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Role of Plectin in Nuclear Mechano- Transduction of Keratinocytes

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Role of Plectin in Nuclear Mechano- Transduction of Keratinocytes Role of Plectin in Nuclear Mechano- transduction of Keratinocytes Submitted in partial fulfilment of the requirements of the Degree of Doctor of Philosophy by Filipe Veiga de Macedo Almeida School of Engineering and Material Sciences Queen Mary, University of London November 2015 I, Filipe Veiga de Macedo Almeida, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: (Filipe Almeida) Date: 27th November of 2015 ii Details of collaboration and publications 1) Filipe V Almeida1,6, Gernot Walko2, James R McMillan3, John A McGrath4, Gerhard Wiche5, Asa H Barber1, John T Connelly6* “The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes”. Journal Cell Science (In press) 2) Urszula Stachewicz, Tuya Qiao, Simon C. F. Rawlinson, Filipe V Almeida, Wei-Qi Li, Michael Cattell and Asa H. Barber. (2015) 3D imaging of cell interactions with electrospun PLGA nanofiber membranes for bone regeneration. Acta Biomaterilia (In press) 3) Costa P, Almeida F V, Connelly JT. (2012) Biophysical signals controlling cell fate decisions: How do stem cells really feel? Int J Biochem Cell Biol., (12) 00314-7 iii Abstract Cell shape dictates nuclear morphology via cytoskeleton remodelling, potentially regulating therefore nuclear architecture and gene expression. Utilising micro- patterned substrates to control cell shape, the physical cell-nuclear integration was investigated. Cells were seeded in patterns with variable sizes and shapes, and the nuclear morphology assessed. The nucleus in spread cells was larger when compared with confined cells as measured by its volume. Actin and keratins are the two main determinants of keratinocytes mechanics and their role in defining nuclear morphology is still unclear. Results here showed that the inhibition of filamentous actin, therefore the formation of stress fibres, was not sufficient to disrupt the physical link between cell shape and nuclear morphology in keratinocytes. Plectin deficient keratinocytes were further investigated as they possess abnormal keratin architecture: their more open network of keratin 14 resulted in a larger and more deformable nucleus. These deformations depended on acto-myosin contractility and the formation of cell-cell contacts. When seeded as cell clusters with a confined adhesive area, Plec KO were able to compact to a higher extent and when allowed to migrate outside this confined area, the nucleus of Plec KO underwent larger and quicker morphological remodelling in comparison to the WT. Changes in nuclear morphology may influence chromatin remodelling. Confined keratinocytes committed to terminal differentiation, had lower levels of transcriptionally active sites (H3K4me3), and re-distributed heterochromatin markers (H3K27me3). In summary, the present thesis demonstrates that cell shape mediates nuclear morphology and chromatin remodelling, and this cell-nucleus coupling is coordinated by the cross-talk between keratins and actin, in which plectin is a key player. iv Acknowledgments I would like to thank, first of all, to Prof. Asa Barber to bring me on board in this PhD, for all the motivating words and support. To my mentor Dr John Connelly, a huge thanks for the active patience, enormous support and trust. A special and warm thanks for all the amazing team of post-docs with whom I learnt a lot: Patricia Costa, Vera Martins, Roanne Jones, Matthew Calley, Viviana Castagna, Krystelle Mafina, Valentina Rapisarda, Supatra Marsh, among others. Also to all my dear PhD colleagues from both SEMS and Blizards who helped in crucial occasions: Georgios Theocharidis, Fiona Kenny, Zoe Drymoussi, Alex Kao, Wenjun, Hao, Feng, Barbara Ricci, Aneta, Francesca, Ana Laly, among others. This intense experience was elevated to a level of enjoyment thanks to you guys, being there every day in good and bad moments made it a quite special journey. One is made of experiences, not only the recent matter. I owe my fascination towards research to Dr. Ana Queiroz and Prof. Jorge Monteiro, the two figures who introduced me in the world of research and most of all believed in me. For that, I’ll be for ever grateful. I could not let my family and friends out, they are the scaffold of my life - they make every effort worth it. To all the kindness, believe and love that was to me provided, an endless thank you. Mae, Pai, Mariana, Sergio, Tita, Zeca, Gaucho, Eric, Carolina, Mauri e Luis um grande sorriso, pois por voces eu sou a pessoa mais feliz e completa do Mundo ! Diana Leandro, Carlos Gomes, Joao Rola, Joao Torres, Liliana Reis, Luis Filipe Rodrigues, Victor Sousa, Ben Johnson, voces tambem sao familia, portanto o mesmo de aplica ehehe. Obrigado a todos. Thank you all. v Table of contents Details of collaboration and publications ............................................................. iii Abstract .................................................................................................................... iv Acknowledgments.................................................................................................... v List of Figure .......................................................................................................... xii List of Tables ......................................................................................................... xiv List of Equations .................................................................................................... xv Abbreviations ........................................................................................................ xvi Chapter 1. Introduction ......................................................................................... 1 1.1 Introduction ................................................................................................ 2 Aims and objectives ................................................................................................. 5 Chapter 2. Literature Review ................................................................................ 6 2.1 The eukaryotic cell ..................................................................................... 7 2.2 The cytoskeleton ........................................................................................ 7 2.2.1 Actin ...................................................................................................... 8 2.2.2 Intermediate Filaments ....................................................................... 13 2.2.3 Cytoskeleton mechanics ..................................................................... 18 2.3 Physical linkages: cell-ECM adhesions, cell-cell junctions and the nucleus ................................................................................................................... 22 2.3.1 Cell-ECM adhesions ........................................................................... 22 2.3.2 Cell-cell junctions ................................................................................ 25 2.3.3 The nucleus ........................................................................................ 27 2.4 Mechano-transduction............................................................................. 32 2.4.1 The cytoskeleton in mechano-transduction ........................................ 33 2.4.2 The Nucleus as mechano-sensor ....................................................... 35 2.4.3 Signalling pathways involved in mechano-transduction ...................... 39 2.5 The skin .................................................................................................... 42 vi 2.5.1 The cytoskeleton in skin homeostasis ................................................ 43 2.6 Plectin and Plectinopathies .................................................................... 46 2.6.1 Keratins and nuclear mechano-transduction in skin ........................... 49 Chapter 3. Materials and Methods ..................................................................... 52 3.1 Antibodies ................................................................................................ 53 3.2 Reagents and buffers .............................................................................. 55 3.3 Tissue and cell isolation.......................................................................... 56 3.3.1 Isolation of primary keratinocytes from neonatal human foreskin tissue . ............................................................................................................ 56 3.3.2 Mouse skin wholemount of plectin transgenic mice ............................ 56 3.4 Cell culture methods ..............................................................................
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