport is completely dependent on its binding to the Rab27A effector Slac2-a/melanophilin. When we forcibly expressed Rab27A(Q78L) on mature Melanosomes by using a novel Melanosome-targeting tag that we developed in this study and named the MST tag, the MST-Rab27A(Q78L) fusion protein behaved in the same manner as wild-type Rab27A. It localized on mature Melanosomes without inducing Melanosome aggregation and restored normal peripheral Melanosome distribution in Rab27A-deficient cells. These findings indicate that the GTPase activity of Rab27A is required for its Melanosome localization but is not required for Melanosome transport.

Qiuyu Gong - One of the best experts on this subject based on the ideXlab platform.

Jin Zhou - One of the best experts on this subject based on the ideXlab platform.

Clare E Futter - One of the best experts on this subject based on the ideXlab platform.

  • coming or going un bloc ing delivery and recycling pathways during Melanosome maturation
    Journal of Cell Biology, 2016
    Co-Authors: Clare E Futter, Daniel F Cutler
    Abstract:

    Melanosome biogenesis requires successive waves of cargo delivery from endosomes to immature Melanosomes, coupled with recycling of the trafficking machinery. Dennis et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201605090) report differential roles for BLOC-1 and BLOC-3 complexes in delivery and recycling of melanosomal biogenetic components, supplying directionality to Melanosome maturation.

  • Regulation of Melanosome number, shape and movement in the zebrafish retinal pigment epithelium by OA1 and PMEL
    Journal of Cell Science, 2015
    Co-Authors: Thomas Burgoyne, M. N. O'connor, D F Cutler, Miguel C. Seabra, Clare E Futter
    Abstract:

    Analysis of Melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of Melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in Melanosome number, recapitulating a key feature of the mammalian disease where reduced Melanosome numbers precede macroMelanosome formation. We further show that PMEL, a key component of mammalian Melanosome biogenesis, is required for the generation of cylindrical Melanosomes in zebrafish, which in turn is required for Melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical Melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical Melanosomes enter the apical processes. Taken together, our findings indicate that Melanosome number and shape are independently regulated and that Melanosome shape controls a function in the RPE that depends on localisation in the apical processes.

  • Melanosome maturation defect in rab38-deficient retinal pigment epithelium results in instability of immature Melanosomes during transient melanogenesis
    MOL BIOL CELL, 2007
    Co-Authors: Clare E Futter
    Abstract:

    Pathways of Melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce Melanosomes. Here, we show that progression from pmel17-positive preMelanosomes to tyrosinase-positive mature Melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of Melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced Melanosome numbers, but a continuing low level of Melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for Melanosome biogenesis. In cht RPE, preMelanosomes form but delivery of tyrosinase is impaired. PreMelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature Melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing Melanosome from the activity of lumenal acid hydrolases.

  • the role of rab27a in the regulation of Melanosome distribution within retinal pigment epithelial cells
    Molecular Biology of the Cell, 2004
    Co-Authors: Clare E Futter, Jose S Ramalho, G B Jaissle, Mathias W Seeliger, Miguel C. Seabra
    Abstract:

    Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of Melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of Melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, Melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, Melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates Melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar Melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.