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Anti-Arpc5 Nanobodies As Potential Inhibitors of Cancer Cell Migration and Invasion

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Anti-Arpc5 Nanobodies As Potential Inhibitors of Cancer Cell Migration and Invasion Faculteit Bio-ingenieurswetenschappen Academiejaar 2009 – 2010 Anti-ArpC5 nanobodies as potential inhibitors of cancer cell migration and invasion. Sofie Perdu Promotor: Prof. Dr. Els Van Damme Co-promotor: Prof. Dr. Jan Gettemans Tutor: Dr. Thomas Hubert Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen, cel- en gen biotechnologie Faculteit Bio-ingenieurswetenschappen Academiejaar 2009 – 2010 Anti-ArpC5 nanobodies as potential inhibitors of cancer cell migration and invasion. Sofie Perdu Promotor: Prof. Dr. Els Van Damme Co-promotor: Prof. Dr. Jan Gettemans Tutor: Dr. Thomas Hubert Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen, cel- en gen biotechnologie “De auteur en de promotor geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit deze scriptie.” Gent, juni 2010 De promotoren: Prof. Dr. Jan Gettemans Prof. Dr. Els Van Damme De auteur: Sofie Perdu woord vooraf Deze masterproef is tot stand gekomen dankzij de kans die ik heb gekregen van Prof. Dr. Els Van Damme en Prof. Dr. Jan Gettemans. Ik wil hen bedanken voor de leerrijke ervaring. De grootste dank van allemaal gaat uit naar de persoon die me gedurende deze maanden rechtstreeks begeleidde: Dr. Thomas Hubert. Ik wil hem bedanken voor zijn bereidheid om me te helpen bij elk probleem dat zich voordeed en voor zijn goede ideeën die mijn onderzoek steeds interessanter maakten. Bovendien moet ik hem bedanken voor zijn positieve en motiverende woorden als het even wat minder ging en voor al het verbeterwerk dat hij aan deze masterproef gehad heeft. Bedankt ook: Aude, Anske, Ariane, Eline, Jonas, Katrien, Sarah, Wouter, Ciska, Eveline, Olivier, Hanne, Isabel en Marco voor de leuke tijd in het Rommelaere complex. Bovendien wil ik mijn ouders en broer bedanken voor hun steun en luisterend oor. En den bompa natuurlijk, voor zijn algemene interesse in het wetenschappelijk onderzoek en de dingen waar ik mee bezig was. Ook Jack McMartin verdient een plaatsje op deze pagina, aangezien hij me geholpen heeft met de Engelse taal. En tenslotte mag ik Jonas zeker niet vergeten. Hij was degene die al mijn labo avonturen moest aanhoren, elke dag opnieuw. Bedankt voor de steun en hulp waar mogelijk. Table of contents SUMMARY ....................................................................................................................................................... 1 NEDERLANDSE SAMENVATTING ....................................................................................................................... 3 INTRODUCTION ................................................................................................................................................ 5 1. NANOBODIES ................................................................................................................................................ 5 1.1. The immune system ................................................................................................................................ 5 1.2. The immune system of the Camelidae .................................................................................................... 7 1.3. Properties of the nanobodies .................................................................................................................. 8 1.4. Generating nanobodies .......................................................................................................................... 9 1.5. Applications of nanobodies ................................................................................................................... 10 2. THE ARP2/3 COMPLEX ................................................................................................................................. 11 2.1. Actin: a building block for the cell cytoskeleton.................................................................................... 11 2.2. The ARP2/3 complex ............................................................................................................................. 12 2.3. The Nucleation process of actin filaments ............................................................................................ 15 2.4. Structures built up by actin and the Arp2/3 complex ........................................................................... 16 2.5. The Arp2/3 complex and its cellular functions ...................................................................................... 18 2.6. Actin and diseases ................................................................................................................................ 19 MATERIALS AND METHODS ........................................................................................................................... 20 1. PLASMIDS AND ANTIBODIES ........................................................................................................................... 20 2. DNA MANIPULATIONS .................................................................................................................................. 20 1.1. Classical ligation dependent cloning ..................................................................................................... 20 1.2. QuikChange site-specific mutagenesis.................................................................................................. 21 1.3. Oligonucleotide (oligo) annealing ......................................................................................................... 22 1.4. Ligation ................................................................................................................................................. 22 1.5. Sequencing ............................................................................................................................................ 23 3. PROTEIN MANIPULATION ............................................................................................................................... 23 2.1. Bio-Rad protein assay for determination of protein concentration ...................................................... 23 2.2. Sodium Dodecyl Sulphate Polyacrylamide Gel Electroforesis (SDS-PAGE) ............................................ 23 2.3. Western blot ......................................................................................................................................... 23 2.4. Production of nanobodies ..................................................................................................................... 23 4. CELL MANIPULATION .................................................................................................................................... 25 3.1. Used cell lines ....................................................................................................................................... 25 3.2. Transfection .......................................................................................................................................... 26 3.3. Immunostaining and immunofluorescence microscopy ....................................................................... 26 3.4. Immunoprecipitation and in vitro binding ............................................................................................ 26 RESULTS ......................................................................................................................................................... 28 1. ANTI-ARPC5 NANOBODY PURIFICATION ........................................................................................................... 28 2. QUALITY CONTROL OF THE ANTI-ARPC5 NANOBODY........................................................................................... 29 2.5. Quality control with purified His6-SUMO-ArpC5 ................................................................................... 30 2.6. Quality control with antigen-containing E. coli extract ........................................................................ 30 2.7. Quality control with HEK-extract .......................................................................................................... 32 3. IN VITRO BINDING ANALYSIS ........................................................................................................................... 33 3.5. Immunoprecipitation with purified bovine Arp2/3 complex ................................................................. 33 4. IN VIVO BINDING ANALYSIS ............................................................................................................................. 35 4.1. Nanobody fused with a Mitochondrial Outer Membrane (MOM) targeting signal ............................. 35 4.2. Nanobody fused with a Nucleolar targeting signal .............................................................................. 37 4.3. Further observations by microscopy that could explain the discrepancy found in the previous experiments ................................................................................................................................................. 38 DISCUSSION ..................................................................................................................................................
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