Advancing Molecular Force Measurements Across Talin
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Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie an der Ludwig-Maximilians-Universität München Advancing molecular force measurements across talin Pia Sabrina Ringer aus Herrenberg, Deutschland 2017 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Reinhard Fässler betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, 07.12.2017 Pia Sabrina Ringer Dissertation eingereicht am 07.12.2017 Erstgutachter: Prof. Dr. Reinhard Fässler Zweitgutachter: Prof. Dr. Martin Biel Mündliche Prüfung am 29.01.2018 i Contents Page Summary vi Abbreviations viii List of Publications1 1 Introduction3 1.1 Mechanobiology . .3 1.1.1 Introduction into the field of mechanobiology . .3 1.1.2 Mechanical forces influence the cell’s function . .4 1.1.3 Conversion of mechanical cues into biochemical signals . .6 1.2 Focal adhesions: Mechanical anchor to the ECM and signaling hub . .8 1.2.1 Structure and components of cell-ECM adhesions . .9 1.2.2 Membrane-spanning integrin receptor family . .9 1.2.3 Activation of integrins by talin and kindlin . 10 1.2.4 Actin cytoskeleton - architecture and mechanics . 12 1.3 Talin is the central adapter protein in FAs . 13 1.3.1 Talin holds a crucial function in tissue physiologie and pathologie 13 1.3.2 Domain structure of talin . 15 1.3.3 Insights into talin binding partners . 15 1.3.4 Talin - recruitment and activation . 16 1.3.5 Talin-2: isoform with almost unknown function . 19 1.4 Talin a putative force sensitive protein . 19 1.5 Techniques to measure cellular and molecular forces . 20 1.5.1 Cell-ECM traction . 21 1.5.2 Probing the mechanical properties of proteins in vitro ...... 21 1.5.3 Förster resonance energy transfer . 22 1.5.4 Microscopical methods for measuring FRET . 22 1.5.5 Genetically-encoded tension sensors: Unraveling molecular forces 23 2 Aim of Thesis 27 iii 3 Short summary of manuscripts 29 3.1 Paper I – How to measure molecular forces in cells: A guide to evaluating genetically-encoded FRET-based tension sensors. 29 3.2 Paper II – Extracellular rigidity sensing by talin isoform-specific mechan- ical linkages. 31 3.3 Paper III – Sensing the mechano-chemical properties of the extracellular matrix . 33 3.4 Paper IV – Multiplexing molecular tension sensors reveals force gradient across talin-1 . 34 Bibliography 35 Acknowledgments 53 Curriculum vitae 56 iv Summary The ability of cells to sense and adapt to mechanical cues is indispensable for proper tissue development and function. Disruption of cells’s mechanotransduction processes lead to diseases, like arteriosclerosis or tumor formation. Mechanical information is diversely transmitted by the cell depending on the mechanical signal and the subcellular structures responsible for the transmission. However, mechanical signals form the extracellular environment are sensed by integrin-dependent, multi-molecular complexes, called focal adhesions (FAs). The integrin family of adhesion receptors plays an essential role in those cell-matrix interactions by spanning the membrane and anchoring to the extracellular matrix (ECM). One key regulator of cell adhesion is the adaptor protein talin, which is able to activate integrins by binding with the N-terminal head domain to the cytoplasmic β-tails and at the same time mediates the link to the actin cytoskeleton via its elongated rod domain. Vertebrates express two very similar talin isoforms, the ubiquitously expressed talin-1, whereas the expression of talin-2 is mainly restricted to heart, skeletal muscle and brain. Even though the two isoforms share similar structures and binding partners, the reason for the diverse expression pattern remained unclear as well as isoform specific functions have been elusive. Several studies showed that integrins bear forces that reach values of a few tens of piconewtons (pNs) and it was hypothesized that talin tansduces those mechanical signals, however, quantitative evidence for force transmission across talin within living cells was still missing. In order to unravel the mechanotransduction process across talin-1 and talin-2 (Paper II), I analyzed both isoforms on a talin double knockout background and could demonstrate that both talin-1 and talin-2 rescue cell spreading and localize to the same subcellular structures. Two single-molecule calibrated Förster resonance energy transfer (FRET)-based tension sensors, sensitive to force ranges of 6-8 pN and 9-11 pN, unraveled an isoform-specific force transduction across talin, which is regulated by actin and vinculin engagement. In addition, it was shown, that talin-2 recruits the stabilizing adapter protein vinculin more efficiently to its N-terminal rod domain, which allow cells v to reinforce FAs even on compliant substrates. By diverse expression of the two talin isoforms cells can modulate their ability of sensing substrate rigidity. The question remained, how the different subdomains and interaction partners, that bind to the elongated talin rod, contribute to force transmission across this major adapter protein. In order to tackle this question and to elucidate the process of force propagation across talin’s rod domain, I generated an additional talin-1 tension sensor, that allowed to distinguish force experienced by different subdomains (Paper IV). Using a novel tension sensor module with a near digital force response sensitive to 3-5 pN at position aa 447 and aa 1973 of the talin rod, it could be demonstrated that mechanical tension is not equally distributed. Instead, talin is subjected to a force gradient, with forces as high as 7 pN at the N-terminal rod domain and lower forces of around 3 pN generated by the very C-terminal actin binding site. This finding was furthermore confirmed by simultaneously evaluating the two talin tension sensors within one cell, using a newly developed multiplexing fluorescence-lifetime imaging microscopy (FLIM) method. Finally, the digital nature of the new sensor module enabled us to determine the fraction of stretched talin molecules per cell. Taken together, this study shows that force transduction across talin is regulated on multiple layers, first of all, talin molecules bear a range of forces, which is mostly modulated by vinculin and actomyosin contractility. In addition, cells tune the amount of mechanically engaged molecules within FAs. During my work I acquired broad insights into the field of biosensors that allow force measurements across single molecules, especially FRET-based tension sensor probes. In Paper I we summarized the diverse techniques to measure molecular forces and suggested a strategy to carefully analyze the currently available FRET-based biosensors. Additionally, in a second review article, the current knowledge about the mechano- chemical signaling hub of cell-ECM contact sites was outlined (Paper III). Here, I described the recent findings about the vertical and horizontal layered structure of FAs and their complex regulation. Furthermore, recent approaches in single molecule resolution micrsocopy, force spectroscopy methods, as well as molecular tension sensors were highlighted, which toghether with theoretical studies had a significant impact on the current understanding of how cells sense and adapt to the mechanical and chemical properties of the ECM. vi Abbreviations AFM atomic force microscopy ECM extracellular matrix FA focal adhesion FLIM fluorescence-lifetime imaging microscopy FRET Förster resonance energy transfer pN piconewton HP35 villin headpiece peptide TSM tension sensor module FL ferredoxin-like TFM traction force microscopy D donor nm nanometer GFP green fluorescent protein VBS vinculin binding site RTK receptor tyrosine kinase MAPK mitogen-activated protein kinase GPCR G-protein-coupled receptors ATP adenosine triphosphate MLC myosin light chain FERM four point one, ezrin, radixin, myosin PTB phosphotyrosine-binding FAK focal adhesion kinase RIAM Rap1-GTP interacting adapter molecule PIPKIγ Phosphatidylinositol phophate kinase type Iγ vii TIAM T-cell lymphoma invasion and metastasis 1 VBS vinculin binding site eq equation viii Publications This thesis is based on the following publications, which are referred in the text by the following roman numeral letters (I-IV): Paper I: Cost A.-L.∗, Ringer P.∗, Chrostek-Grashoff A., Grashoff C., (2015). How to measure molecular forces in cells: A guide to evaluating genetically-encoded FRET- based tension sensors. Cellular And Molecular Bioengineering, 8(1), 96-105. ∗ equal contribution Paper II: Austen K.∗, Ringer P.∗, Mehlich A.∗, Chrostek-Grashoff A., Kluger C., Klingner C. Sabass B., Zent R., Rief M., Grashoff C., (2015). Extracellular rigidity sensing by talin isoform-specific mechanical linkages. Nature Cell Biology, 17, 1597-1606. ∗ equal contribution Paper III: Ringer P., Colo G., Fässler R., Grashoff C., (2017). Sensing the mechano- chemical properties of the extracellular matrix. Matrix Biology, 1336, 0945-053X. Paper IV: Ringer P., Weißl A., Cost A.-L., Freikamp A., Sabass B., Mehlich A., Tramier M., Rief M., Grashoff C., (2017). Multiplexing molecular tension sensors reveals force gradient across talin-1. Nature Methods, 14, 1090-1096. 1 1 Introduction 1.1 Mechanobiology 1.1.1 Introduction into the field of mechanobiology Essentially all organisms from bacteria to human are subjected to diverse mechanical forces from a wide variety of sources. For instance gravity is an ubiquitous force influencing the whole organism, whereas compressive loads