Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Myosin VIshort is selectively expressed in ovarian cancer cells and is critical for cell migration.
Figure 2: The RRL binder optineurin affects cell migration and selectively binds myosin VIshort.
Figure 3: Clathrin specifically interacts with myosin VIlong.
Figure 4: Myosin VI isoforms have mutually exclusive interactors.
Figure 5: Cancer cells that selectively express only myosin VIshort are addicted to myosin VI in cell migration.

Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

References

  1. Foth, B.J., Goedecke, M.C. & Soldati, D. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. USA 103, 3681–3686 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Richards, T.A. & Cavalier-Smith, T. Myosin domain evolution and the primary divergence of eukaryotes. Nature 436, 1113–1118 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Wells, A.L. et al. Myosin VI is an actin-based motor that moves backwards. Nature 401, 505–508 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Buss, F., Spudich, G. & Kendrick-Jones, J. Myosin VI: cellular functions and motor properties. Annu. Rev. Cell Dev. Biol. 20, 649–676 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Hasson, T. Myosin VI: two distinct roles in endocytosis. J. Cell Sci. 116, 3453–3461 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Geisbrecht, E.R. & Montell, D.J. Myosin VI is required for E-cadherin-mediated border cell migration. Nat. Cell Biol. 4, 616–620 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Hicks, J.L., Deng, W.M., Rogat, A.D., Miller, K.G. & Bownes, M. Class VI unconventional myosin is required for spermatogenesis in Drosophila. Mol. Biol. Cell 10, 4341–4353 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rogat, A.D. & Miller, K.G. A role for myosin VI in actin dynamics at sites of membrane remodeling during Drosophila spermatogenesis. J. Cell Sci. 115, 4855–4865 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Petritsch, C., Tavosanis, G., Turck, C.W., Jan, L.Y. & Jan, Y.N. The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts. Dev. Cell 4, 273–281 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Erben, V. et al. Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI. J. Cell Sci. 121, 1403–1414 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Tumbarello, D.A., Kendrick-Jones, J. & Buss, F. Myosin VI and its cargo adaptors - linking endocytosis and autophagy. J. Cell Sci. 126, 2561–2570 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Penengo, L. et al. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin. Cell 124, 1183–1195 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Spudich, G. et al. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2 . Nat. Cell Biol. 9, 176–183 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Bunn, R.C., Jensen, M.A. & Reed, B.C. Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton. Mol. Biol. Cell 10, 819–832 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sahlender, D.A. et al. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis. J. Cell Biol. 169, 285–295 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Morriswood, B. et al. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion. J. Cell Sci. 120, 2574–2585 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Buss, F., Arden, S.D., Lindsay, M., Luzio, J.P. & Kendrick-Jones, J. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. EMBO J. 20, 3676–3684 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dance, A.L. et al. Regulation of myosin-VI targeting to endocytic compartments. Traffic 5, 798–813 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Tomatis, V.M. et al. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin. J. Cell Biol. 200, 301–320 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Au, J.S., Puri, C., Ihrke, G., Kendrick-Jones, J. & Buss, F. Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells. J. Cell Biol. 177, 103–114 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yoshida, H. et al. Lessons from border cell migration in the Drosophila ovary: a role for myosin VI in dissemination of human ovarian cancer. Proc. Natl. Acad. Sci. USA 101, 8144–8149 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dunn, T.A. et al. A novel role of myosin VI in human prostate cancer. Am. J. Pathol. 169, 1843–1854 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Puri, C. et al. Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis. Oncogene 29, 188–200 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Szczyrba, J. et al. The microRNA profile of prostate carcinoma obtained by deep sequencing. Mol. Cancer Res. 8, 529–538 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Chibalina, M.V., Poliakov, A., Kendrick-Jones, J. & Buss, F. Myosin VI and optineurin are required for polarized EGFR delivery and directed migration. Traffic 11, 1290–1303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Majewski, L., Sobczak, M., Havrylov, S., Jozwiak, J. & Redowicz, M.J. Dock7: a GEF for Rho-family GTPases and a novel myosin VI-binding partner in neuronal PC12 cells. Biochem. Cell Biol. 90, 565–574 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Morris, S.M. et al. Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton. Traffic 3, 331–341 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Yu, C. et al. Myosin VI undergoes cargo-mediated dimerization. Cell 138, 537–548 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Buss, F. & Kendrick-Jones, J. How are the cellular functions of myosin VI regulated within the cell? Biochem. Biophys. Res. Commun. 369, 165–175 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Inoue, A., Sato, O., Homma, K. & Ikebe, M. DOC-2/DAB2 is the binding partner of myosin VI. Biochem. Biophys. Res. Commun. 292, 300–307 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Lu, Q., Li, J. & Zhang, M. Cargo recognition and cargo-mediated regulation of unconventional myosins. Acc. Chem. Res. 47, 3061–3070 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Sweeney, H.L. & Houdusse, A. Myosin VI rewrites the rules for myosin motors. Cell 141, 573–582 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Tumbarello, D.A. et al. Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome. Nat. Cell Biol. 14, 1024–1035 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chibalina, M.V., Puri, C., Kendrick-Jones, J. & Buss, F. Potential roles of myosin VI in cell motility. Biochem. Soc. Trans. 37, 966–970 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Maddugoda, M.P., Crampton, M.S., Shewan, A.M. & Yap, A.S. Myosin VI and vinculin cooperate during the morphogenesis of cadherin cell cell contacts in mammalian epithelial cells. J. Cell Biol. 178, 529–540 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pinheiro, E.M. & Montell, D.J. Requirement for Par-6 and Bazooka in Drosophila border cell migration. Development 131, 5243–5251 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. He, F. et al. Myosin VI contains a structural motif that binds to ubiquitin chains. Cell Rep. (in the press).

  38. Singh, R.K. & Cooper, T.A. Pre-mRNA splicing in disease and therapeutics. Trends Mol. Med. 18, 472–482 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Biamonti, G., Catillo, M., Pignataro, D., Montecucco, A. & Ghigna, C. The alternative splicing side of cancer. Semin. Cell Dev. Biol. 32, 30–36 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Keller, A., Nesvizhskii, A.I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    Article  CAS  PubMed  Google Scholar 

  41. Nesvizhskii, A.I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Kobayashi, N. et al. KUJIRA, a package of integrated modules for systematic and interactive analysis of NMR data directed to high-throughput NMR structure studies. J. Biomol. NMR 39, 31–52 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  44. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

    Article  CAS  PubMed  Google Scholar 

  47. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Bauer, D.F. Constructing confidence sets using rank statistics. J. Am. Stat. Assoc. 67, 687–690 (1972).

    Article  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Simona Polo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3187

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer