309 Dynlt3, a novel fundamental regulator of melanosome movement, distribution, acidity and transfer
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Melanosome
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Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation.
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Recycling embosoms (REs) are transient intermediates of endosomal network, constantly generated from early/sorting endosomes (EEs/SEs). Conventionally, these organelles function in recycling of many growth/nutrient/signalling receptors from SEs to the cell surface and maintain the cellular homeostasis in all cell types. Recent studies have shown that REs slightly diverted their function in specialized cells such as melanocytes for the delivery of melanogenic cargo to a set of lysosome-related organelles (LROs) called melanosomes. However, it is unknown how melanocytes modulate the trafficking routes of REs towards the biogenesis of melanosomes. Any alterations in this process result in occulocutaneous albinism, commonly observed in autosomal recessive disorder, Hermansky-Pudlak Syndrome (HPS). HPS is caused by mutations in nine genes in human and fifteen genes in mouse and the protein products of these genes were grouped in multiple endosomal protein complexes; BLOC (Biogenesis of Lysosome-related Organelles Complex)-1, -2, -3, AP (Adaptor Protein)-3 and HOPS (homotypic fusion and protein sorting). Studies from our laboratory and others have shown that REs deliver the melanin-synthesizing enzymes to melanosome in BLOC-1 and BLOC-2 dependent manner. On the other side, studies in fibroblasts have shown that the adaptor AP-1 and microtubule-dependent motor, KIF13A also regulates the formation of REs. In these studies, it was proposed that AP-1 binds to the cargo tails and interacts with motor KIF13A to generate the RE tubules, where BLOC-1 initiates the biogenesis. Nevertheless, the mechanism behind the biogenesis of REs and how these molecules synergistically control these processes is largely unknown. Additionally, the role of BLOC-2 in REs biogenesis never been implicated. Here we have attempted to study the mechanism of RE biogenesis and their role in pigment granule formation using HeLa and mouse melanocytes as model systems. In general, Rab GTPases (Rabs) regulate the several process of membrane trafficking including cargo sorting, membrane domain organization, tethering and fusion. We hypothesized that the biogenesis of RE is also regulated by one of the endosome localized Rab GTPases. Our RNAi screening against Rabs involved in regulating the RE length/number showed Rab22A as a potential candidate. Thus, we aim to study the role of Rab22A in RE biogenesis and its regulation in melanocyte pigmentation.
The current study entitled as “Understanding the role of Rab22A in recycling endosome biogenesis and melanocyte pigmentation” is divided into five chapters. Chapter-I outlines the review of literature on cell biology of intracellular organelles such as endocytic network and melanosomes. Chapter-II details the experimental procedures used in the study. Chapter-III to Chapter-V describes the results and discussion.
Chapter-III: Identification of endosomal Rab GTPases required for the dynamics of recycling endosomes
Endosomal Rabs are known to regulate various functions such as vesicle biogenesis, transport,…
Melanosome
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Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.
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Myosin VI has been implicated in various steps of organelle dynamics. However, the molecular mechanism by which this myosin contributes to membrane traffic is poorly understood. Here, we report that myosin VI is associated with a lysosome-related organelle, the melanosome. Using an actin-based motility assay and video microscopy, we observed that myosin VI does not contribute to melanosome movements. Myosin VI expression regulates instead the organization of actin networks in the cytoplasm. Using a cell-free assay, we showed that myosin VI recruited actin at the surface of isolated melanosomes. Myosin VI is involved in the endocytic-recycling pathway, and this pathway contributes to the transport of a melanogenic enzyme to maturing melanosomes. We showed that depletion of myosin VI accumulated a melanogenic enzyme in enlarged melanosomes and increased their melanin content. We confirmed the requirement of myosin VI to regulate melanosome biogenesis by analysing the morphology of melanosomes in choroid cells from of the Snell's waltzer mice that do not express myosin VI. Together, our results provide new evidence that myosin VI regulates the organization of actin dynamics at the surface of a specialized organelle and unravel a novel function of this myosin in regulating the biogenesis of this organelle.
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Organelle biogenesis
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MFN2
Organelle biogenesis
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Melanosome
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Biogenesis of lysosome-related organelles (LROs) complex-1 (BLOC-1) is an eight-subunit complex involved in lysosomal trafficking. Interacting proteins of these subunits expand the understanding of its biological functions. With the implementation of the naïve Bayesian analysis, we found that a human uncharacterized 20 kDa coiled-coil KxDL protein, KXD1, is a BLOS1-interacting protein. In vitro binding assays confirmed the interaction between BLOS1 and KXD1. The mouse KXD1 homolog was widely expressed and absent in Kxd1 knockout (KO) mice. BLOS1 was apparently reduced in Kxd1-KO mice. Mild defects in the melanosomes of the retinal pigment epithelia and in the platelet dense granules of the Kxd1-KO mouse were observed, mimicking a mouse model of mild Hermansky-Pudlak syndrome that affects the biogenesis of LROs.
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Melanosome
Rab
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ABSTRACT Sorting endosomes (SEs) are the regulatory hubs for sorting cargo to multiple organelles, including lysosome-related organelles, such as melanosomes in melanocytes. In parallel, melanosome biogenesis is initiated from SEs with the processing and sequential transport of melanocyte-specific proteins toward maturing melanosomes. However, the mechanism of cargo segregation on SEs is largely unknown. Here, RNAi screening in melanocytes revealed that knockdown of Rab4A results in defective melanosome maturation. Rab4A-depletion increases the number of vacuolar endosomes and disturbs the cargo sorting, which in turn lead to the mislocalization of melanosomal proteins to lysosomes, cell surface and exosomes. Rab4A localizes to the SEs and forms an endosomal complex with the adaptor AP-3, the effector rabenosyn-5 and the motor KIF3, which possibly coordinates cargo segregation on SEs. Consistent with this, inactivation of rabenosyn-5, KIF3A or KIF3B phenocopied the defects observed in Rab4A-knockdown melanocytes. Further, rabenosyn-5 was found to associate with rabaptin-5 or Rabip4/4′ (isoforms encoded by Rufy1) and differentially regulate cargo sorting from SEs. Thus, Rab4A acts a key regulator of cargo segregation on SEs. This article has an associated First Person interview with the first author of the paper.
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