Max-Planck-Institut für Biochemie Abteilung Membran- und Neurophysik Cell-substrate distance measurement in correlation with distribution of adhesion molecules by uorescence microscopy Yoriko Iwanaga Vollständiger Abdruck der von der Fakultät für Physik der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. J.L. van Hemmen Prüfer der Dissertation: 1. Hon.-Prof. Dr. P. Fromherz 2. Univ.-Prof. Dr. E. Sackmann Die Dissertation wurde am 12.07.2000 bei der Technischen Universität München eingereicht und durch die Fakultät für Physik am 29.08.2000 angenommen. 2 Acknowledgements The work presented here was completed in the Abteilung für Membran- und Neurophysik at the Max-Planck-Institut für Biochemie, Martinsried under the supervision of Prof. Dr. Peter Fromherz. I would like to thank Prof. Dr. Peter Fromherz for the opportunity to work in his department and for his guidance in completing this thesis. I am grateful to have worked in this excellent research condition with wonderful colleagues. The collaboration with other groups have also given me numerous scientically and personally valuable experiences. Mein Dank geht an alle Mitglieder der Abteilung Membran- und Neurophysik für ihre Hilfs- bereitschaft, ihre Unterstützung und das angenehme Arbeitsklima. Mein ganz besonderer Dank gebürt Dr. Dieter Braun, der mir immer mit zahlreichem fachkundigen und hilfreichem Rat be- treuend zur Seite stand. Bei Dr. Jürgen Kupper möchte ich mich dafür bedanken, dass er mir Techniken der Molekularbiologie beigebracht hat. Bei Prof. Reinhard Fässler und Dr. Cord Brakebusch möchte ich mich für die Hilfe und die Un- terstützung bei Umgang mit der Zellkultur und bei der komplizierten Konstruktion des GFP-ß1 Integrin Fusions Proteins bedanken. Allen Mitarbeitern von Prof. Fässler an der Universität Lund danke ich für fruchtbare Diskussionen und für ihre Hilfsbereitschaft während meines zweimonati- gen Aufenthaltes. Für die harmonische Zusammenarbeit während der Arbeiten mit der TIRAF Mikroskopie und IRM möchte ich Peter Geggier, Prof. Günther Fuhr und seiner Abteilung an der Humboldt Uni- versität zu Berlin herzlich danken. Außerdem möchte ich Karl-Friedrich Giebel and Dr. Martin Bastmeyer für die Zusammenarbeit auf dem Gebiet der SPR Mikroskopie danken. I am grateful to my uncle Prof. Sadaaki Iwanaga for the idea and his support to initially come to Max-Planck-Institute. I would also like to thank both of my grand fathers for their support and incentives. Above all, I am grateful to my parents and my sister for their continuous support and inspirations. 3 Summary Geometry of cell to solid substrate interface was studied by optical techniques and through local- ization of the cell adhesion sites by molecular biological techniques. Measurement of cell-substrate distance with uorescence interference contrast (FLIC) microscopy performed on various extracellular matrix (ECM) protein-coated silicon chip has yielded in a large range between 15-100 nm, depending on the size as well as on the conformation of the ligand. The cellular morphology and the efciency of adhesion were specic to each ECM protein. Focal contacts in broblasts localized by vinculin tagged with green uorescent protein (GFP) observed by FLIC microscopy did not exhibit the expected sharp close cell-substrate adhesion. The classical stripes of vinculin clustering in response to bronectin induced rufing of the mem- brane parallel to but not exactly at the focal contacts. The cells in neuronal culture with smooth membrane recruited vinculin mainly at cell periphery. Sites of ligand-receptor interaction was visualized by tagging ß1 integrin subunit with GFP and correlated simultaneously with FLIC microscopy. The presence of grainy distribution of ß1 in- tegrin in broblasts adhered to bronectin corresponded to the region where the membrane was held at dominating cell-substrate separation. The point contact-like dots of the fusion protein did not induce any membrane deformation in cells of neuronal culture. Focal contacts in broblasts were observed concurrently by total internal reection aqueous uo- rescence (TIRAF) microscopy and interference reection microscopy (IRM) to compare the cell- substrate distance analysis by each technique. Dark patches interpreted as sites of close contact in these images precisely matched the vinculin distribution localized by tagging with the uo- rescence protein. Calculation of the cell-substrate distance at focal contacts by both techniques reveal sensitivity to local variations in optical parameters, which explains the discrepancies at these sites from the estimations by FLIC microscopy. 5 Table of contents 1. INTRODUCTION .................................................................11 1.1 Cell-chip junction ..............................................................11 1.2 Approach .......................................................................12 1.2.1 Distance measurements ................................................12 1.2.2 FLIC microscopy and GFP-tagging .....................................13 1.3 Biology of extracellular matrix ................................................14 1.3.1 Fibronectin and its recombinant modules ...............................15 1.3.2 Laminin and its fragments ...............................................15 1.3.3 Collagen type IV and its fragment CB3 .................................17 1.3.4 Vitronectin ...............................................................17 1.4 Biology of cell adhesion ......................................................18 1.4.1 Focal adhesion ..........................................................18 1.4.2 Integrins .................................................................19 2. MATERIALS AND METHODS .................................................23 2.1 Generation of fusion constructs ..............................................23 2.1.1 Basic steps ..............................................................23 2.1.2 EGFP/ECFP-vinculin ....................................................24 2.1.3 EGFP/ECFP-integrin ß1 ................................................25 2.2 Cell culture .....................................................................28 2.2.1 Fibroblasts ..............................................................28 2.2.2 Primary neuronal cell culture ............................................28 2.2.3 Substrates ...............................................................29 2.2.4 Transient transfection ...................................................29 2.3 FLIC microscopy ...............................................................30 2.3.1 Theory...................................................................31 2.4 Total internal reection aqueous uorescence microscopy ................37 2.4.1 Theory...................................................................38 2.5 Interference reection microscopy ...........................................42 2.5.1 Theory...................................................................42 7 Table of contents 2.6 TIRAF-IRM-uorescence microscopy ........................................43 3. CELL-SUBSTRATE SEPARATION ...........................................45 3.1 Fibronectin .....................................................................45 3.2 Laminin .........................................................................48 3.3 Collagen Type IV ...............................................................54 3.4 Vitronectin ......................................................................56 3.5 Polylysine ......................................................................56 3.6 Discussion .....................................................................56 4. DISTANCE MAPS AND VINCULIN ...........................................61 4.1 Fibroblasts .....................................................................61 4.1.1 Fibronectin ..............................................................61 4.1.2 Laminin ..................................................................66 4.2 Neuronal culture ...............................................................67 4.2.1 Fibronectin and laminin .................................................67 4.3 Discussion .....................................................................72 5. DISTANCE MAPS AND INTEGRIN ß1 .......................................75 5.1 Fibroblasts .....................................................................75 5.1.1 Fibronectin ..............................................................75 5.1.2 Laminin ..................................................................78 5.2 Neuronal culture ...............................................................81 5.2.1 Fibronectin and laminin .................................................81 5.3 Discussion .....................................................................87 5.3.1 Cell-substrate distance at vinculin and integrin ß1 ......................87 6. GFP-SUBSTRATE DISTANCE ................................................91 6.0.2 GFP-vinculin to substrate ...............................................92 6.0.3 GFP-ß1 integrin to substrate ............................................93 7. COMPARISON OF DISTANCE MEASUREMENTS........................95 7.1 Dependence on cell parameters ..............................................95 8 7.1.1 TIRAF microscopy ......................................................96 7.1.2