The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes

Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Rab27a is an essential component of melanosome receptor for myosin Va

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution in dilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5'-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.     

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin–myosin Va interaction

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan‐a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan‐a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan‐a melanocytes were co‐cultured with SP‐1 keratinocytes or stimulated by α‐MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.     

The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological processes, including adaptive immunity, innate immunity, and bone metabolism. Importantly, it is essential for activating NF-kappaB signaling pathway in response to interleukin-1 and Toll-like receptor ligands. Previously, we characterized TRAF6 to be a ubiquitin ligase. In combination with the ubiquitin conjugating enzyme complex Ubc13/Uev1A, TRAF6 could catalyze the formation on itself of unique Lys-63 linked polyubiquitin chain that positively regulated NF-kappaB signaling pathway. However, it remains unknown how this auto-ubiquitination process is regulated. In this study, we found that the coiled-coil domain of TRAF6 was essential for its auto-ubiquitination and activating NF-kappaB signaling pathway. This domain served not as the specific target where the polyubiquitin chain was linked, but as a specific bridge to recruit Ubc13/Uev1A.     

Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.     

Rab27a: A key to melanosome transport in human melanocytes

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.     

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.     

A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins.

In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242). The amino acid sequence of domain 1 indicates that it may be a coiled-coil oligomerization domain. We show here that domain 1 of Bcr forms a homotetramer. Tetramerization of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase and F-actin-binding functions of Abl. Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function as well as the ability of Bcr-Abl to transform Rat-1 fibroblasts or to abrogate interleukin-3 dependence in lymphoid cells. These results strongly suggest that Bcr-Abl oligomers are the active entities in transformation.     

A tissue-specific exon of myosin Va is responsible for selective cargo binding in melanocytes

Class V myosins are molecular motors used for intracellular transportation and organelle tethering. The mouse Myosin Va (MyoVa) is encoded by the dilute locus, which is alternatively spliced to generate several tissue specific isoforms. The tail of MyoVa is the putative cargo-binding domain. To determine the functions of different isoforms of MyoVa and the minimal cargo-binding region, we tagged various isoforms and different portions of the mouse MyoVa tail with a green fluorescent protein and examined their intracellular localizations in the mouse melan-a cells. We found that the amino acid sequence encoded by an alternatively spliced exon, exon F, is necessary for the selective binding of MyoVa to melanosome. The MyoVa isoforms lacking this amino acid sequence are not targeted to the melanosomes, but localized to the perinuclear region instead. These findings suggested that MyoVa is able to bind to more than one types of cargos, with the selectivities determined by alternative spliced sequences.     

Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.     

In vitro reconstitution of a transport complex containing Rab27a, melanophilin and myosin Va

Rab27a and melanophilin (Mlph) are required in vivo to form a melanosome receptor for myosin Va in which Rab27a anchored in the melanosome membrane recruits Mlph, which in turn recruits myosin Va. Here, we show by reconstitution using purified proteins that Rab27a and Mlph are sufficient to form a transport complex with myosin Va in vitro. These results suggest that additional proteins are not required in vivo for assembly of the myosin Va receptor, although other proteins may associate with this tripartite complex to regulate its activity and/or to assist Rab27a in anchoring the complex to the melanosome membrane.     

Myosin Va is required for P body but not stress granule formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.     

A novel sequence in the coiled-coil domain of Stat3 essential for its nuclear translocation

Stat3 is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation is unclear. In this study, by systematic deletion and site-directed mutagenesis we identified Arg-214/215 in the alpha-helix 2 region of the coiled-coil domain of Stat3 as a novel sequence element essential for its nuclear translocation, stimulated by epidermal growth factor as well as by interleukin-6. Furthermore, we identified Arg-414/417 in the DNA binding domain as also required for the nuclear localization of Stat3. This sequence element corresponds to Lys-410/413 of Stat1, a reported sequence for Stat1 nuclear translocation. On the other hand, Leu-411 of Stat3, corresponding to Leu-407 of Stat1, a necessary residue for Stat1 nuclear transport, is not essential for Stat3 nuclear import. The mutant of Arg-214/215 or Arg-414/417 was shown to be tyrosyl-phosphorylated normally but failed to enter the nucleus in response to epidermal growth factor or interleukin-6. The defect, however, can be rescued by the wild-type Stat3 but cannot be compensated by these two mutants. Mutations on Arg-414/417, but not Arg-214/215, destroy the DNA binding activity of Stat3. Our data for the first time identified a sequence element located in the coiled-coil domain that is involved in the ligand-induced nuclear translocation of Stat3. This novel sequence together with a conserved sequence element in the DNA binding domain coordinates to mediate the nuclear translocation of Stat3.     

Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells

To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.     

Toll‐like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll‐like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100‐positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.     

Dimerization of the human E3 ligase CHIP via a coiled-coil domain is essential for its activity

The Hsp70-interacting E3-ubiquitin ligase CHIP has been implicated in the decision as to whether a target protein enters the refolding or the degradation pathway. To further characterize the activity of CHIP we purified untagged Homo sapiens and Drosophila melanogaster CHIP (hCHIP, dCHIP). In contrast to other E3-ubiquitin ligases, both hCHIP and dCHIP proteins formed homodimers at physiological concentrations. We identified a predicted coiled-coil region in a mixed charge segment of the hCHIP and dCHIP sequence and found it to be necessary and sufficient for dimer formation. A mutant of hCHIP lacking this segment (hCHIPdelta-(128-229)) was incapable of dimer formation, but the segment by itself (hCHIP-(128-229)) readily dimerized. Furthermore, we demonstrated that dimerization is a prerequisite for activity of hCHIP in the reconstituted ubiquitination assay. Control of dimerization may thus provide a mechanism for regulation of CHIP activity.     

Making sense of melanosome dynamics in mouse melanocytes

Molecular motors drive most if not all organelle movements in Eukaryotic cells. These proteins are thought to bind to the organelle surface and, through the action of their mechanochemical domains, to translocate the organelle along a cytoskeletal track. In the case of the myosin family of molecular motors, the cytoskeletal track is filamentous actin. Microtubules serve as the cytoskeletal track for the kinesins and dyneins. While a considerable amount is known about the motors and tracks responsible for the bi-directional movement of pigment granules in fish and frog melanophores, relatively little is known about how melanosomes in mammalian melanocytes are transported out the cells dendritic arbor, accumulated at the ends of these dendrites, and transferred to keratinocytes. In this short review, we focus on the use of video microscopy to address these questions in mouse melanocytes, and we describe how an analysis of melanosome dynamics within wild type and dilute melanocytes shaped our thinking regarding the role of an unconventional myosin in melanosome transport and distribution.     

A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport

The Rab27a GTPase regulates diverse processes involving lysosome-related organelles, including melanosome motility in melanocytes, and lytic granule release in cytotoxic T lymphocytes. Toward an understanding of Rab27a function, we searched for proteins that interact with Rab27a(GTP) using the yeast two-hybrid system and identified JFC1/Slp1, a protein of unknown function. JFC1/Slp1 and related proteins, including melanophilin, contain a conserved amino-terminal domain similar to the Rab3a-binding domain of Rabphilin-3. We used several methods to demonstrate that this conserved amino-terminal domain is a Rab27-binding domain. We show that this domain interacts directly, and in a GTP-dependent manner with Rab27a. Furthermore, overexpression of this domain in melanocytes results in perinuclear clustering of melanosomes, suggesting that this region is sufficient for interaction with, and perturbation of function of, Rab27a in a physiological context. Thus, we identified a novel family of Rab27-binding proteins. We also show that melanophilin associates with Rab27a and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va. Thus, melanophilin can associate simultaneously with activated Rab27a and myosin Va via distinct regions, and serve as a linker between these proteins.