Functional Analysis of Slac2-c/MyRIP as a Linker Protein between Melanosomes and Myosin VIIa*[boxs]
2005; Elsevier BV; Volume: 280; Issue: 30 Linguagem: Inglês
10.1074/jbc.m501465200
ISSN1083-351X
AutoresTaruho S. Kuroda, Mitsunori Fukuda,
Tópico(s)Signaling Pathways in Disease
ResumoSlac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c·myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A·Slac2-c·myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells. Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c·myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A·Slac2-c·myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells. Rab27A is a member of the small GTPase Rab family (reviewed in Refs. 1Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2662) Google Scholar and 2Segev N. Sci. STKE. 2001; 100: RE11Google Scholar) and has recently been identified as a critical regulator of various types of membrane trafficking, including vesicle exocytosis in some secretory cells and melanosome transport in melanocytes (reviewed in Refs. 3Izumi T. Gomi H. Kasai K. Mizutani S. Torii S. Cell Struct. Funct. 2003; 28: 465-474Crossref PubMed Scopus (101) Google Scholar, 4Seabra M.C. Coudrier E. Traffic. 2004; 5: 393-399Crossref PubMed Scopus (159) Google Scholar, 5Fukuda M. J. Biochem. (Tokyo). 2005; 137: 9-16Crossref PubMed Scopus (186) Google Scholar). Eleven effector-type Rab27A-binding proteins have been identified to date: Slac2-a/melanophilin, Slac2-b, Slac2-c/MyRIP, Slp1/JFC1, Slp2-a, Slp3-a, Slp4/granuphilin, Slp5, rabphilin, Noc2, and Munc13-4 (5Fukuda M. J. Biochem. (Tokyo). 2005; 137: 9-16Crossref PubMed Scopus (186) Google Scholar). The members of the Slp and Slac2 families of these Rab27A-binding proteins contain a common Rab27A-binding domain at the N terminus (referred to as the Slp homology domain (SHD) 1The abbreviations used are: SHD, Slp homology domain; ABD, actin-binding domain; CFP, cyan fluorescent protein; GFP, green fluorescent protein; MBD, myosin-binding domain; RPE, retinal pigment epithelium; siRNA, small interfering RNA; YFP, yellow fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate. 1The abbreviations used are: SHD, Slp homology domain; ABD, actin-binding domain; CFP, cyan fluorescent protein; GFP, green fluorescent protein; MBD, myosin-binding domain; RPE, retinal pigment epithelium; siRNA, small interfering RNA; YFP, yellow fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate.) (6Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. 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Based on these observations, Slac2-c has been proposed to function as a linker protein between myosin VIIa and retinal melanosomes in RPE cells, although involvement of Rab27A·Slac2-c·myosin VIIa complex in melanosome transport in living cells has never been investigated. Despite its proposed function, Slac2-c has recently been shown to regulate granule exocytosis independently of myosin Va and myosin VIIa in pancreatic β cell lines (35Waselle L. Coppola T. Fukuda M. Iezzi M. El-Amraoui A. Petit C. Regazzi R. Mol. Biol. Cell. 2003; 14: 4103-4113Crossref PubMed Scopus (132) Google Scholar), PC12 cells (36Desnos C. Schonn J.S. Huet S. Tran V.S. El-Amraoui A. Raposo G. Fanget I. Chapuis C. Ménasché G. de Saint Basile G. Petit C. Cribier S. Henry J.P. Darchen F. J. Cell Biol. 2003; 163: 559-570Crossref PubMed Scopus (131) Google Scholar), and parotid acinar cells (37Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (92) Google Scholar), and thereby the function of Slac2-c as a myosin receptor has been questioned. To address the question of whether Slac2-c actually acts as a functional myosin receptor, we first investigated the function of the Slac2-c·myosin VIIa complex in the transport of Rab27A-bound melanosomes in skin melanocytes as a model. Slac2-c had a much higher affinity for myosin VIIa than for myosin Va and colocalized with myosin VIIa on melanosomes. The expression of Slac2-c together with myosin VIIa in Slac2-a-depleted melanocytes restored the normal distribution of melanosomes from perinuclear aggregation, whereas the expression of Slac2-c alone did not. We discuss the possible function of Slac2-c·myosin VIIa complex in retinal melanosome transport based on these findings. Plasmid Construction—Deletion mutants of Slac2-c (pEF-T7-Slac2-c-ΔSHD/ΔABD and -MBD) were constructed by conventional PCR using the following oligonucleotides with a BamHI site (underlined) or a stop codon (in boldface type): 5′-CGGATCCCGTCTGGAGAGCGGTGCCTG-3′ (ΔSHD primer, sense), 5′-TCACAGGTACACGTTTTCTTCCA-3′ (ΔABD primer, antisense), 5′-CGGATCCTTGAGTAAGCTGTGCCCACC-3′ (MBD-5′ primer, sense), and 5′-CTACTGGTCCTCCCCAGAAG-3′ (MBD-3′ primer, antisense). Site-directed mutagenesis and construction of Slac2-c point mutants (pEF-T7-Slac2-cE14A and pEF-T7-Slac2-cEA) and small interfering RNA (siRNA)-resistant Slac2-a mutant (pEGFP-C1-Slac2-aSR) were also performed by PCR as described previously (38Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) using the following oligonucleotides with a restriction enzyme site (underlined) and substituted nucleotides (in boldface type): 5′-GGATCCATGGGGAGGAAGCTGGACCTGTCGGGTCTGACCGACGATGCGAC-3′ (E14A primer, sense), 5′-GAATTCCCGGTCCAGCACGGCCTCAGTAGCGGCGAAGCT-3′ (EA primer, sense), and 5′-AAGCTTGGAGGAGGGTAACGGTGATAGAGAGCAGACTGATGA-3′ (SR primer, sense). Other pEF expression vectors (pEF-FLAG-Rab27A, pEF-T7-Slac2-a, pEF-T7-Slac2-c, pEF-T7-Slac2-c-ΔSHD, pEF-FLAG-MC-myosin Va-tail (melanocyte-type), 3Myosin Va-tail used in this study contains melanocyte-specific exon F. and pEF-FLAG-myosin VIIa-tail) were prepared as described previously (7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 10Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Construction of pEGFP-C1-Slac2-c, pECFP-C1-Slac2-c, and pEYFP-C1-myosin VIIa was performed by transfer of cDNA inserts from the above pEF vectors with appropriate restriction enzymes. In Vitro Binding Assays—In vitro T7-Slac2 and FLAG-myosin binding assays in COS-7 cells were performed as described previously (39Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). SDS-PAGE and immunoblot analyses with horseradish peroxidase-conjugated anti-FLAG-tag antibody (Sigma) and anti-T7-tag antibody (Novagen, Madison, WI) were also performed as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 38Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The immunoreactive bands were visualized by means of enhanced chemiluminescence systems (Amersham Biosciences). The intensity of the bands on x-ray film was quantified with Lane Analyzer (version 3.0) (ATTO, Tokyo, Japan) as described previously (39Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). The statistical analyses and curve fitting were performed with the GraphPad PRISM computer program (version 4.0). The blots shown in this paper are representative of at least two or three independent experiments. Recombinant T7-Slac2-c (or T7-Slac2-a) expressed in COS-7 cells was affinity-purified with anti-T7 tag antibody-conjugated agarose (Novagen) as described previously (7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). B16-F1 cells (one confluent 10-cm dish) were harvested and homogenized in a buffer containing 1 ml of 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 0.5 mm GTPγS, and protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, and 10 μm pepstatin A) in a glass-Teflon Potter homogenizer by 10 strokes at 900-1000 rpm, and the proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removing insoluble material by centrifugation at 15,000 rpm for 10 min, the supernatant was incubated with the above agarose beads coupled with T7-Slac2-a or T7-Slac2-c (wet volume 20 μl) for 1 h at 4 °C. After washing the beads five times with 10 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 0.2% Triton X-100, and protease inhibitors, proteins trapped by the beads were subjected to 10% SDS-PAGE, followed by immunoblotting with anti-Rab27A mouse monoclonal antibody (BD Transduction Laboratories, Lexington, KY), anti-myosin Va rabbit polyclonal antibody (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), and horseradish peroxidase-conjugated anti-T7 tag antibody. Cell Culture, Transfections, and Immunofluorescence Analysis—Melan-a cells, an immortal black mouse-derived melanocyte cell line (40Bennett D.C. Cooper P.J. Hart I.R. Int. J. Cancer. 1987; 39: 414-418Crossref PubMed Scopus (394) Google Scholar), were cultured in RPMI 1640 medium (Sigma) supplemented with 2.7 mm HCl, 10% fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin, 7.5 μg/ml phenol red, and 0.1 mm 2-mercaptoethanol at 37 °C under 10% CO2. Immediately prior to use, 200 nm phorbol 12-myristate 13-acetate (Sigma) was added to the medium. COS-7 cells and B16-F1 cells were cultured at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin. FuGENE6 (1.5 μl/μg plasmid DNA; Roche Applied Science) and Lipofectamine PLUS reagents (Invitrogen) were used for transfection into melan-a and COS-7 cells, respectively, according to the manufacturers' instructions. For plasmid transfection and microscopic analysis, melan-a cells (7.5 × 104 cells, the day before transfection) were seeded on 35-mm glass bottom dishes (MatTek Corp.), and the following amounts of plasmids were transfected into melan-a cells: 2 μg of green fluorescent protein (GFP) expression vector (see Fig. 3, D-J) or 1 μg each of cyan fluorescent protein (CFP) expression vector, and yellow fluorescent protein (YFP) expression vector (see Fig. 5, A-I). At 48-72 h after transfection, cells were fixed with 4% paraformaldehyde (catalogue number 168-20955; Wako Pure Chemicals, Osaka, Japan) for 20 min. For immunostaining of myosin Va and Rab27A, melan-a cells were permeabilized with 0.3% Triton-X-100 for 2 min and blocked with blocking buffer (1% bovine serum albumin and 0.1% Triton-X-100 in phosphate-buffered saline) for 1 h. The cells were then immunostained with anti-myosin Va antibody (1:100 dilution) and anti-Rab27A antibody (1:50 dilution), followed by Alexa Fluor 568 and 633 secondary IgG, respectively (1:5000 dilution; Molecular Probes, Inc., Eugene, OR). Fluorescence and bright field images were acquired and pseudocolored with a confocal laser-scanning microscope (Fluoview; OLYMPUS, Tokyo, Japan), and the images were processed with Adobe Photoshop software (version 7.0).Fig. 5Melanosome transport through Rab27A·Slac2-c·myosin VIIa complex.A-H, YFP-myosin VIIa alone (A and B), CFP-Slac2-c together with YFP-myosin VIIa (C-E), or CFP-Slac2-c together with YFP-myosin VIIa-tail (F-H) was expressed in melan-a cells. CFP/YFP fluorescence and the corresponding bright field images of the cells were acquired with a confocal laser scan microscope. The insets show 3-fold magnified views of the boxed area (A-E). The arrowheads in A and B indicate the absence of myosin VIIa from melanosomes, whereas those in C-E indicate accumulation of Slac2-c and myosin VIIa on melanosomes. The cell showing perinuclear melanosome aggregation has been outlined in yellow (H). Bars in A, C, and F, 10 μm. I, melanosome distribution in the Slac2-a-depleted melan-a cells expressing CFP-Slac2-c and YFP-myosin VIIa. Melan-a cells were transfected with pECFP-C1-Slac2-c and pEYFP-C1-myosin VIIa or empty vectors, followed by treatment with siRNA directed against Slac2-a. The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing CFP and YFP fluorescence. Data are expressed as means ± S.D. of three independent experiments (n > 100). Note that simultaneous expression of CFP-Slac2-c and YFP-myosin VIIa considerably, but not fully, rescued the perinuclear melanosome aggregation phenotype induced by the siRNA directed against Slac2-a.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Melanosome Distribution Assay—Melanosome distribution was assayed as described previously (12Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (101) Google Scholar, 17Kuroda T.S. Fukuda M. Nat. Cell Biol. 2004; 6: 1195-1203Crossref PubMed Scopus (129) Google Scholar). In brief, bright field images of melan-a cells transfected with fluorescent vector(s) were obtained at random (more than 50 cells/dish, three independent dishes for each plasmid). Cells in which more than 50% of the melanosomes were located around the nucleus were judged to be "aggregated." Data were expressed as means ± S.D. of three independent experiments. siRNA—The siRNA expression vector against mouse Slac2-a (target site 5′-GAAGGAAATGGAGACAGTG-3′) was prepared as described previously (17Kuroda T.S. Fukuda M. Nat. Cell Biol. 2004; 6: 1195-1203Crossref PubMed Scopus (129) Google Scholar), using pSilencer™ 1.0-U6 vector (Ambion, Austin, TX), which expresses short hairpin RNA under the control of the U6 promoter. Subconfluent COS-7 cells seeded on 10-cm dishes were transfected with 2 μg of pEGFP-C1-Slac2-a, pEGFP-C1-Slac2-aSR, or pEGFP-C1-Slac2-c together with 2 μg of either siRNA expression vector against Slac2-a or a control vector. Cell lysates were prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 39Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar) and subjected to 7.5% SDS-PAGE. Immunoblotting analysis with anti-GFP rabbit polyclonal antibody (1:1000 dilution; MBL Co., Ltd., Nagoya, Japan) and anti-actin (I-19) goat polyclonal antibody (1:300 dilution; Santa Cruz Biotechnology, Inc.) was performed as described above. Melan-a cells seeded on 35-mm glass bottom dishes were transfected with 1 μg each of siRNA expression vector and GFP-tagged protein expression vector and analyzed for melanosome distribution as described above. In the experiment shown in Fig. 5I, melan-a cells were first transfected with 1 μg each of pECFP-C1-Slac2-c and pEYFP-C1-myosin VIIa (or their control vectors) using FuGENE6. One day after transfection, the cells were then transfected with 2 μg of a chemically synthesized siRNA against the same site of mouse Slac2-a (5′-GAAGGAAAUGGAGACAGUGdTdT-3′, sense; B-Bridge International Inc., Sunnyvale, CA) using X-tremeGENE siRNA transfection reagent (Roche Applied Science) to knock down endogenous Slac2-a molecules. The cells were fixed the next day and analyzed for melanosome distribution as described above. Slac2-c Preferentially Interacts with Myosin VIIa Rather than Myosin Va in Vitro—Although we and others previously showed that the tail domain of myosin Va and the tail domain of myosin VIIa interact with the C-terminal region of Slac2-c (10Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 26El-Amraoui A. Schonn J.S. Kussel-Andermann P. Blanchard S. Desnos C. Henry J.P. Wolfrum U. Darchen F. Petit C. EMBO Rep. 2002; 3: 463-470Crossref PubMed Scopus (149) Google Scholar, 36Desnos C. Schonn J.S. Huet S. Tran V.S. El-Amraoui A. Raposo G. Fanget I. Chapuis C. Ménasché G. de Saint Basile G. Petit C. Cribier S. Henry J.P. Darchen F. J. Cell Biol. 2003; 163: 559-570Crossref PubMed Scopus (131) Google Scholar), the precise myosin-binding domain of Slac2-c has not yet been identified; nor has it been determined whether the two myosins bind the same domain of Slac2-c. We therefore attempted to determine the myosin-binding domain of Slac2-c by a co-transfection assay using COS-7 cells. Consistent with the previous reports, Slac2-c interacted with the tail domain of both myosin Va and myosin VIIa irrespective of the presence of the N-terminal SHD (Fig. 1, B and C, middle panels, lanes 1 and 2). First we tested the myosin binding activity of the putative myosin Va-binding domain of Slac2-c (amino acid residues 409-602), which corresponds to the previously characterized myosin Va-binding domain of Slac2-a (amino acid residues 241-405) (10Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (101) Google Scholar). Unlike Slac2-a, however, this domain alone was insufficient for the interaction with myosin Va to occur (i.e. weak interaction between T7-MBD and myosin Vatail; Fig. 1B, middle panel, lane 4), suggesting that amino acid residues 409-602 may be the minimal essential myosin Va-binding domain but that an additional sequence is required for full myosin Va binding. We then tested the myosin Va binding activity of the whole middle domain of Slac2-c (ΔSHD/ΔABD; amino acid residues 146-701). To our surprise, however, the ΔSHD/ΔABD mutant did not interact with myosin Va-tail at all (Fig. 1B, middle panel, lane 3), indicating that the C-terminal ABD of Slac2-c is required for full interaction with myosin Va. On the other hand, the myosin VIIa-tail preferentially interacted with the ΔSHD/ΔABD mutant of Slac2-c rather than the full-length Slac2-c or the Slac2-c-ΔSHD mutant (Fig. 1C, middle panel, lane 3), but the putative MBD alone did not interact with myosin VIIa-tail at all (Fig. 1C, middle panel, lane 4). Additionally, since we previously showed that a conserved acidic cluster in the MBD of Slac2-a is required for myosin Va binding activity (12Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (101) Google Scholar), we prepared a similar mutant of Slac2-c (referred to as Slac2-cEA; see Fig. 1A) and tested its myosin Va/VIIa-tail binding activity. In contrast to the reduced interaction between Slac2-cEA and myosin Va-tail, how
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