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Nanobody Derivatization for Molecular Study and Perturbation of Cancer Cell Invadopodia

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Nanobody Derivatization for Molecular Study and Perturbation of Cancer Cell Invadopodia Nanobody derivatization for molecular study and perturbation of cancer cell invadopodia Tim Hebbrecht Promotor: Prof. Dr. Jan Gettemans 2019 - 2020 Thesis submitted to fulfil the requirements for the degree of Doctor (PhD) of Biomedical Sciences Department of Biomolecular Medicine Faculty of Medicine and Health Sciences Ghent University Nanobody derivatization for molecular study and perturbation of cancer cell invadopodia By Ir. Tim Hebbrecht Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences Campus Rommelaere Albert Baertsoenkaai 3 9000 Gent Belgium Cover illustration Photographs were taken by Tim Hebbrecht at the Rommelaere complex. The picture in front is the AF488 signal detected inside the FastGene FAS-digi Geldoc device (NIPPON genetics, Dueren, Germany). The picture at the back is a view through a window showing the historical charms of the Rommelaere complex. Promotor Prof. Dr. Jan Gettemans Nanobody lab Department of Biomolecular Medicine Faculty of Medicine and Health Sciences Campus Rommelaere Ghent University Dean Prof. dr. Piet Hoebeke Rector Prof. dr. ir. Rik Van de Walle The author and promotor give the authorization to consult and copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Tim Hebbrecht was supported by the Concerted Actions Program (GOA) of Ghent University and an Emmanuel van der Schueren grant of the Flemish cancer society (Kom op tegen kanker). Advisory Committee Prof. Dr. Marleen Van Troys Department Biomolecular Medicine Ghent University Prof. Dr. Ir. An Hendrix Department of Human Structure and Repair Ghent University Examination Committee: Chairman Prof. Dr. Bruno Verhasselt Department of Diagnostic Sciences Ghent University Members Dr. Cécile Vincke Department of Bioengineering Sciences VUB Prof. Dr. Frank Peelman Department of Biomolecular Medicine Ghent University Prof. Andre Skirtach Department of Biotechnology Ghent University Dr. Stephan Stremersch Department of Pharmaceutics Ghent University Prof. Dr. Nadine Van Roy Department of Biomolecular Medicine Ghent University Table of Contents Table of Contents .......................................................................................................... I List of Abbreviations .................................................................................................... V List of Tables ............................................................................................................... XI List of Videos ............................................................................................................... XI List of Figures ............................................................................................................ XIII Summary .................................................................................................................. XV Samenvatting .......................................................................................................... XVII Part I General introduction ........................................................................................... 1 Chapter 1 A nanobody or VHH ............................................................................... 3 1.1 The discovery of heavy chain antibodies ............................................... 3 1.2 Nanobodies .......................................................................................... 5 1.2.1 VH versus VHH ........................................................................................................ 5 1.2.2 Advantages and disadvantages of nanobodies ...................................................... 7 1.2.3 Nanobody generation ........................................................................................... 11 1.2.4 Nanobody interference with protein functions .................................................... 13 1.3 Nanobody applications ....................................................................... 14 1.3.1 Research purposes ................................................................................................ 14 1.3.2 Therapeutic and diagnostic purposes ................................................................... 18 1.4 Non-immunoglobulin based molecules ............................................... 20 I Chapter 2 Labelling and microscopy ................................................................... 23 2.1 Fusion proteins ................................................................................... 23 2.2 Coupling mechanisms for the labelling of nanobodies ......................... 25 2.2.1 Self-labelling enzymes ........................................................................................... 25 2.2.2 Enzymatic recognition tags ................................................................................... 26 2.2.3 Chemical ways for labelling nanobodies ............................................................... 28 2.2.4 Other strategies .................................................................................................... 30 2.2.5 An alternative method: the genetic incorporation of unnatural amino acids ..... 32 2.2.5.1 Non-canonical amino acid options ............................................................ 34 2.2.5.2 Optional chemical reactions after the incorporation of an unnatural amino acid .................................................................................................. 36 2.2.6 A label-free strategy .............................................................................................. 37 Chapter 3 Cancer, metastasis and cellular protrusions ........................................ 39 3.1 Cancer and metastasis ........................................................................ 39 3.2 The actin cytoskeleton ........................................................................ 40 3.3 Actin-based protrusions, a way to move the cell ................................. 42 3.3.1 Lamellipodia and filopodia .................................................................................... 43 3.3.2 Invadosomes: podosomes and invadopodia ........................................................ 43 3.3.3 Focal adhesions ..................................................................................................... 45 3.3.4 Formation of an invadopodium ............................................................................ 45 3.3.4.1 Initiation ..................................................................................................... 46 3.3.4.2 Overview of the initiation .......................................................................... 47 3.3.4.3 The initiation leads to the invadopodium precursor ................................. 49 3.3.4.4 Maturation and ECM degradation ............................................................. 51 3.4 Neural Wiskott-Aldrich syndrome protein or N-WASp ........................ 54 3.4.1 Wiskott-Aldrich syndrome .................................................................................... 54 3.4.2 Wiskott-Aldrich syndrome protein family ............................................................ 54 3.4.3 N-WASp ................................................................................................................. 55 3.4.3.1 The domains of WASp and N-WASp .......................................................... 56 3.4.3.2 From an auto-inhibition state to an activated protein.............................. 57 II Part II Scope ............................................................................................................... 59 Chapter 4 Scope .................................................................................................. 61 Part III Results ............................................................................................................ 63 Chapter 5 VCA nanobodies target N-WASp to reduce invadopodium formation and functioning ......................................................................................... 65 5.1 Introduction ........................................................................................ 65 5.2 Research paper ................................................................................... 66 5.2.1 Abstract ................................................................................................................. 66 5.2.2 Introduction .......................................................................................................... 67 5.2.3 Results ................................................................................................................... 68 5.2.3.1 N-WASp recognising of VCA nanobodies .................................................. 68 5.2.3.2 Mitochondrial outer membrane anchoring and intracellular displacement of N-WASp ........................................................................... 69 5.2.3.3 Affinity and stoichiometry of 4 selected VCA Nbs .................................... 73 5.2.3.4 The VCA Nbs interfere in the binding between N-WASp and its direct interaction partner Arp2/3, but not actin ....................................... 74 5.2.3.5 VCA nanobodies reduce cancer cell invadopodium formation ................. 75 5.2.3.6 The VCA Nbs reduce overall matrix degradation ...................................... 77 5.2.4 Discussion .............................................................................................................
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