Motility and force generation based on the dynamics of actin gels [Elektronische Ressource] / vorgelegt von Stephan Schmidt

Schmidtst

Sujets

• Biologie

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Aus der Universität Bayreuth 




Motility and Force          
Generation Based on the    
Dynamics of Actin Gels 
 
Dissertation  


zur Erlangung des akademischen Grades  
Doktor der Naturwissenschaften  
– Dr. rer. nat. –  
im Fach Chemie der Fakultät Biologie, Chemie, Geowissenschaften     
der Universität Bayreuth
 
 
 
vorgelegt von 
Stephan Schmidt 
geboren in Potsdam 





Bayreuth, im Februar 2009 Erklärung
Die vorliegende Arbeit wurde in der Zeit vom Dezember 2005 bis Mai 2007
im Max-Planck-Institut für Kolloid und Grenzflächenforschung in Golm,
danach bis zum Februar 2009 in der Universiät Bayreuth angefertigt. Die
Betreuung an beiden Instituten erfolgte durch Prof. Dr. Andreas Fery.




Vollständiger Abdruck der von der Fakultät für Biologie / Chemie / Geo-
wissenschaften der Universität Bayreuth genehmigten Dissertation zur
Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.).





Dissertation eingereicht am: 27.02.2009
Zulassung durch die Prüfungskommision: 11.03.2009
Wissenschaftliches Kolloquium: 18.05.2009
Amtierender Dekan: Prof. Dr. Axel H.E. Müller








Prüfungsausschuss:

Prof. Dr. Andreas Fery (Erstgutachter)
Prof. Dr. Andreas Bausch (TUM, Zweitgutachter)
Prof. Dr. Walter Zimmermann
Prof. Dr. Franz Schmid (Vorsitz)

Table of Contents

1 Table of Contents 
2 Introduction .............................................................................................. 5
3 Status of the Field ..................................................................................... 8
3.1 Structure and Properties of Actin ..................................................... 10
3.2 Actin Polymerization ....................................................................... 11
3.2.1 Actin Treadmilling ..................................................................... 12
3.2.2 The Effect of Regulatory Proteins ............................................. 13
3.2.3 Formin Based Actin Polymerization ......................................... 16
3.2.4 Reconstruction In Vitro ............................................................. 17
3.3 Actin Force Assays .......................................................................... 19
3.4 Force Generation of Actin Filaments: Microscopic View ............... 22
3.4.1 Elastic Brownian Ratchet .......................................................... 22
3.4.2 Tethered Ratchet Model ............................................................ 23
3.5 Force Generation of Actin Gels: Mesoscopic Elastic Model .......... 28
3.5.1 Role of Stresses in Listeria Motility .......................................... 30
3.5.2 Effect of Stresses on Gel Growth and Gel Symmetry Breaking
33
3.6 Microscopy ....................................................................................... 37
3.6.1 Light Microscopy Basics ........................................................... 37
3.6.2 Phase Contrast Microscopy ....................................................... 38
3.6.3 Fluorescence Microscopy .......................................................... 40
3.7 Atomic Force Microscopy ............................................................... 43
3.7.1 AFM Working Principle ............................................................ 44
3.7.2 AFM Force Measurements ........................................................ 46
3.7.3 The Colloidal Probe ................................................................... 50
4 Preparation Procedures ........................................................................... 52
4.1 Preparing the Actin In Vitro Medium .............................................. 52
4.2 Preparation of the Bead Trajectory Assay ....................................... 55
4.3 Force Assay Preparation Procedures ............................................... 57 2. Introduction

5 Results and Discussion ........................................................................... 62
5.1 Trajectories of Actin Propelled Beads ............................................. 62
5.1.1 Curvature Distribution of the Bead Trajectories ....................... 64
5.1.2 Trajectory Analysis in Confining Channels .............................. 76
5.2 AFM Force Measurements............................................................... 83
5.2.1 Development of the AFM-Experiment ...................................... 84
5.2.2 AFM Force Measurements for Varying Gel Size and Curvature ..
.................................................................................................... 91
5.2.3 Effect of the Medium Composition ......................................... 117
5.2.4 Formin Based Actin Polymerization and Generation of Force ..... .................................................................................................. 126
5.3 Measuring Forces In-Vivo: Capsule Deformation in Cells ........... 129
6 Conclusion ............................................................................................ 132
7 Zusammenfassung ................................................................................ 136
8 Appendix .............................................................................................. 140
8.1 Parameters and Abbreviations ....................................................... 140
8.2 Force measurement on actin comets at the colloidal probe ........... 142
8.3 Working up g-Actin ....................................................................... 146
8.4 Grey Value Normalization in Image Stacks Using an Internal
Reference ........................................................................................ 146
8.5 Automated Linear Fits for AFM Force-Distance Curves .............. 147
9 References ............................................................................................ 152


4 2. Introduction
2 Introduction 
It was in 1675 when van Leeuwenhoek discovered motile microscopic crea-
tures in rainwater. He observed that these cellular microorganisms would “put
forth little horns, extended and contracted, and had pleasing and nimble mo-
tions” [1]. Even after centuries of scientific development the significance of this
observation remains. The key point of this discovery was that cells drive them-
selves actively by extension and contraction of their body. This mode of active
motion is called cell crawling and it is an important part of fundamental biolog-
ical and medical phenomena, such as: morphogenesis, wound healing, immune
response and cancer spread. The basic concept of cell crawling has been estab-
lished already almost 40 years ago, but it is the molecular details and the me-
chanism of the driving force that are subject of intense research until today.
Crawling cells generate their driving force by expanding the cytoskeleton
against the leading edge of the cell. The cytoskeleton is almost solely comprised
of a gel-like actin filament network. As the cell moves, actin filaments elongate
by polymerization so that they collectively grow against the membrane. From a
broader perspective the process appears as supramolecular self-assembly where
the structure of the network and the polarity of the filaments establish an “auto-
pilot” that directs the involved biomolecular reactions into forward motion of
the cell. Even though the process of actin network formation seems to be
straight forward, there are many unclear aspects, in particular concerning the
generation of force. For example, the response to external forces, the regulation
of the moving direction, and even the nature of the propulsive force are not un-
derstood. In this work we study these phenomena and focus on the following
questions.
• What is the magnitude of forces generated by the actin gel and how does the
gel morphology affect the generation of force?
• What are the mechanical properties of the actin network and how are these
properties regulated?
• How is the direction and distribution of the force in the gel regulated? What
are the implications for motility?
• How can we quantitatively measure forces in cells?
5
2. Introduction

In order to analyze these problems we mainly address these problems using
an in vitro approach: Here gel-motility and force measurements are conducted
in stripped-down model systems. These are comprised of only purified proteins
that reconstitute actin polymerization in solution. Second, measurements in vi-
vo: Here force measurements are performed directly in living cells.
It is tempting to speculate about how the mesoscopic actin based motion is
generated by just molecular sel