In vitro studies of actin-microtubule
coordination
Magdalena Preciado López
This thesis was reviewed by:
Prof.dr. Marcel E. Janson
Wageningen UR
Dr. Lukas C. Kapitein
Universiteit Utrecht
Prof.dr. Erwin J.G. Peterman Vrije Universiteit Amsterdam
Dr. Cornelis Storm
Technische Universiteit Eindhoven
Dr. Clare M. Waterman
National Institutes of Health
The research described in this thesis was performed at the FOM Institute AMOLF,
Science Park 104, 1098 XG, Amsterdam, the Netherlands. This work is part of the
resarch program of the Foundation for Fundamental Research on Matter (FOM), which
is financially supported by the Netherlands Organization for Scientific Research (NWO).
© Magdalena Preciado López 2015
ISBN/EAN: 978-90-77209-89-9
A digital version of this thesis is available at www.amolf.nl/publications/theses and
www.ubvu.vu.nl/dissertations. Printed copies can be requested to the library of the
FOM Institute AMOLF (library@amolf.nl).
VRIJE UNIVERSITEIT
In vitro studies of actin-microtubule coordination
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. F.A. van der Duyn Schouten,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Exacte Wetenschappen
op maandag 9 maart 2015 om 11.45 uur
in het auditorium van de universiteit,
De Boelelaan 1105
door
Magdalena Preciado López
geboren te Mexico City, Mexico
promotor:
prof.dr. G.H. Koenderink
copromotor:
prof.dr. M. Dogterom
“The true biologist deals with life, with teeming boisterous life, and learns something from
it, learns that the first rule of life is living.”
– John Steinbeck, The Log from the Sea of Cortez
Abstract
Eukaryotic cellular life critically relies on cell division, growth and migration, all highly
dynamic processes that are organized and powered by the microtubule and actin
cytoskeletons. Although it is now clear that these two cytoskeletal systems must be
coordinated, it remains unclear how the activity of actin-microtubule cross-linkers
enables their functional co-organization in different cellular contexts. In particular,
it is unknown how cross-linker mediated cytoskeletal coordination is influenced by the
diversity of filamentous actin (F-actin) architectures found in cells (i.e. free, cross-linked
and bundled), whose distinct mechanical properties are likely to impact the outcome
of their encounters with growing microtubules.
Here, with the use of a minimal model system reconstituted from purified proteins,
we elucidate how linking growing microtubule ends to F-actin structures can help
direct cytoskeletal organization. To establish actin-microtubule interactions in vitro, we
engineered a model actin-binding, microtubule plus-end tracking protein that we call
TipAct. This simple cross-linking system recapitulates the in vivo ability of stiff actin
bundles to capture and guide microtubule growth, which is highly dependent on their
encounter angle and the concentration of cross-linking protein both at the microtubule
tip and lattice. In a different context, the same cross-linking system conversely enables
growing microtubules to globally dictate F-actin organization, as they can pull, stretch
and bundle single actin filaments. To explain these effects, we developed a model of
biased diffusion at microtubule tips, which recapitulates the dependency of actin-filament
transport on both EB and tubulin concentration, and which also predicts that growing
microtubules can potentially generate picoNewton forces through this mechanism.
We conclude that, independently of biochemical regulation, a variety of cytoskeletal
organizations can arise from the interplay between physical cross-links and the mechanical properties of F-actin and microtubule structures. And finally, that cross-linkers can
establish a mechanical feedback between actin and microtubule organization which is
likely to be relevant in diverse biological contexts.
Contents
Abstract
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Contents
ix
1 Introduction: The eukaryotic cytoskeleton and actin-microtubule
ordination
1.1 The eukaryotic cytoskeleton . . . . . . . . . . . . . . . . . . . . . . .
1.2 Cytoskeletal interactions . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Multiple roles for the cytoskeletal coordination toolbox . . . . . . . .
1.4 Motivation and thesis outline . . . . . . . . . . . . . . . . . . . . . .
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2 General experimental methods
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Flow cell preparation and surface functionalization . . . . . . . . . . .
2.3 Buffer conditions to work with actin filaments and dynamic microtubules
2.4 Microtubule polymerization and tip tracking assays . . . . . . . . . . .
2.5 Proteins used in this thesis . . . . . . . . . . . . . . . . . . . . . . . . .
2.6 Buffers and stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7 Total internal reflection fluorescence (TIRF) microscopy . . . . . . . .
2.8 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3 TipAct – An engineered actin-binding microtubule +TIP
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 TipAct localization in mammalian cultured cells . . . . . . .
3.3 In vitro characterization of TipAct . . . . . . . . . . . . . .
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 Materials and methods . . . . . . . . . . . . . . . . . . . . .
3.6 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . .
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4 Guidance of microtubule growth and organization by F-actin
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 TipAct and EB3 couple microtubule growth to F-actin bundles . . . . .
4.3 EB3 and TipAct have reduced off-rates at actin-microtubule overlaps .
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4.4
4.5
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4.8
Actin bundles capture and redirect growing microtubules . . . . . . . .
Ordered arrays of F-actin bundles can globally dictate microtubule organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 F-actin organization by dynamic microtubules
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Growing microtubules deform and reposition F-actin bundles . .
5.3 Growing microtubules exert forces on single actin filaments . . .
5.4 Growing microtubules organize F-actin networks . . . . . . . . .
5.5 Closing the loop: growing microtubules induce F-actin bundling
5.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7 Materials and methods . . . . . . . . . . . . . . . . . . . . . . .
5.8 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6 Transport and force generation by microtubule +TIPs
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Model of biased actin filament diffusion at microtubule tips . . . . . . .
6.3 Gillespie-based simulations of actin filament transport by growing microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4 Simulation results: effects of variable actin filament length, EB and
tubulin concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5 Comparison between simulation and experimental data . . . . . . . . .
6.6 Further predictions of the model . . . . . . . . . . . . . . . . . . . . . .
6.7 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.8 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.9 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7 Conclusions and outlook
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Bibliography
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Samenvatting
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List of publications
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Acknowledgements
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