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Characterization of Kindlin3 in Integrin Functions

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Characterization of Kindlin3 in Integrin Functions This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Characterization of kindlin3 in Integrin functions Feng, Chen 2013 Feng, C. (2013). Characterization of kindlin3 in Integrin functions. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/54828 https://doi.org/10.32657/10356/54828 Downloaded on 27 Sep 2021 23:43:18 SGT Characterization of kindlin3 in Integrin functions Feng Chen School of Biological Sciences 2013 Characterization of kindlin3 in Integrin functions Feng Chen School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2013 Acknowledgements First, I would like to thank my supervisor Dr. Tan Suet Mien, for his guidance during the course of my studies. He gave me many helpful suggestions for my project and has been supportive of me for trying out new ideas. I’m grateful to Dr. Yau Yin Hoe and Assoc Prof. Shochat for helping me with the SPR analyses. I wish to thank my lab mates Man Li, Yan Feng, Zhi Hong, Xiao Yan, Rhoda, Henni, Sze Wah, Manisha, Joel, Li Teng, and Jessie for their helpful suggestions and assistance. Special thanks to Dr. Tang Man Li for her guidance at the beginning of my study and Tang Xiao Yan and Ong Li Teng for the support they gave me when I encounter problems in my studies. Finally, I am indebted to my parents, husband, and friends. They provided me selfless help and encouragement during the course of my Ph.D. study. Special thanks to my husband Jarrett Dumond for his endless love and support. Contents Acknowledgements Contents Abstract Abbreviations Chapter 1 Introduction ............................................................................................ 2 1.1 Integrins: heterodimeric cell adhesion molecules ..............................................2 1.2 General domain-organization and structure of integrins ...................................5 1.2.1 Extracellular domains of the integrin α subunit ....................................... 6 1.2.2 Extracellular domains of the integrin β subunit ....................................... 8 1.2.3 The transmembrane domains of integrins .............................................. 10 1.2.4 Cytoplasmic tails of integrins ................................................................ 12 1.2.5 Conformation changes in integrin during its activation ......................... 13 1.3 The β2 integrins ...............................................................................................15 1.3.1 Integrin αLβ2 ........................................................................................ 16 1.3.2 Inside-out activation of integrin αLβ2 .................................................. 17 1.3.3 Outside-in signaling of integrin αLβ2 ................................................... 19 1.4 Talin .................................................................................................................20 1.5 Kindlins as co-activators of integrins ..............................................................22 1.6 Receptor for activated C kinase 1 (RACK1) ...................................................26 1.7 Integrin α5β1 ....................................................................................................28 1.8 Aims of this study ............................................................................................30 Chapter 2 Materials and Methods ......................................................................... 31 2.1 General reagents.............................................................................................31 2.2 Vectors and cDNA .........................................................................................31 2.3 Antibodies ......................................................................................................32 2.4 Expression plasmids of integrins and talin 1 head domain (HD) ..................34 2.5 Expression plasmids of kindlin2 and kindlin3 ...............................................34 2.6 Expression plasmids of RACK1 ....................................................................38 2.7 Medium ..........................................................................................................40 2.8 Solutions ........................................................................................................40 2.9 Miniprep and midiprep of plasmid DNA .......................................................42 2.10 Quantitation of DNA......................................................................................43 2.11 DNA electrophoresis ......................................................................................43 2.12 Purification of DNA fragments agarose gel ...................................................43 2.13 DNA ligation ..................................................................................................44 2.14 Preparation of Escherichia coli competent cells ............................................44 2.15 Transformation of plasmid DNA ...................................................................44 2.16 Standard PCR protocol ..................................................................................45 2.17 mRNA extraction and reverse transcription PCR ..........................................45 2.18 Expression and purification of recombinant kindlin2 and kindlin3 protein ..46 2.19 Expression and purification of recombinant RACK1 and RACK1-W-5-7 ...46 2.20 Mammalian cell culture .................................................................................47 2.20.1 Culture of HEK293T cells .................................................................. 47 2.20.2 Culture of COS-7 cells ........................................................................ 47 2.20.3 Culture of K562 and SKW 3.0 cells ................................................... 48 2.20.4 Culture of HUVECs ............................................................................ 48 2.21 Cell storage in liquid nitrogen........................................................................48 2.22 Cell recovery from liquid nitrogen ................................................................49 2.23 Isolation of human blood primary T cells ......................................................49 2.24 Transfection of 293T or COS-7 cells .............................................................49 2.25 Transfection of K562 cells .............................................................................50 2.26 Generation of stable K562 cells with reduced kindlin3 expression ...............50 2.27 Coating of microtitre plates, glass bottom, Ibidi® µ-slide | 0.4 Luer chambers or 16-well E-plate dishes with ICAM-1, fibrinogen or fibronectin ......................................................................................................51 2.28 Preparation of protein A-sepharose with or without rabbit anti-rat IgG conjugation ....................................................................................................52 2.29 Western blot ...................................................................................................53 2.30 Immunoprecipitation ......................................................................................55 2.31 Real-time electric cell-substrate impedance sensing (ECIS) measurments ...56 2.32 GST pull-down assays ...................................................................................56 2.33 Surface plasmon resonance (SPR) analyses ..................................................57 2.34 Flow cytometry ..............................................................................................58 2.35 Shear flow ......................................................................................................58 2.36 FRET analyses ...............................................................................................59 2.37 Fluorescence microscopy ...............................................................................60 2.38 Statistical analysis ..........................................................................................61 Chapter 3 Characterization of kindlin3 monoclonal antibodies ........................... 62 3.1 Introduction ......................................................................................................62 3.2 kindlin3 monoclonal antibodies .......................................................................62 3.3 The epitopes of the four mAbs in kindlin3 ......................................................63 3.4 Discussion ........................................................................................................68 Chapter 4 Kindlin3 is important for integrin αLβ2 ouside-in signaling and it interacts with the scaffold protein receptor for activated-C kinase 1 (RACK1) .............................................................................................. 70 4.1 Introduction ......................................................................................................70 4.2 Kindlin3 plays an important role in integrin αLβ2 outside-in signaling..........70 4.3 Kindlin3 over-expression induced integrin αLβ2 micro-clustering in K562 cells. ...............................................................................................................73 4.4 Kindlin3 interacts with the scaffold protein RACK-1 .....................................76
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