Quantitative analysis of haptotactic cell migration Dissertation Thesis

Author(s): Schwarz, Jan
Advisor(s): Sixt, Michael
Committee Chair(s): Wagner, Uli
Committee Member(s): Siekhaus, Daria; Janovjak, Harald; Legler, Daniel
Title: Quantitative analysis of haptotactic cell migration
Affiliation IST Austria
Abstract: Directed cell migration is a hallmark feature, present in almost all multi-cellular organisms. Despite its importance, basic questions regarding force transduction or directional sensing are still heavily investigated. Directed migration of cells guided by immobilized guidance cues - haptotaxis - occurs in key-processes, such as embryonic development and immunity (Middleton et al., 1997; Nguyen et al., 2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues comprise adhesive ligands, such as collagen and fibronectin (Barczyk et al., 2009), or chemokines - the main guidance cues for migratory leukocytes (Middleton et al., 1997; Weber et al., 2013). While adhesive ligands serve as attachment sites guiding cell migration (Carter, 1965), chemokines instruct haptotactic migration by inducing adhesion to adhesive ligands and directional guidance (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis of the cellular response to immobilized guidance cues requires in vitro assays that foster cell migration, offer accurate control of the immobilized cues on a subcellular scale and in the ideal case closely reproduce in vivo conditions. The exploration of haptotactic cell migration through design and employment of such assays represents the main focus of this work. Dendritic cells (DCs) are leukocytes, which after encountering danger signals such as pathogens in peripheral organs instruct naïve T-cells and consequently the adaptive immune response in the lymph node (Mellman and Steinman, 2001). To reach the lymph node from the periphery, DCs follow haptotactic gradients of the chemokine CCL21 towards lymphatic vessels (Weber et al., 2013). Questions about how DCs interpret haptotactic CCL21 gradients have not yet been addressed. The main reason for this is the lack of an assay that offers diverse haptotactic environments, hence allowing the study of DC migration as a response to different signals of immobilized guidance cue. In this work, we developed an in vitro assay that enables us to quantitatively assess DC haptotaxis, by combining precisely controllable chemokine photo-patterning with physically confining migration conditions. With this tool at hand, we studied the influence of CCL21 gradient properties and concentration on DC haptotaxis. We found that haptotactic gradient sensing depends on the absolute CCL21 concentration in combination with the local steepness of the gradient. Our analysis suggests that the directionality of migrating DCs is governed by the signal-to-noise ratio of CCL21 binding to its receptor CCR7. Moreover, the haptotactic CCL21 gradient formed in vivo provides an optimal shape for DCs to recognize haptotactic guidance cue. By reconstitution of the CCL21 gradient in vitro we were also able to study the influence of CCR7 signal termination on DC haptotaxis. To this end, we used DCs lacking the G-protein coupled receptor kinase GRK6, which is responsible for CCL21 induced CCR7 receptor phosphorylation and desensitization (Zidar et al., 2009). We found that CCR7 desensitization by GRK6 is crucial for maintenance of haptotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. In the context of the organism, immobilized haptotactic guidance cues often coincide and compete with soluble chemotactic guidance cues. During wound healing, fibroblasts are exposed and influenced by adhesive cues and soluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly, migrating DCs are exposed to both, soluble chemokines (CCL19 and truncated CCL21) inducing chemotactic behavior as well as the immobilized CCL21. To quantitatively assess these complex coinciding immobilized and soluble guidance cues, we implemented our chemokine photo-patterning technique in a microfluidic system allowing for chemotactic gradient generation. To validate the assay, we observed DC migration in competing CCL19/CCL21 environments. Adhesiveness guided haptotaxis has been studied intensively over the last century. However, quantitative studies leading to conceptual models are largely missing, again due to the lack of a precisely controllable in vitro assay. A requirement for such an in vitro assay is that it must prevent any uncontrolled cell adhesion. This can be accomplished by stable passivation of the surface. In addition, controlled adhesion must be sustainable, quantifiable and dose dependent in order to create homogenous gradients. Therefore, we developed a novel covalent photo-patterning technique satisfying all these needs. In combination with a sustainable poly-vinyl alcohol (PVA) surface coating we were able to generate gradients of adhesive cue to direct cell migration. This approach allowed us to characterize the haptotactic migratory behavior of zebrafish keratocytes in vitro. Furthermore, defined patterns of adhesive cue allowed us to control for cell shape and growth on a subcellular scale.
Publication Title: IST Dissertation
Degree Granting Institution: IST Austria  
Degree: PhD
Degree Date: 2016-07-01
Start Page: 1
Total Pages: 178
Notes: First, I would like to thank Michael Sixt for being a great supervisor, mentor and scientist. I highly appreciate his guidance and continued support. Furthermore, I am very grateful that he gave me the exceptional opportunity to pursue many ideas of which some managed to be included in this thesis. I owe sincere thanks to the members of my PhD thesis committee, Daria Siekhaus, Daniel Legler and Harald Janovjak. Especially I would like to thank Daria for her advice and encouragement during our regular progress meetings. I also want to thank the team and fellows of the Boehringer Ingelheim Fond (BIF) PhD Fellowship for amazing and inspiring meetings and the BIF for financial support. Important factors for the success of this thesis were the warm, creative and helpful atmosphere as well as the team spirit of the whole Sixt Lab. Therefore I would like to thank my current and former colleagues Frank Assen, Markus Brown, Ingrid de Vries, Michelle Duggan, Alexander Eichner, Miroslav Hons, Eva Kiermaier, Aglaja Kopf, Alexander Leithner, Christine Moussion, Jan Müller, Maria Nemethova, Jörg Renkawitz, Anne Reversat, Kari Vaahtomeri, Michele Weber and Stefan Wieser. We had an amazing time with many legendary evenings and events. Along these lines I want to thank the in vitro crew of the lab, Jörg, Anne and Alex, for lots of ideas and productive discussions. I am sure, some day we will reveal the secret of the ‘splodge’. I want to thank the members of the Heisenberg Lab for a great time and thrilling kicker matches. In this regard I especially want to thank Maurizio ‘Gnocci’ Monti, Gabriel Krens, Alex Eichner, Martin Behrndt, Vanessa Barone,Philipp Schmalhorst, Michael Smutny, Daniel Capek, Anne Reversat, Eva Kiermaier, Frank Assen and Jan Müller for wonderful after-lunch matches. I would not have been able to analyze the thousands of cell trajectories and probably hundreds of thousands of mouse clicks without the productive collaboration with Veronika Bierbaum and Tobias Bollenbach. Thanks Vroni for countless meetings, discussions and graphs and of course for proofreading and advice for this thesis. For proofreading I also want to thank Evi, Jörg, Jack and Anne. I would like to acknowledge Matthias Mehling for a very productive collaboration and for introducing me into the wild world of microfluidics. Jack Merrin, for countless wafers, PDMS coated coverslips and help with anything micro-fabrication related. And Maria Nemethova for establishing the ‘click’ patterning approach with me. Without her it still would be just one of the ideas… Many thanks to Ekaterina Papusheva, Robert Hauschild, Doreen Milius and Nasser Darwish from the Bioimaging Facility as well as the Preclinical and the Life Science facilities of IST Austria for excellent technical support. At this point I especially want to thank Robert for countless image analyses and technical ideas. Always interested and creative he played an essential role in all of my projects. Additionally I want to thank Ingrid and Gabby for welcoming me warmly when I first started at IST, for scientific and especially mental support in all those years, countless coffee sessions and Heurigen evenings. #BioimagingFacility #LifeScienceFacility #PreClinicalFacility
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