Cover Page The handle http://hdl.handle.net/29816 holds

Cover Page
The handle http://hdl.handle.net/1887/29816 holds various files of this Leiden University
dissertation
Author: Hoorn, Hedde van
Title: Cellular forces : adhering, shaping, sensing and dividing
Issue Date: 2014-11-26
Cellular Forces
Adhering, Shaping, Sensing and
Dividing
PROEFSCHRIFT
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof. mr. C.J.J.M. Stolker,
volgens besluit van het College voor Promoties
te verdedigen op 26 november 2014
klokke 10:00 uur
door
Hedde van Hoorn
geboren te Marum, Nederland
in 1983
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Promotiecommissie
Promotor:
Prof. dr. T. Schmidt
Overige leden: Dr. E.H. Danen
Prof. dr. E.R. Eliel
Prof. dr. B. Ladoux (Université Paris Diderot, France)
Dr. ir. S.J.T. van Noort
Prof. dr. H. Schiessel
Dr. C. Storm (TU Eindhoven)
©2014 Hedde van Hoorn. All rights reserved.
Cover: HeLa cell at the end of mitosis, visualized with tubulin-GFP and
"Vitruvian Man" by Leonardo da Vinci.
Casimir PhD Series, Delft-Leiden, 2014-28
ISBN 978-90-8593-201-7
An electronic version of this thesis can be found at
https://openaccess.leidenuniv.nl
Het onderzoek beschreven in dit proefschrift is onderdeel van het wetenschappelijke programma van de Stichting voor Fundamenteel Onderzoek
der Materie (FOM), die financieel wordt gesteund door de Nederlandse
organisatie voor Wetenschappelijk Onderzoek (NWO).
ii
"Through measurement to knowledge."
Heike Kamerlingh Onnes
Voor Bart, Melissa, Inez, Peter en Vivian.
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Contents
1 Introduction
1.1 Mechanics matters . . . . . . . . . . . . . . . . . . . . .
1.2 Stiffness varies . . . . . . . . . . . . . . . . . . . . . . .
1.3 Techniques advance . . . . . . . . . . . . . . . . . . . . .
1.3.1 Active deformation . . . . . . . . . . . . . . . . .
1.3.2 Passive cell mechanics . . . . . . . . . . . . . . .
1.3.3 Micropillars . . . . . . . . . . . . . . . . . . . . .
1.4 Combining imaging and mechanics . . . . . . . . . . . .
1.4.1 Inversion improves resolution . . . . . . . . . . .
1.5 From mechanics to biology (and back) . . . . . . . . . .
1.5.1 Mechanosensing through network reorganization
1.5.2 Local activation through force . . . . . . . . . . .
1.5.3 P130Cas as a mechanosensor . . . . . . . . . . .
1.5.4 Force exertion during cell division . . . . . . . . .
1.6 Outline of this thesis . . . . . . . . . . . . . . . . . . . .
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2 Focal adhesions and forces
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1 Cell biology . . . . . . . . . . . . . . . . . . . . . .
2.2.2 Confocal microscopy . . . . . . . . . . . . . . . . .
2.2.3 Force measurement on micropillar arrays . . . . . .
2.2.4 Super-resolution imaging on micropillar arrays . .
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1 High-resolution measurement of cellular forces. . .
2.3.2 Super-resolution imaging of focal-adhesion proteins
on micropillar arrays. . . . . . . . . . . . . . . . . .
2.4 Discussion and conclusion . . . . . . . . . . . . . . . . . .
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CONTENTS
2.5
2.6
Supplemental methods . . . . . . . . . . .
2.5.1 Micropillar fabrication and coating
2.5.2 Force-deflection calibration . . . .
2.5.3 Deflection map . . . . . . . . . . .
2.5.4 Focal adhesion analysis . . . . . .
2.5.5 Super resolution imaging . . . . . .
Supplemental figures . . . . . . . . . . . .
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3 Actin orientation and cell shape
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1 Cell biology . . . . . . . . . . . . . . . . . . . . . .
3.2.2 Force measurement . . . . . . . . . . . . . . . . . .
3.2.3 Microscopy . . . . . . . . . . . . . . . . . . . . . .
3.2.4 Cellular curvature fitting . . . . . . . . . . . . . . .
3.2.5 Actin stress fiber orientation . . . . . . . . . . . . .
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1 Actin stress-fibers co-orient with local force exertion
3.3.2 Cortical stress fibers suggest a homogeneous contractility . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3 Contractile stress increases depending on the local
stress fiber orientation . . . . . . . . . . . . . . . .
3.4 Discussion and conclusion . . . . . . . . . . . . . . . . . .
3.5 Supplemental figures . . . . . . . . . . . . . . . . . . . . .
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4 P130Cas in mechanosensing
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1 Cell biology . . . . . . . . . . . . . . . . . . . .
4.2.2 PolyAcrylamide gels . . . . . . . . . . . . . . .
4.2.3 Substrate stiffness . . . . . . . . . . . . . . . .
4.2.4 Immunostaining . . . . . . . . . . . . . . . . .
4.2.5 FA analysis . . . . . . . . . . . . . . . . . . . .
4.2.6 Force measurement . . . . . . . . . . . . . . . .
4.2.7 Microscopy . . . . . . . . . . . . . . . . . . . .
4.2.8 Force measurement . . . . . . . . . . . . . . . .
4.2.9 Force dynamics fitting . . . . . . . . . . . . . .
4.2.10 Comparing stiffness of PA-gel and micropillars
4.2.11 Force and p130cas dynamics . . . . . . . . . . .
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4.3
4.4
4.5
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Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1 Stiffness-dependent FA formation depends on
p130Cas . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2 P130Cas localization depends on stiffness . . . . .
4.3.3 Force exertion dynamics depend on substrate stiffness and p130Cas localization . . . . . . . . . . . .
4.3.4 P130Cas localizes before and during force exertion
Discussion and conclusion . . . . . . . . . . . . . . . . . .
Supplemental figures . . . . . . . . . . . . . . . . . . . . .
5 Cell division forces
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1 Cell culture . . . . . . . . . . . . . . . . . . . .
5.2.2 Micropillars . . . . . . . . . . . . . . . . . . . .
5.2.3 Microscopy . . . . . . . . . . . . . . . . . . . .
5.2.4 Radial force interpretation . . . . . . . . . . . .
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.1 From pulling to pushing forces in prometaphase
5.3.2 Force plateau during anaphase and telophase .
5.3.3 Outward pushing is vital for succesful mitosis .
5.4 Discussion and conclusion . . . . . . . . . . . . . . . .
5.5 Supplemental figures . . . . . . . . . . . . . . . . . . .
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Summary
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Samenvatting
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Publications
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Curriculum Vitae
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Acknowledgements
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viii
CONTENTS

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