Elin Torvaldson Elin Models to Study Intermediate Filament Dynamics and Functions Models Study Intermediate to Filament Models to Study Intermediate Filament Dynamics and Functions Elin Torvaldson Cell Biology, Faculty of Science and Engineering Åbo Akademi University Turku Centre for Biotechnology, University of Turku and Åbo Akademi University 2015 ISBN 978-952-12-3330-2 Painosalama Oy – Turku, Finland 2015 2015 Models to Study Intermediate Filament Dynamics and Functions Elin Torvaldson Cell Biology, Faculty of Science and Engineering Åbo Akademi University Turku Centre for Biotechnology, University of Turku and Åbo Akademi University 2015 From Cell Biology, Faculty of Science and Engineering, Åbo Akademi University, and Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Supervised by Professor John Eriksson Cell Biology, Faculty of Science and Engineering, Åbo Akademi University, and Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Turku, Finland Reviewed by Adjunct Professor Jeroen Pouwels Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Turku, Finland Professor Yosef Gruenbaum The Alexander Silberman Institute of Life Sciences The Edmond Safra Campus The Hebrew University of Jerusalem Jerusalem, Israel Opponent Professor Roy Quinlan School of Biological and Biomedical Sciences, Durham University Durham, United Kingdom ISBN 978-952-12-3330-2 Painosalama Oy – Turku, Finland 2015 River, river have mercy Take me down to the sea For if I perish on these rocks My love no more I'll see -Philip Chevron Table of Contents List of Original Publications ......................................................................................... 6 Abbreviations ................................................................................................................. 8 Svensk sammanfattning .............................................................................................. 10 Abstract ......................................................................................................................... 11 1 Introduction ............................................................................................................... 12 2 Review of the Literature ........................................................................................... 14 2.1 Intermediate filaments ..................................................................................... 14 2.1.1 Cellular functions of IFs .......................................................................... 16 2.1.2 Dynamics of IFs ........................................................................................ 20 2.1.3 Signalling properties of IFs ..................................................................... 22 2.2 Lamin A/C ......................................................................................................... 23 2.2.1 Splicing and processing of lamin A ....................................................... 23 2.2.2 Lamin PTMs ............................................................................................. 25 2.2.3 Structural and mechanical functions of lamin A/C ............................. 27 2.2.4 Nucleoplasmic lamin A ........................................................................... 28 2.2.5 Laminopathies - peculiar diseases that give insights into lamin functions .................................................................................................... 29 2.3 Nestin ................................................................................................................. 31 2.3.1 Nestin structure and polymerisation ..................................................... 31 2.3.2 Regulation of nestin gene and protein .................................................. 31 2.3.3 Functions of nestin................................................................................... 34 2.3.4 Nestin in muscle ....................................................................................... 35 2.4 Models to study IFs .......................................................................................... 36 2.4.1 Transgenic mice as models to study IFs ................................................ 36 2.4.2 Invertebrate models to study IFs ............................................................ 41 2.5 Cdk5 – an IF interacting kinase ...................................................................... 42 2.6 Satellite cells and muscle regeneration .......................................................... 45 2.6.1 Models to study muscle differentiation and regeneration .................. 46 3 Aims ............................................................................................................................. 48 4 Materials and Methods ............................................................................................. 49 4.1 Standard methods and Cell lines used ............................................................ 49 4.2 GFP-tagged lamin constructs .......................................................................... 49 4.3 Primary myoblast and myofiber cultures ....................................................... 50 4.4 Additional methods .......................................................................................... 50 5 Results and Discussion .............................................................................................. 52 5.1 Phosphorylation determines lamin A/C localization and mobility (I) ...... 52 5.2 The physiological relevance of lamin A/C phosphorylation ....................... 54 5.3 Cdk5 phosphorylates lamin A/C (unpublished) ........................................... 57 5.4 Depletion of nestin has no obvious effect on muscle stem cell behaviour (II) ....................................................................................................................... 60 5.5 Nestin influences body composition (II) ....................................................... 61 5.6 The role of nestin in muscle differentiation and regeneration (II) ............. 62 5.7 Nestin affects male characteristics (unpublished) ........................................ 63 5.8 Expression of human vimentin in Drosophila (III) ...................................... 66 6 Concluding Remarks ................................................................................................. 68 7 Acknowledgments ..................................................................................................... 70 8 References ................................................................................................................... 72 Original Publications ................................................................................................... 89 List of Original Publications List of Original Publications This thesis is based upon the following original publications and manuscripts. They are referred to in the text by their roman numerals. The original publications have been reproduced with permission of the copyright holders. In addition, this thesis includes unpublished data. I. Kochin V.*, Shimi T.*, Torvaldson E., Adam S.A., Goldman A., Pack C.-G., Melo-Cardenas J., Imanishi S.Y., Goldman R.D. and Eriksson J.E. (2014). Interphase phosphorylation of lamin A. J. Cell Sci. 127, 2683–2696. II. Lindqvist J.*, Torvaldson E.*, Gullmets J., Karvonen H., Taimen P. and Eriksson J.E. Nestin is required for skeletal muscle homeostasis and regeneration. Manuscript. III. Gullmets J., Torvaldson E., Lindqvist J., Imanish S.Y., Taimen P., Meinander A. and Eriksson J.E. Transgenically expressed human vimentin forms a network in epithelial Drosophila tissues. Manuscript. *Equal contribution Related publications Virtakoivu R., Mai A., Mattila E., De Franceschi N., Imanishi S.Y., Corthals G., Kaukonen R., Saari M., Cheng F., Torvaldson E., et al. (2015). Vimentin-ERK signaling uncouples Slug gene regulatory function. Cancer Res. 75, 2349–2362 Lindqvist J., Imanishi S.Y.*, Torvaldson E.*, Malinen M., Remes M., Örn F., Palvimo J., Eriksson J.E. (2015). Cyclin-dependent kinase 5 acts as a critical determinant of AKT-dependent proliferation and regulates differential gene expression by the androgen receptor in prostate cancer cells. Mol. Biol. Cell 26, 1971–1984. Ferraris S.E., Isoniemi K., Torvaldson E., Anckar J., Westermarck J., and Eriksson J.E. (2012). Nucleolar AATF regulates c-Jun-mediated apoptosis. Mol Biol Cell 23, 4323-4332. 6 List of Original Publications Pallari H-M., Lindqvist J., Torvaldson E., Ferraris S., He T., Sahlgren C., Eriksson J.E. (2011). Nestin as a regulator of Cdk5 in differentiating myoblasts. Mol Biol Cell 22, 1539-1549. Peuhu E., Rivero-Müller A., Stykki H., Torvaldson E., Holmbom T., Unkila M., Eklund P., Sjöholm R., Eriksson J.E. (2010). Inhibition of Akt signalling by the lignan matairesinol sensitizes prostate cancer cells to TRAIL- induced apoptosis. Oncogene 29, 898–908. Review articles the author has contributed to that are not included in this thesis: Torvaldson, E., Kochin, V. and Eriksson, J.E. (2015). Phosphorylation of lamins determine their structural properties and signaling functions. Nucl. Austin Tex 6, 166–171. Hyder, C. L., Isoniemi, K.O.*, Torvaldson, E.*, and Eriksson, J.E. (2011). Insights into intermediate filament regulation from development to ageing. J. Cell Sci. 124, 1363–1372. 7 Abbreviations Abbreviations Cdk Cyclin dependent kinase CHX Cycloheximide CLN Centrally