Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Kank family proteins comprise a novel type of talin activator Zhiqi Sun aus Anshun, Guizhou, China 2015 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 selbstständig, ohne unerlaubte Hilfe erarbeitet. München, ………………………….. ______________________ (Zhiqi Sun) Dissertation eingereicht am 03.07.2015 1. Gutachterin / 1. Gutachter: Prof. Dr. Reinhard Fässler 2. Gutachterin / 2. Gutachter: Prof. Dr.med. Markus Sperandio Mündliche Prüfung am Table of contents| 3 Table of contents Table of contents ........................................................................................................................................ 3 Abbreviations .............................................................................................................................................. 5 1. Summary ............................................................................................................................................. 7 2. Introduction .......................................................................................................................................... 9 2.1. Integrin receptors ....................................................................................................................... 9 2.1.1. Integrin structure ................................................................................................................. 9 2.1.2. Allosteric integrin activation ............................................................................................ 15 2.2. Talin-dependent integrin activation ........................................................................................ 16 2.2.1. Talin structure and autoinhibition ................................................................................... 17 2.2.2. Talin activation .................................................................................................................. 19 2.2.3. Structural basis of Talin-dependent integrin activation ............................................... 21 2.2.4. Connections between talin and actin ............................................................................ 23 2.2.5. Post-translational regulation of talin function ............................................................... 24 2.3. Kindlin-dependent integrin activation .................................................................................... 25 2.4. A molecular clutch between integrins and actomyosin required for migration ................ 27 2.4.1. Actin structures and dynamics in mesenchymal migration ........................................ 27 2.4.2. Molecular clutch between integrin and actomyosin .................................................... 29 2.4.3. Mechano-sensing and transduction .............................................................................. 31 2.5. Molecular organization of integrin-based adhesions .......................................................... 34 2.5.1. Nascent adhesion (NA) ................................................................................................... 35 2.5.2. Focal adhesion (FA) ......................................................................................................... 37 2.5.3. Fibrillar adhesion (FB) ..................................................................................................... 40 2.5.4. Adhesion sliding ............................................................................................................... 41 2.5.5. Focal adhesion disassembly .......................................................................................... 42 2.5.6. Microtubules target FAs .................................................................................................. 44 2.5.7. Cortical complexes around FAs ..................................................................................... 47 2.6. Integrin adhesome analysis .................................................................................................... 49 2.7. Kank family proteins ................................................................................................................. 52 Table of contents| 4 3. Aim of the thesis ............................................................................................................................... 58 4. Short summary of manuscripts ...................................................................................................... 59 4.1. Kank family proteins comprise a novel type of talin activator ............................................ 59 4.2. β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments ............................................................................................... 59 4.3. A firm grip does not always pay off: a new Phact(r) 4 integrin signaling ......................... 60 4.4. Nascent Adhesions: From Fluctuations to a Hierarchical Organization .......................... 60 5. References ........................................................................................................................................ 61 6. Acknowledgement ............................................................................................................................ 80 7. Curriculum vitae ................................................................................................................................ 81 8. Appendix ............................................................................................................................................ 83 8.1. Publication 1 .............................................................................................................................. 83 8.2. Publication 2 .............................................................................................................................. 83 8.3. Publication 3 .............................................................................................................................. 83 8.4. Publication 4 .............................................................................................................................. 83 Abbreviations| 5 Abbreviations ADMIDAS metal ion-dependent adhesion site APC adenomatous polyposis coli ARP2/3 actin-related protein 2/3 ATAT1 α-tubulin N-acetyltransferase 1 CDK5 cyclin-dependent kinase 5 CFEOM1 congenital fibrosis of the extraocular muscles type 1 CLASP cytoplasmic linker associated protein CRAPome contaminant repository for affinity purification Dab2 disabled homolog 2 DD dimerization domain DLC1 deleted in liver cancer 1 DOK1 docking protein 1 ECM extracellular matrix EGF epidermal growth factor EM electron microscopy Eps8 epidermal growth factor receptor pathway substrate 8 EZH2 enhancer of zeste homolog 2 FA focal adhesion FAEI focal adhesion enrichment index FAK focal adhesion kinase FB fibrillar adhesion FCHO2 FCH domain only 2 FERM 4.1/ezrin/radixin/moesin FN fibronectin FRAP fluorescence recovery after photobleaching GEF guanine nucleotide exchange factor HDAC6 histone deacetylase 6 hMSC human mesenchymal stem cell IBS integrin binding site ICAP1 integrin cytoplasmic domain-associated protein 1 ILK integrin-linked kinase IMC inner membrane clasp IRSp53 insulin receptor substrate p53 Kank KN motif and ankyrin repeat domains LADIII leukocyte adhesion deficiency type III LATS large tumor suppressor LIMBS or ligand-induced metal ion binding site MDGI mammary-derived growth inhibitor METTL8 methyltransferase-like protein 8 MIDAS metal ion-dependent adhesion site MRL Mig-10/RIAM/Lamellipodin MST STE20-like protein kinase MT1-MMP membrane type 1 matrix metalloprotease MYPT1 myosin phosphatase-targeting subunit 1 NA nascent adhesion NMR nuclear magnetic resonance OMC outer membrane clasp Abbreviations| 6 PH pleckstrin homology PIPKIγ90 phosphatidylinositol 4-phosphate 5-kinase Type Iγ 90 PP2A protein phosphatase 2A PSI plexin-semaphorin-integrin PTB phospho-tyrosine binding RA Ras-association RIAM Rap1–GTP-interacting adapter molecule ROCK Rho-associated coiled-Coil containing protein kinase SAXS small angle X‑ray scattering SHARPIN SHANK-associated RH domain-interacting protein Smurf1 SMAD ubiquitination regulatory factor 1 SNX sorting nexin SRC steroid receptor coactivator SRF serum-responsive factor SyMBS synergistic metal ion binding site TAZ transcriptional co-activator with PDZ-binding motif TEAD TEA domain THATCH talin/HIP1R/Sla2p actin tethering C-terminal homology TMD transmembrane domain VASP vasodilator-stimulated phosphoprotein VASP vasodilator-stimulated phosphoprotein VBS vinculin-binding site WAVE2 WASP-family verprolin-homologous protein-2 YAP Yes-associated protein Summary| 7 1. Summary Cell adhesion to the extracellular matrix (ECM) is mainly mediated by integrins, which are heterodimeric transmembrane receptors composed of α and β subunits. 18 different α subunits and 8 different β subunits assemble into 24 different integrin heterodimers recognizing specific ECM ligands. Prior to the assembly of an adhesion site, integrins have to be activated. Integrin activation is characterized by switching the unbound integrin from a low affinity (inactive) to a high affinity (active) state,