Nano-mechanics of biomimetic models of the actin based cytoskeleton [Elektronische Ressource] : from single molecules to complex composite structures / Alexander Roth
198 pages
English

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Nano-mechanics of biomimetic models of the actin based cytoskeleton [Elektronische Ressource] : from single molecules to complex composite structures / Alexander Roth

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198 pages
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Physik-Department der Technischen Universität München Lehrstuhl für Biophysik E22 - Univ.-Prof. Dr. M. Rief Nano-mechanics of biomimetic models of the actin based cytoskeleton From single molecules to complex composite structures Alexander Roth Vollständiger Abdruck der von der Fakultät für Physik der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Univ. Prof. Dr. Manfred Kleber Prüfer der Dissertation: 1. Univ. Prof. Dr. Matthias Rief (schriftliche Beurteilung) Univ. Prof. Dr. Erich Sackmann (mündliche Prüfung) 2. Univ. Prof. Dr. Michael Schleicher (Ludwigs-Maximilians-Universität München) Die Dissertation wurde am 8. 7. 2004 bei der Technischen Universität München eingereicht und durch die Fakultät für Physik am 2. 8. 2004 angenommen. ContentsAbstract............................... 81 Introduction 121.1 Motorproteins ...........................151.2 Dissociationofsinglespecificbonds................171.2.1 Thermalbonddissociation.181.2.2 Force induced bond dissociation . . . . . . . . . . . . . . 181.3 Introduction to semi-flexible polymers . . . . . . . . . . . . . . . 201.3.1 Oscillations of semi-flexible polymers . . . . . . . . . . . 221.3.2 Solutions of semi-flexible actin polymers . . . . . . . . . 231.4 Linearviscoelastictheory......................231.4.1 Linearviscoelasticmodels.

Informations

Publié par
Publié le 01 janvier 2004
Nombre de lectures 31
Langue English
Poids de l'ouvrage 8 Mo

Extrait


Physik-Department
der Technischen Universität München
Lehrstuhl für Biophysik E22 - Univ.-Prof. Dr. M. Rief




Nano-mechanics of biomimetic models
of the actin based cytoskeleton
From single molecules to complex composite structures

Alexander Roth


Vollständiger Abdruck der von der Fakultät für Physik der Technischen Universität
München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.




Vorsitzender: Univ. Prof. Dr. Manfred Kleber

Prüfer der Dissertation:

1. Univ. Prof. Dr. Matthias Rief (schriftliche Beurteilung)
Univ. Prof. Dr. Erich Sackmann (mündliche Prüfung)
2. Univ. Prof. Dr. Michael Schleicher
(Ludwigs-Maximilians-Universität München)




Die Dissertation wurde am 8. 7. 2004 bei der Technischen Universität München

eingereicht und durch die Fakultät für Physik am 2. 8. 2004 angenommen. Contents
Abstract ............................... 8
1 Introduction 12
1.1 Motorproteins ........................... 15
1.2 Dissociationofsinglespecificbonds ................ 17
1.2.1 Thermalbonddissociation . 18
1.2.2 Force induced bond dissociation . . . . . . . . . . . . . . 18
1.3 Introduction to semi-flexible polymers . . . . . . . . . . . . . . . 20
1.3.1 Oscillations of semi-flexible polymers . . . . . . . . . . . 22
1.3.2 Solutions of semi-flexible actin polymers . . . . . . . . . 23
1.4 Linearviscoelastictheory. ..................... 23
1.4.1 Linearviscoelasticmodels . ................ 25
2 Materials and Methods 28
2.1 BiochemicalMaterials . ...................... 28
2.1.1 Proteins . .......................... 28
2.1.2 Phospholipids . ....................... 37
2.1.3 Chemicals 38
2.2 SamplePreparation......................... 39
2.2.1 Surface coating with thin polymer layers . . . . . . . . . 39
2.2.2 Pillar array preparation . . . . . . . . . . . . . . . . . . 40
2.2.3 Giantphospholipidvesiclepreparation .......... 41
2.2.4 Magnetic bead functionalization . . . . . . . . . . . . . . 42
2.3 ExperimentalTechniques...................... 42
2.3.1 OpticalMicroscopy ..................... 42
2.3.2 Image analysis and processing . . . . . . . . . . . . . . . 48
2.3.3 Magnetic colloidal force transducer
(Magnetic Tweezers) . . . . . . . . . . . . . . . . . . . . 55
12 CONTENTS
3 Results and Discussion 63
3.1 MyosinVsinglemoleculeexperiments .............. 63
3.1.1 ParticletransportbymyosinV 63
3.1.2 Averagestepsize ...................... 65
3.1.3 Force spectroscopy of the actin binding potential of mov-
ingmyosinVmolecules. .................. 68
3.1.4 Myosin V movement under forward and backward forces 83
3.2 Actin cortex models on micro pillar arrays . . . . . . . . . . . . 91
3.2.1 Self assembly of a freely suspended quasi two dimen-
sionalactinnetwork .................... 92
3.2.2 Mechanical properties of the quasi two dimensional actin
network ........................... 98
3.2.3 Elastic properties of actin filaments . . . . . . . . . . . . 101
3.2.4 Elastic properties of actin - filamin bundles . . . . . . . . 107
3.3 Structure and mechanics of actin cortex vesicles . . . . . . . . . 112
3.3.1 Structureoftheactincortex . ...............113
3.3.2 Dataacquisition.......................119
3.3.3 Analysisofthecreepcompliance . ............120
3.3.4 Calculation of the strain relaxation function G(t) . . . . 126
3.3.5 Photochemical alternation of the actin cortex . . . . . . 129
3.3.6 Workhardening131
3.3.7 Analysisofthermalbeadfluctuations ...........132
3.3.8 Strainfieldmapping ....................134
3.4 Drop evaporation of entangled actin solutions . . . . . . . . . . 139
3.4.1 Reconstructionofthedropletshape ............139
3.4.2 Filament bending under hydrodynamic pressure . . . . . 143
3.5 Contraction forces in active actin-myosin networks . . . . . . . . 152
3.5.1 Activeactin-myosinnetworks . ..............152
3.5.2 Percolation and viscoelastic properties . . . . . . . . . . 154
3.5.3 Three dimensional reconstruction of vesicles . . . . . . . 160
3.5.4 Contractionforces .....................163
4 Conclusion and Outlook 170
A Appendix 177
A.1 Chemicals ..............................177
A.1.1 Ascorbicacid . .......................177CONTENTS 3
A.1.2 GlucoseOxidase . .....................177
A.1.3 Catalase . ..........................177
A.2 Buffercomposition . ........................178
A.2.1 Abuffer ...........................178
A.2.2 Bbuffer178
A.2.3 G*buffer178
A.2.4 Fbuffer179
A.3 Abbreviations .179
Bibliography 180List of Figures
1.1 Imagesoftheactincytoskeletoninsidecells ........... 14
1.2 Electronmicrographsofmotorproteins .............. 16
1.3 Hypothetic binding potential of a molecular bond . . . . . . . . 19
1.4 Geometry of a schematic semi-flexible polymer . . . . . . . . . . 21
1.5 Modelsforviscoelasticmaterials. ................. 26
2.1 Actin................................. 29
2.2 Theprocessofactinpolymerization ................ 30
2.3 StructureofmyosinV ....................... 32
2.4 Electron micrograph and schematics of smooth muscle . . . . . 34
2.5 formation of pillar array substrates from silicon . . . . . . . . . 40
2.6 SEM images of pillar array substrates made from silicon and
PDMS ................................ 41
2.7 Schematics of the principle of a fluorescence microscope. . . . . 45
2.8 Principle of the Reflection Interference Contrast Microscope . . 46
2.9 Threedimensionaltrackingalgorithm ............... 50
2.10 Calibration curve for the three dimensional particle tracking
algorithm .............................. 51
2.11 Resolution of the particle tracking algorithm as function of the
velocityoftheparticle . ...................... 52
2.12 Actinfilamentshapetracing .................... 53
2.13 Orderanalysisofanactinassembly ................ 55
2.14 Electron micrographs of super-paramagnetic colloidal particles . 56
2.15 Vertical magnetic tweezers . . . . . . . . . . . . . . . . . . . . . 58
2.16 Vertical m tw calibration . . . . . . . . . . . . . . . 59
2.17 Vertical magnetic tweezers magnetization . . . . . . . . . . . . . 60
2.18 Horizontalforcetransducer..................... 61
2.19 Horizontal force transducer magnetic field and field gradient . . 61
4LIST OF FIGURES 5
2.20 Horizontal force transducer magnetization and calibration curve 62
3.1 Movement of a myosin V coated bead along an actin filament . 65
3.2 Histograms of the image to image displacement of a moving bead. 66
3.3 Snapshots of a force induced bond rupture of a myosin V molecule
movingalonganactinfilament . ................. 70
3.4 Rupture process a of single myosin V molecule from an actin
filament . .............................. 71
3.5 Rupture forces of single myosin V molecules from actin filaments 73
3.6 Force - time relation of a moving motor molecule . . . . . . . . 75
3.7 Rupture forces of single myosin V molecules with a least square
fit .................................. 78
3.8 The probability N(F) of an intact bond and the bond rupture
N(F)
distribution − ......................... 79
dF
3.9 Stochasticdeviationsoftheruptureforces ............ 80
3.10 Combined probability density P (f ,k ) for a myosin V bondg 0 0
rupture as a function of the parameters k and f. . ...... 810 0
3.11 Movement of a magnetic bead transported by a single myosin
V molecule under increasing backward load . . . . . . . . . . . . 85
3.12 Displacement of myosin V along actin under backward load . . . 85
3.13 Schematics of the geometry when myosin V is examined under
a load parallel to its direction of movement. . . . . . . . . . . . 86
3.14 Displacement of myosin V along actin under forward load . . . . 88
3.15 Electron micrographs of moving myosin V molecules . . . . . . 89
3.16 Fluorescence micrograph of actin filaments attached to a silicon
pillar array substrate . . . . . . . . . . . . . . . . . . . . . . . . 93
3.17 micrograph of a cross-linked quasi two dimensional
actin network on a silicon pillar array substrate . . . . . . . . . 94
3.18 Actin network cross linked with filamin on a flat patterned sub-
strate. . ............................... 96
3.19 A 40 nm polystyrene bead being transported by myosin V on
an quasi two dimensional actin network. . . . . . . . . . . . . . 97
3.20 Probing the elastic properties of a freely suspended quasi two
dimensional actin network on a pillar array substrate. . . . . . 99
3.21 Thermally undulating filament on a photo resin pillar substrate 101
3.22 Fit of the first four Eigenmodes for the contour of an undulating
actinfilament ............................1036 LIST OF FIGURES
3.23 Bending stiffness of an actin filament determined from the first
four undulation Eigenmodes . . . . . . . . . . . . . . . . . . . . 104
3.24 Principle of the superposition mechanism to determine the bend-
ingrigidityofanactinfila

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