Impact of Keratin Network Regulation on Migrating Cells Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Anne Pora, Ingénieur, Master aus Rueil-Malmaison, Frankreich Berichter: Univ.-Prof. Dr. Björn Kampa Univ.-Prof. Dr. med. Rudolf Leube Tag der mündlichen Prüfung: 02.04.19 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. This work was performed at the Institute for Molecular and Cellular Anatomy at University Hospital RWTH Aachen by the mentorship of Prof. Dr. med. Rudolf E. Leube. It was exclusively performed by myself, unless otherwise stated in the text. 1. Reviewer: Univ.-Prof. Dr. Björn Kampa 2. Reviewer: Univ.-Prof. Dr. med. Rudolf E. Leube Toulouse (FR), 30.11.18 2 Table of Contents Table of Contents 3 Chapter 1: Introduction 6 1. Cell migration 6 A key process in physiological and pathological conditions 6 Different kinds of migration 6 Influence of the environment 7 2. The cytoskeleton: a key player in cell migration 9 The cytoskeleton 9 Actin and focal adhesions 9 Microtubules 12 Keratin intermediate filaments and hemidesmosomes 13 Cross-talk between keratin intermediate filaments and others 22 cytoskeletal components Imaging cytoskeletal dynamics in migrating cells 24 3. Objectives 26 Chapter 2: Materials and Methods 27 1. Cell culture conditions 27 2. Keratin extraction and immunoblotting 28 3. Immunofluorescence 32 4. Plasmid constructs and DNA transfection into cultured cells 34 5. Drug treatments 35 6. Micropatterning 35 7. Preparation of elastic substrates 36 8. Imaging conditions 36 3 9. Image analysis 37 10. Statistical analysis 43 Chapter 3: Results 44 A. Influence of the mechanophysical environment on keratin 44 network dynamics in migrating cells 1. K5-YFP is a reliable reporter to measure keratin dynamics in migrating 44 normal human epidermal keratinocytes 2. The keratin flow pattern in migrating keratinocytes differs between the 47 cell front, center, and back with respect to speed and direction of keratin flow 3. High keratin flow correlates with high migration speed 50 4. The keratin flow pattern mirrors the trajectory of cell migration 52 5. Increased ECM coating density leads to decreased keratin flow 56 6. Decreased substrate stiffness increases keratin flow 59 7. Confinement of migrating normal human epidermal keratinocytes 62 reduces keratin flow 8. Keratins only mildly affect the mechanophysical-dependent regulation of 64 migration 9. Keratin flow lags behind actin flow in migrating keratinocytes 67 B. Hemidesmosomes and focal adhesions form treadmilling 72 arrays in migrating primary keratinocytes 1. Hemidesmosomal proteins cluster in chevron-shaped arrays in migrating 72 primary human keratinocytes 2. Hemidesmosomal chevron arrays and focal adhesion sites are spatially 75 linked but segregated 3. Focal adhesion-decorated hemidesmosomal chevrons are formed at the 78 cell front and are removed in the cell rear in migrating keratinocytes 4 4. Hemidesmosomal chevron patterns form during cell adhesion and 80 spreading 5. Focal adhesions and hemidesmosomes affect each other's distribution 83 during chevron pattern formation Chapter 4: Discussion 90 1. Keratin flow pattern during migration 90 2. Impact of the mechanophysical environment 92 3. Actin as an upstream regulator of keratin dynamics 93 4. Highly ordered chevron-like hemidesmosomal structures 95 5. Cross-talk between hemidesmosomes and focal adhesions 96 6. Cell-matrix adhesions as key players in actin-keratin cross-talks 98 7. Conclusion and future work 100 What we learn about the complexity of cytoskeletal cross-talk 100 during cell migration Future work 103 Summary 106 References 108 List of Figures 118 List of Tables 120 List of Movies 121 List of Abbreviations 123 Acknowledgements 125 Curriculum Vitae 127 Bibliography 129 5 Chapter 1: Introduction 1. Cell migration A key process in physiological and pathological conditions Cell migration is a highly complex process that is crucial in various processes, either physiological or pathological. Physiological processes include organ development during embryogenesis, maintenance of tissue homeostasis at every stage of life, as well as movement of immune cells in the body. Pathological processes include wound-healing but also migration of cancer cells away from the initial tumor to form metastases (Doyle et al., 2013). Different kinds of migration Among animal cells, migration behaviors are very diverse. Two main modes of migration are described in the literature for single cells: mesenchymal and amoeboid migration. Mesenchymal migration is characterized by a low speed, cells with irregular shapes, strong cell-matrix adhesions, and the development of flat protrusions at the leading-edge called lamellipodium and filipodium (Friedl and Wolf, 2010; Welch et al., 2015). Amoeboid migration is characterized by a higher speed, cells with round shapes, weak cell-matrix adhesions, and the formation of blebs (Gardel et al., 2010). Depending on the composition of the blebs (pseudopods or stable blebs), amoeboid migration can be subdivided into two sub-kinds of migration (Lämmermann and Sixt, 2009). Mesenchymal migration is typically adopted by fibroblasts and keratinocytes, while amoeboid migration is adopted by dendritic cells. Nevertheless, the cell behavior is very plastic, so that depending on the environment, cells can switch from one mode to another mode of migration. Typically, upon confinement fibroblasts can change in favor of amoeboid migration, in a process called mesenchymal-to-amoeboid migration (Friedl and Wolf, 2010). 6 This study focuses on epithelial cell migration. In the next parts, I will only focus on migration in the mesenchymal mode. Influence of the environment Migration is highly dependent on the cellular environment: cells are subjected to a high variety of biochemical signals, but also to different mechanical properties of their surroundings (Fig. 1.1) (Doyle et al., 2013). These two types of signal must be perceived and integrated for cellular responses. The ability of cells to sense the mechanical characteristics of their environment is called mechanosensation, while their ability to respond to it is called mechanotransduction (Bukoreshtliev et al., 2013). Relevant cell components and mechanisms for such processes are discussed in the next parts. Additionally, cells not only respond to their environment but also modify their environment; this bi-directional cross-talk is called dynamic reciprocity (Helvert et al., 2018). The most relevant physical parameters to consider in the physiological environment for cells are the dimensionality and degree of confinement, the topography, and the extracellular matrix (ECM) composition, density and stiffness (Charras and Sahai, 2014). The way cells respond to these different stimuli is highly context dependent. Experimentally, when preparing a given matrix, changing one parameter might well affect several others. For example, the composition of 3D gels affects their elastics response: fibronectin fibers are covalently linked to each other so that they form a linear elastic gel, whereas collagen fibers can move independently so that collagen gels do not have a linear elastic behavior (Pedersen et al., 2005). In terms of dimensionality, 2D planar substrates are the most common geometry used in cell culture, even though they are poorly representative of physiological conditions encountered in the human body where cells are confronted with a 3D environment. Cells are mostly confined in physiological conditions, and 1D models (e.g., cells migrating on 7 fibers) can be representative of such situations. 2D geometries promote cell spreading while 1D and 3D models inhibit cell spreading due to their fibrillar or porous topography (Doyle et al., 2013). Confinement promotes blebbing when friction is enough to generate motion without the need of cell-matrix adhesions (Welch et al., 2015). Figure 1.1: Influence of the environment on cell migration. Scheme depicting environmental factors affecting intracellular regulators and ultimately determining the migratory phenotype of cells. Adapted from Doyle et al. (2013). ECM-ligand interactions also strongly affect cell migration. Different ECM proteins potentially interact with different ligands. The coating density as well as the substrates stiffness affect migration speed in a biphasic manner in 2D. At very low density, cell- matrix adhesions are hardly formed, while at very high density, cells fail at retracting in the rear (Palecek et al., 1997; Palecek et al., 1998). At fixed ECM concentration, migration speed is reduced on stiffer substrates, except for very soft substrates (i.e. in the sub-kPa range where migration speed is reduced) (Lo et al., 2000; Peyton and Putnam, 2005; Yeung et al., 2005; Zhong and Ji, 2013). Adjusting both parameters in parallel allows modulation of the migration speed. The environment acts as an extracellular regulator of cell migration. At the intracellular level, these signals are integrated by a highly complex system called the cytoskeleton. 8 2. The cytoskeleton: a key player in cell migration The cytoskeleton In eukaryotic cells, a network of filaments extending throughout the cytoplasm is found and called the cytoskeleton. It has various roles including defining the cell shape, organizing the cytoplasm, maintenance of mechanical stability, and regulation of a wide range