Delft University of Technology On the role of microtubules in cell polarity A reconstituted minimal system Vendel, K.J.A. DOI 10.4233/uuid:34f174d5-f8ad-4bf2-86f7-47660a84fe64 Publication date 2020 Document Version Final published version Citation (APA) Vendel, K. J. A. (2020). On the role of microtubules in cell polarity: A reconstituted minimal system. https://doi.org/10.4233/uuid:34f174d5-f8ad-4bf2-86f7-47660a84fe64 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. ON THE ROLE OF MICROTUBULES IN CELL POLARITY A RECONSTITUTED MINIMAL SYSTEM ON THE ROLE OF MICROTUBULES IN CELL POLARITY A RECONSTITUTED MINIMAL SYSTEM Proefschrift ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus Prof. dr. ir. T. H. J. J. van der Hagen, voorzitter van het College voor Promoties, in het openbaar te verdedigen op 21 september 2020 om 12:30 uur door Kim Jikke Anna VENDEL Master in physics, Universiteit Leiden, Nederland geboren te Amsterdam, Nederland. Dit proefschrift is goedgekeurd door de promotor: prof. dr. M. Dogterom Samenstelling promotiecommissie: Rector Magnificus, voorzitter Prof. dr. M. Dogterom, Technische Universiteit Delft, promotor Onafhankelijke leden: Prof. dr. G.H. Koenderink, Technische Universiteit Delft Prof. dr. T. Mitchison, Harvard Medical School Prof. dr. S. Grill, MPI-CBG and TU Dresden Prof. dr. B. Mulder, AMOLF and Universiteit Utrecht Dr. E. Spruijt, Radboud Universiteit Prof. dr. ir. S. Tans, Technische Universiteit Delft, reservelid Overige leden: Dr. ir. L. Laan, Technische Universiteit Delft Keywords: cell polarity, emulsion droplets, microtubules, reconstitution Printed by: Gildeprint Cover: Mirte Vendel Copyright © 2020 by K.J.A. Vendel Casimir PhD series 2020-20 ISBN 978-90-8593-447-9 An electronic version of this dissertation is available at http://repository.tudelft.nl/. Voor mijn ouders Magalhaes voer de wereld rond, langs de Bosporus en de Hellespont, maar hij zag veel minder dan wij. Hij zag veel minder dan wij. Ivo de Wijs - Vroege Vogels Vliegen CONTENTS Summary ix Samenvatting xi 1 Introduction1 1.1 The cytoskeleton.........................2 1.2 Microtubule-based cell polarity.................. 10 1.3 Towards a minimal system for microtubule-based cell polarity.. 17 1.3.1 The artificiality of minimal systems............ 18 1.3.2 Outline of this thesis.................... 19 2 Methods and optimization steps 21 2.1 Water-in-oil emulsion droplets.................. 23 2.2 Dynamic MT asters with protein comets............. 27 2.2.1 Centrosomes........................ 28 2.2.2 Artificial MTOCs: Aurora kinase A beads.......... 28 2.2.3 Visualizing MTs...................... 30 2.2.4 Proteins.......................... 33 2.3 Functionalized lipids....................... 34 2.3.1 Nonionic surfactants and functionalized lipids...... 37 2.4 Imaging emulsion droplets.................... 38 2.5 Flow channels........................... 40 2.5.1 Preparation........................ 40 2.6 Kymograph analysis........................ 41 3 The influence of MAP7 on MT dynamics and structure 43 3.1 Introduction........................... 44 3.2 Results.............................. 45 3.2.1 Possible hypotheses.................... 51 3.3 Discussion............................ 53 3.4 Materials and methods...................... 56 3.4.1 Reaction mix........................ 56 3.4.2 Image Acquisition and data analysis............ 56 4 EB3-mediated interaction between MTs and the droplet cortex 57 4.1 Introduction........................... 58 4.2 Influence of confinement on MT dynamics............ 60 4.3 MT capture at the droplet cortex................. 63 4.3.1 Characterization of MACF peptides............ 63 4.3.2 MT tip tethered to the cortex through MACF18...... 66 4.3.3 MT lattice tethered to the cortex through MACF43..... 70 vii viii CONTENTS 4.4 Discussion............................ 74 4.5 Materials and methods...................... 78 4.5.1 Protein purification.................... 78 4.5.2 Emulsion droplet preparation and imaging........ 79 4.5.3 Data analysis........................ 80 5 Optogenetic tools inside emulsion droplets 87 5.1 Introduction........................... 88 5.2 Background............................ 89 5.3 Results.............................. 92 5.3.1 iLID and SspB binding pair................ 92 5.3.2 Photo-dissociable ¼-EB1.................. 94 5.4 Discussion............................ 104 5.5 Methods............................. 109 5.5.1 Protein purification.................... 109 5.5.2 Emulsion droplets..................... 109 5.6 Data analysis........................... 109 5.6.1 Reaction scheme of PD-EB1................ 109 6 Theoretical models on cell polarity 113 6.1 Introduction........................... 114 6.2 Theoretical models on microtubule-based cell polarity...... 114 6.3 Implications for our in vitro minimal system........... 118 6.3.1 Fission yeast model.................... 118 6.3.2 Models for a spherical minimal system.......... 119 6.4 Conclusion............................ 121 7 Discussion and outlook 123 Bibliography 131 Acknowledgements 151 Curriculum Vitæ 155 List of Publications 157 SUMMARY VERY living organism consists of cells. Even for the simplest single-cell organism, E this cell is extremely complex. Thousands of components (such as DNA, cytoskele- tal filaments, proteins, lipids, nutrients and energy) are organized both spatially and temporally to ensure proper functioning of vital cellular processes. One of those pro- cesses is pattern formation, or cell polarity. Cell polarity is defined as the morpho- logical and functional differentiation of cellular compartments in a directional man- ner. This directionality is crucial for processes like cell division, cell migration and cell growth, which require an asymmetric action of the cell. When polarity is inhibited by silencing of cell polarity proteins, cells become deformed and have trouble to func- tion, if viable at all. It is well-known that cell polarity is the result of reaction-diffusion and cytoskeleton-based mechanisms, but the exact mechanisms remain unknown. In this thesis, we focus on the latter, and more specifically on one type of cytoskeletal filaments: microtubules (MTs). Our goal is to study the role of MTs in cell polarity establishment. Because cells form the basis of life, a question that drives many researchers is “how does a cell work?”. Even though this question is short and simple, the answer is not. A wealth of in vivo, in vitro and theoretical studies have been performed and we are just starting to understand the essential cellular mechanisms like cell polarity. One relatively new research approach are in vitro minimal systems. A minimal system is a bottom-up approach where a certain cellular function is reconstituted with the mini- mal number of components. It provides a way to check hypotheses in an experimental setting which is less complex than a live cell yet more similar to a cell than conven- tional in vitro reconstitutions. Our aim was to employ the minimal system approach to investigate the minimal re- quirements for MT-based cell polarity. We defined polarity in a minimal system as an asymmetric accumulation of proteins at the periphery of a 3D confinement induced by MTs. We focused on the general mechanisms of cell polarity establishment, rather than to mimic the specific polarity system from a certain organism. To that end, we first investigated what is known about MT-based cell polarity from previous in vivo, in vitro and theoretical studies. From this, we concluded that the main ingredients of our minimal system had to be: 1. radially organized MTs in a spherical, cell-sized confinement with a cortex: water- in-oil emulsion droplets, 2. concentration of polarity proteins on the cortex by MTs, 3. positive feedback between cortical polarity proteins and the MT stability. ix x SUMMARY The next step towards reconstitution of such a system was to optimize existing meth- ods for MT networks inside emulsion droplets. After optimization of the MT visibil- ity and the interaction of polarity proteins with the droplet cortex, we reconstituted MT asters inside water-in-oil emulsion droplets. The MT tips could interact with the droplet cortex through MT-associated proteins (MAPs). We studied the effect of this MT capture on MT stability and MT organization in the droplets, and investigated whether this was enough for an asymmetric accumulation of proteins on the cortex. Our experiments showed that merely the interaction between MT tips and droplet cor- tex through MAPs sufficed to locally accumulate the proteins and to stabilize the MT aster position. However, this accumulation was not sustainable and disappeared as soon as a MT detached from the cortex. Several potential improvements were formu- lated with the help of theoretical models and knowledge from