Abstract
Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles.
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Acknowledgements
We thank F. Buss for critically reading the manuscript and for helpful discussions and advice and M. Ladwein for performing initial wound healing experiments. We also thank E. Hirsch (Università di Torino), S. Maddika (Centre for DNA Fingerprinting and Diagnostics), A. Israel (Institute Pasteur), F. Buss (Cambridge Institute for Medical Research), G. Serini (Università di Torino) and F. Randow (Medical Research Council) for DNA constructs. This work was supported by the Association for International Cancer Research (AICR), grant 11-0051 (S.P.); from Italian Ministry of Education, Universities and Research, grant PRIN 20108MXN2J (S.P.) and the Intramural Research Program of the US National Cancer Institute (K.J.W.). H.-P.W. was supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC) cofunded by Marie Curie Actions. M.B. was supported by a fellowship from the Fondazione Umberto Veronesi (FUV).
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H.-P.W., M.B. and E. Magistrati designed and performed the experiments and analyzed the data; F.H. and K.J.W. designed and interpreted CD and NMR experiments, which F.H. performed and analyzed; P.S. carried out MS analysis; M.L. and U.C. generated primary cells from high-grade ovarian cancer; E. Molteni and U.P. conducted exon analysis; K.R. participated in setting up and interpreting the migration assays; M.M. participated in the experimental design and data analysis; S.P. conceived the project, interpreted the results and wrote the paper with contributions from all authors.
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Integrated supplementary information
Supplementary Figure 1 Myosin VI sequence conservation and genomic organization.
(a) Genomic organization of the three main isoforms of the human myosin VI gene. Whole transcripts are reported in the panel on top while the alternatively spliced region, together with the flanking exons, are depicted in the magnified pink box. (b) Sequence alignment of myosin VI 983–1140 region (Homo sapiens, isoform 3), color-coded according to sequence conservation. ClustalW was used to create a sequence alignment of myosin VI tail domains from various organisms. Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Ss, Sus scrofa; Xt, xenopus tropicalis; Gg, Gallus gallus; Dr, Danio rerio. Secondary structure elements, predicted using http://www.predictprotein.org and confirmed by structural data are depicted above the sequence. Exons are reported at the bottom with a box encompassing the corresponding amino acid sequence.
Supplementary Figure 2 Cancer cells are addicted to myosin VIshort in cell migration.
(a,b) Wound healing assay. The indicated cell lines were knocked down for myosin VI (KD, using oligo 2) or mock treated. Left panel, sample images: T0 first frame, T1 and T2 arbitrary points identical for control and KD of the same cell lines. Scale bars, 200μm. Central panel: quantification of the wound closure speed relative to control. Error bars, s.d. (n=10 movies per condition, from three independent experiments) **** P<0.0001 by two-tailed T-test. Right panel: IB anti-myosin VI performed at T0.
Supplementary Figure 3 R1050–R1131 of myosin VIlong is the minimal clathrin-binding surface.
(a, b, c) GST pull-down assay with the indicated deletion constructs incubated with HEK293T cellular lysate. The largest fragment used is depicted on top of each panel with LI represented in orange. IB and ponceau as indicated. (d) Stereoview of twenty calculated structures of myosin VI R1050–R1131 displayed as a backbone trace with amino acids T1054–R1068 and Y1084–S1126 superimposed. (e) Helical wheel for α2-linker that shows its amphipathic nature. (f) Regions from a 3D 13C-dispersed NOESY spectrum displaying myosin VI L1118 NOE interactions, with those involving α2-linker labelled in orange. This spectrum was recorded on 0.8 mM 13C, 15N labelled myosin VI 1050–1131 at 700 MHz on a spectrometer equipped with a cryogenically cooled probe. (g) Circular dichroism data of the myosin VI998–1131 and with the R1117A mutation incorporated. (h) Selected region of 1H-15N HSQC spectra displaying K1090 and L1086 of myosin VIshort (998–1099, black), myosin VIlong (998–1131) wild-type protein (red), and myosin VIlong (998–1131) with alanine substituted at position L1118 (green). Many signals, including the shown ones (green versus black and red), appear at similar positions in myosin VIlongL1118 and myosin VIshort due to the conformational change induced by the mutation.
Supplementary Figure 4 An isoform-specific binding surface drives the interaction of myosin VIlong with clathrin.
(a) HeLa cells stably knocked down for myosin VI were transiently transfected with the indicated GFP-myosin VI tail constructs. Endogenous CLTC is in red. Scale bars, 10μm, 2μm for the magnifications. (b) Quantification of the co-localization using Pearson’s coefficient. Error bars, s.e.m. (n=16 cells for Long, Short, W1192L; n=19 cells for L1118A, from two independent experiments) **** P<0.0001 by two-tailed T-test. (c) HeLa cells stably knocked down for myosin VI were transfected as in a. Lysates were immunoprecipitated with GFP-trap (Chromotek). IB as indicated.
Supplementary Figure 5 Relative abundance of exon E31 in different cancer types for which a normal counterpart was available.
Exon E31 relative abundance at the single tumour level compared with their normal counterparts. E31RA has been calculated for each sample dividing E31 RPKMS by the average of the flanking constitutive exons (i.e. E27, E28, E32, E33). All values have been normalized by their median in the tumour matched control samples. Bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC).
Supplementary Figure 6 Drosophila Jaguar has an alternative-splicing region in the same position as that identified in humans.
Amino acid sequence alignment covering the region of the MIU, the α1-linker and the clathrin-binding domains, color-coded according to sequence conservation. ClustalW was used to create a sequence alignment of myosin VI998–1131 between human and drosophila isoforms. Secondary structure elements for Jaguar, predicted using http://www.predictprotein.org, are depicted above the alignments. The alternatively spliced region codifies for an alpha helix (α1-linker) with length and position similar to the α2-linker in human.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Tables 2 and 3 (PDF 1122 kb)
Supplementary Table 1
Complete list of proteins that differentially interact with isoform 1, isoform 2 and isoform 3 versus GST control (XLSX 486 kb)
Supplementary Data Set 1
Original uncropped images of PCR gels and blots (PDF 10786 kb)
Phase-contrast (PC) time-lapse analysis (8.5 h, time interval 5 min) of the wound closure of SKOV-3 knocked down for myosin VI (lower panel) or mock treated (upper panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 2838 kb)
PC time-lapse analysis (6.5 h, time interval 5 min) of the wound closure of HEY knocked down for myosin VI (lower panel) or mock treated (upper panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 2269 kb)
PC time-lapse analysis (28 h, time interval 10 min) of the wound closure of OVCAR-5 knocked down for myosin VI (lower panel) or mock treated (upper panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 3598 kb)
PC time-lapse analysis (18h, time interval 5 min) of the wound closure of SKOV-3 mock treated (upper panel), knocked down for myosin VI (second from the top), optineurin (third from the top), myosin VI and optineurin together (lower panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 764 kb)
PC time-lapse analysis (16 h, time interval 5 min) of the wound closure of MDA-MB-231 knocked down for myosin VI (lower panel) or mock treated (upper panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 5516 kb)
PC time-lapse analysis (10 h, time interval 5 min) of the wound closure of MCF-10 knocked down for myosin VI (lower panel) or mock treated (upper panel)
Time (min) is indicated on the top left corner. The movie is representative of three independent experiments. (MOV 3673 kb)
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Wollscheid, HP., Biancospino, M., He, F. et al. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain. Nat Struct Mol Biol 23, 300–308 (2016). https://doi.org/10.1038/nsmb.3187
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DOI: https://doi.org/10.1038/nsmb.3187
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