---
OA_place: publisher
_id: '1129'
abstract:
- lang: eng
  text: "Directed cell migration is a hallmark feature, present in almost all multi-cellular\r\norganisms.
    Despite its importance, basic questions regarding force transduction\r\nor directional
    sensing are still heavily investigated. Directed migration of cells\r\nguided
    by immobilized guidance cues - haptotaxis - occurs in key-processes,\r\nsuch as
    embryonic development and immunity (Middleton et al., 1997; Nguyen\r\net al.,
    2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues\r\ncomprise
    adhesive ligands, such as collagen and fibronectin (Barczyk et al.,\r\n2009),
    or chemokines - the main guidance cues for migratory leukocytes\r\n(Middleton
    et al., 1997; Weber et al., 2013). While adhesive ligands serve as\r\nattachment
    sites guiding cell migration (Carter, 1965), chemokines instruct\r\nhaptotactic
    migration by inducing adhesion to adhesive ligands and directional\r\nguidance
    (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis\r\nof the
    cellular response to immobilized guidance cues requires in vitro assays\r\nthat
    foster cell migration, offer accurate control of the immobilized cues on a\r\nsubcellular
    scale and in the ideal case closely reproduce in vivo conditions. The\r\nexploration
    of haptotactic cell migration through design and employment of such\r\nassays
    represents the main focus of this work.\r\nDendritic cells (DCs) are leukocytes,
    which after encountering danger\r\nsignals such as pathogens in peripheral organs
    instruct naïve T-cells and\r\nconsequently the adaptive immune response in the
    lymph node (Mellman and\r\nSteinman, 2001). To reach the lymph node from the periphery,
    DCs follow\r\nhaptotactic gradients of the chemokine CCL21 towards lymphatic vessels\r\n(Weber
    et al., 2013). Questions about how DCs interpret haptotactic CCL21\r\ngradients
    have not yet been addressed. The main reason for this is the lack of\r\nan assay
    that offers diverse haptotactic environments, hence allowing the study\r\nof DC
    migration as a response to different signals of immobilized guidance cue.\r\nIn
    this work, we developed an in vitro assay that enables us to\r\nquantitatively
    assess DC haptotaxis, by combining precisely controllable\r\nchemokine photo-patterning
    with physically confining migration conditions. With this tool at hand, we studied
    the influence of CCL21 gradient properties and\r\nconcentration on DC haptotaxis.
    We found that haptotactic gradient sensing\r\ndepends on the absolute CCL21 concentration
    in combination with the local\r\nsteepness of the gradient. Our analysis suggests
    that the directionality of\r\nmigrating DCs is governed by the signal-to-noise
    ratio of CCL21 binding to its\r\nreceptor CCR7. Moreover, the haptotactic CCL21
    gradient formed in vivo\r\nprovides an optimal shape for DCs to recognize haptotactic
    guidance cue.\r\nBy reconstitution of the CCL21 gradient in vitro we were also
    able to\r\nstudy the influence of CCR7 signal termination on DC haptotaxis. To
    this end,\r\nwe used DCs lacking the G-protein coupled receptor kinase GRK6, which
    is\r\nresponsible for CCL21 induced CCR7 receptor phosphorylation and\r\ndesensitization
    (Zidar et al., 2009). We found that CCR7 desensitization by\r\nGRK6 is crucial
    for maintenance of haptotactic CCL21 gradient sensing in vitro\r\nand confirm
    those observations in vivo.\r\nIn the context of the organism, immobilized haptotactic
    guidance cues\r\noften coincide and compete with soluble chemotactic guidance
    cues. During\r\nwound healing, fibroblasts are exposed and influenced by adhesive
    cues and\r\nsoluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly,\r\nmigrating
    DCs are exposed to both, soluble chemokines (CCL19 and truncated\r\nCCL21) inducing
    chemotactic behavior as well as the immobilized CCL21. To\r\nquantitatively assess
    these complex coinciding immobilized and soluble\r\nguidance cues, we implemented
    our chemokine photo-patterning technique in a\r\nmicrofluidic system allowing
    for chemotactic gradient generation. To validate\r\nthe assay, we observed DC
    migration in competing CCL19/CCL21\r\nenvironments.\r\nAdhesiveness guided haptotaxis
    has been studied intensively over the\r\nlast century. However, quantitative studies
    leading to conceptual models are\r\nlargely missing, again due to the lack of
    a precisely controllable in vitro assay. A\r\nrequirement for such an in vitro
    assay is that it must prevent any uncontrolled\r\ncell adhesion. This can be accomplished
    by stable passivation of the surface. In\r\naddition, controlled adhesion must
    be sustainable, quantifiable and dose\r\ndependent in order to create homogenous
    gradients. Therefore, we developed a novel covalent photo-patterning technique
    satisfying all these needs. In\r\ncombination with a sustainable poly-vinyl alcohol
    (PVA) surface coating we\r\nwere able to generate gradients of adhesive cue to
    direct cell migration. This\r\napproach allowed us to characterize the haptotactic
    migratory behavior of\r\nzebrafish keratocytes in vitro. Furthermore, defined
    patterns of adhesive cue\r\nallowed us to control for cell shape and growth on
    a subcellular scale."
acknowledged_ssus:
- _id: Bio
- _id: PreCl
- _id: LifeSc
acknowledgement: "First, I would like to thank Michael Sixt for being a great supervisor,
  mentor and\r\nscientist. I highly appreciate his guidance and continued support.
  Furthermore, I\r\nam very grateful that he gave me the exceptional opportunity to
  pursue many\r\nideas of which some managed to be included in this thesis.\r\nI owe
  sincere thanks to the members of my PhD thesis committee, Daria\r\nSiekhaus, Daniel
  Legler and Harald Janovjak. Especially I would like to thank\r\nDaria for her advice
  and encouragement during our regular progress meetings.\r\nI also want to thank
  the team and fellows of the Boehringer Ingelheim Fond\r\n(BIF) PhD Fellowship for
  amazing and inspiring meetings and the BIF for\r\nfinancial support.\r\nImportant
  factors for the success of this thesis were the warm, creative\r\nand helpful atmosphere
  as well as the team spirit of the whole Sixt Lab.\r\nTherefore I would like to thank
  my current and former colleagues Frank Assen,\r\nMarkus Brown, Ingrid de Vries,
  Michelle Duggan, Alexander Eichner, Miroslav\r\nHons, Eva Kiermaier, Aglaja Kopf,
  Alexander Leithner, Christine Moussion, Jan\r\nMüller, Maria Nemethova, Jörg Renkawitz,
  Anne Reversat, Kari Vaahtomeri,\r\nMichele Weber and Stefan Wieser. We had an amazing
  time with many\r\nlegendary evenings and events. Along these lines I want to thank
  the in vitro\r\ncrew of the lab, Jörg, Anne and Alex, for lots of ideas and productive\r\ndiscussions.
  I am sure, some day we will reveal the secret of the ‘splodge’.\r\nI want to thank
  the members of the Heisenberg Lab for a great time and\r\nthrilling kicker matches.
  In this regard I especially want to thank Maurizio\r\n‘Gnocci’ Monti, Gabriel Krens,
  Alex Eichner, Martin Behrndt, Vanessa Barone,Philipp Schmalhorst, Michael Smutny,
  Daniel Capek, Anne Reversat, Eva\r\nKiermaier, Frank Assen and Jan Müller for wonderful
  after-lunch matches.\r\nI would not have been able to analyze the thousands of cell
  trajectories\r\nand probably hundreds of thousands of mouse clicks without the productive\r\ncollaboration
  with Veronika Bierbaum and Tobias Bollenbach. Thanks Vroni for\r\ncountless meetings,
  discussions and graphs and of course for proofreading and\r\nadvice for this thesis.
  For proofreading I also want to thank Evi, Jörg, Jack and\r\nAnne.\r\nI would like
  to acknowledge Matthias Mehling for a very productive\r\ncollaboration and for introducing
  me into the wild world of microfluidics. Jack\r\nMerrin, for countless wafers, PDMS
  coated coverslips and help with anything\r\nmicro-fabrication related. And Maria
  Nemethova for establishing the ‘click’\r\npatterning approach with me. Without her
  it still would be just one of the ideas…\r\nMany thanks to Ekaterina Papusheva,
  Robert Hauschild, Doreen Milius\r\nand Nasser Darwish from the Bioimaging Facility
  as well as the Preclinical and\r\nthe Life Science facilities of IST Austria for
  excellent technical support. At this\r\npoint I especially want to thank Robert
  for countless image analyses and\r\ntechnical ideas. Always interested and creative
  he played an essential role in all\r\nof my projects.\r\nAdditionally I want to
  thank Ingrid and Gabby for welcoming me warmly\r\nwhen I first started at IST, for
  scientific and especially mental support in all\r\nthose years, countless coffee
  sessions and Heurigen evenings. #BioimagingFacility #LifeScienceFacility #PreClinicalFacility"
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Jan
  full_name: Schwarz, Jan
  id: 346C1EC6-F248-11E8-B48F-1D18A9856A87
  last_name: Schwarz
citation:
  ama: Schwarz J. Quantitative analysis of haptotactic cell migration. 2016.
  apa: Schwarz, J. (2016). <i>Quantitative analysis of haptotactic cell migration</i>.
    Institute of Science and Technology Austria.
  chicago: Schwarz, Jan. “Quantitative Analysis of Haptotactic Cell Migration.” Institute
    of Science and Technology Austria, 2016.
  ieee: J. Schwarz, “Quantitative analysis of haptotactic cell migration,” Institute
    of Science and Technology Austria, 2016.
  ista: Schwarz J. 2016. Quantitative analysis of haptotactic cell migration. Institute
    of Science and Technology Austria.
  mla: Schwarz, Jan. <i>Quantitative Analysis of Haptotactic Cell Migration</i>. Institute
    of Science and Technology Austria, 2016.
  short: J. Schwarz, Quantitative Analysis of Haptotactic Cell Migration, Institute
    of Science and Technology Austria, 2016.
corr_author: '1'
date_created: 2018-12-11T11:50:18Z
date_published: 2016-07-01T00:00:00Z
date_updated: 2026-07-29T11:30:22Z
day: '01'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: MiSi
- _id: GradSch
doi_confirm: '1'
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has_accepted_license: '1'
language:
- iso: eng
month: '07'
oa: 1
oa_version: Published Version
page: '178'
publication_identifier:
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
publist_id: '6231'
status: public
supervisor:
- first_name: Michael K
  full_name: Sixt, Michael K
  id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87
  last_name: Sixt
  orcid: 0000-0002-6620-9179
title: Quantitative analysis of haptotactic cell migration
type: dissertation
user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9
year: '2016'
...