How kinesin-1 deals with roadblocks [Elektronische Ressource] : biophysical description and nanotechnological application / by Till Korten

Description

How Kinesin-1 Deals WithRoadblocks: BiophysicalDescription andNanotechnological Applicationdissertationsubmitted for the degree ofDoctor rerum naturalium(Dr. rer. nat.)at theFakult at fur Mathematik und NaturwissenschaftenTechnische Universit at DresdenbyTill Kortenthborn on April 6 , 1978 in Berlin, GermanythSeptember 30 , 20091. Reviewer: Prof. Dr. Anthony Hyman2. Reviewer: Prof. Dr. Henry HessthDay of the defense: December 10 2009Signature from head of PhD committee:AbstractProteins have been optimized by evolution for billions of years to work on ananometer scale. Therefore, they are extremely promising for nanotechnologicalapplications. Cytoskeletal laments propelled by surface-attached motor proteinshave been recently established as versatile transport platforms for nano-sized cargoin molecular sorting and nano-assembly devices. However, in this gliding motilitysetup, cargo and motors share the lament lattice as a common substrate for theiractivity. Therefore, it is important to understand the in uence of cargo-loadingon transport properties.By performing single molecule stepping assays on biotinylated microtubules, itwas shown that kinesin-1 motors rst stop and then detach when they encountera streptavidin obstacle on their path along the microtubule.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2009
• Nombre de lectures: 152
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

How Kinesin1 Deals With Roadblocks: Biophysical Description and Nanotechnological Application

dissertation submitted for the degree of Doctor rerum naturalium (Dr. rer. nat.) at the Fakultät für Mathematik und Naturwissenschaften Technische Universität Dresden by Till Korten th born on April 6 , 1978 in Berlin, Germany

th September 30 , 2009

1. Reviewer: Prof. Dr. Anthony Hyman

2. Reviewer: Prof. Dr. Henry Hess

th Day of the defense: December 10 2009

Signature from head of PhD committee:

Abstract

Proteins have been optimized by evolution for billions of years to work on a nanometer scale. Therefore, they are extremely promising for nanotechnological applications. Cytoskeletal filaments propelled by surfaceattached motor proteins have been recently established as versatile transport platforms for nanosized cargo in molecular sorting and nanoassembly devices. However, in this gliding motility setup, cargo and motors share the filament lattice as a common substrate for their activity. Therefore, it is important to understand the influence of cargoloading on transport properties. By performing single molecule stepping assays on biotinylated microtubules, it was shown that kinesin1 motors first stop and then detach when they encounter a streptavidin obstacle on their path along the microtubule. Consequently, the deceleration of streptavidin coated microtubules in gliding assays could be at tributed to an obstruction of kinesin1 ’s path on the microtubule rather than to ”frictional” streptavidinsurface interactions. The insights gained by studying kinesin’s behavior at obstacles were then used to demonstrate a novel sensing application: Using a mixture of two distinct microtubule populations that each bind a different kind of protein, the presence of these proteins was detectedviaspeed changes in the respective microtubule populations. In future applications, this detection scheme could be combined with other recent advancements in the field, creating highly integrated labonachip devices that use microtubule based transport to detect, sort and concentrate analytes. It has been envisioned that the kinesin1 microtubule system could be used for even more complex appliances like nanoassembly lines. However, currently available control mechanisms for kinesin1 based transport are not precise enough. Therefore, improved temporal control mechanisms for kinesin1 were investigated: Using a polymer that changes its size in solution with temperature, starting and stopping of gliding microtubules was demonstrated. In combination with local heating by light, this effect could be used to control the gliding of single micro tubules. Finally, a strategy to create photoswitchable kinesin1 was developed and tested for feasibility using molecular modeling.

To my wife without whose love and support I would not have managed.

Acknowledgements

I am grateful for the help of many people and it is with great pleasure, that I take this opportunity to thank them! First and foremost Dr. Stefan Diez: With his extremely positive nature he always encouraged and motivated me to try new ideas and to not give up when things looked bad. I was great fun to be part of his extremely interdisciplinary group, learning to look at science from the physicists perspective.

Prof. Dr. Christoph Thiele and Prof. Dr. Francis Stewart for being on my thesis advisory committee always ready for lively discussions and giving valuable input. Oliver Wüseke for really getting absorbed with the roadblock ex periments and always having a joke up his sleeve to cheer everyone up. Prof. Dr. Joachim Knölker for his kind gift of a library of potential kinesin1 inhibitors and Anja Henning for her help with screening and analyzing that library.

Felix Ruhnow and David Zwicker for their excellent tracking software. Dr. Stefan Diez, Dr. Bert Nitzsche and René Schneider for their comments on my writing. They managed to straighten my sentences, when I had strung them up and lost the thread.

Corina Bräuer and Doreen Naumburger for all their help and for being the fairy godmothers keeping the lab together.

Dr. Claire Friel and Dr Chris Gell for their very kind and patient help as native speaking counselors for the nuances of the English language.

The fantastic working conditions that I experienced are to a great extend due to the members of the research group of Dr. Stefan Diez and Prof. Jonathon Howard. As far as I have not already done so above I would like to thank all these people, in particular, Bert Nitzsche, Rene Schneider, Felix Ruhnow, Gero Fink, Regine Hartmann, Volker Bormuth, Leonid Ionov and Cordula Reuther for help and for many inspiring and moreover pleasant discussions. My parents for always supporting me and being there when I needed them and my parentsinlaw and especially my wife for pampering me during the time of writing. Finally, I would like to acknowledge the thousands of individuals who have coded for the LaTeX project for free. It is due to their efforts that we can generate professionally typeset PDFs now.

Contents

List of Figures

List of Tables

Glossary

1 Introduction 1.1 Biomolecular transportin vivo. . . . . . . . . . . . . . . . . . . . 1.2 Biomolecular recognition . . . . . . . . . . . . . . . . . . . . . . . 1.3 Biomolecules for nanotechnology . . . . . . . . . . . . . . . . . . .

2In vitroreconstitution of transport on crowded microtubules 2.1 Single motor proteins at obstacles . . . . . . . . . . . . . . . . . . 2.2 Effects of obstacles on multimotor transport . . . . . . . . . . . . 2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 A novel method for molecular detection 3.1 Detection of streptavidin and antirhodamine antibodies . . . . . 3.2 A generalized detection method . . . . . . . . . . . . . . . . . . . 3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 Towards temporal motility control 4.1 Switchable roadblocks . . . . . . . . . . . . . . . . 4.2 Towards photoswitchable kinesin1 . . . . . . . . .

i

. . . . . . . . . . . . . . . .

iii

v

vii

1 3 10 15

23 24 35 39

49 49 52 56

59 59 64

CONTENTS

5

6

Summary and outlook 5.1 Kinesin1’s behavior at roadblocks . . . . . . . . . . . . . . . . . . 5.2 Refining molecular detection . . . . . . . . . . . . . . . . . . . . . 5.3 Improving spatiotemporal control . . . . . . . . . . . . . . . . . . 5.4 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . .

Materials and methods 6.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3In vitro. . . . . . . . . . . . . . . . . . . . . . . .motility assays 6.4 Image acquisition and data analysis . . . . . . . . . . . . . . . . .

References

ii

73 73 75 77 79

81 81 84 87 93

97

List

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10

of

Figures

Fluorescence image of filaments of the cytoskeleton . . . . . . . . 4 Structure of a tubulin heterodimer . . . . . . . . . . . . . . . . . 5 Overview of microtubule structure and dynamics . . . . . . . . . . 7 Structure of a kinesin1 heavy chain dimer . . . . . . . . . . . . . 8 Current model of the kinesin1 stepping mechanism . . . . . . . . 9 Composition of an immunoglobulin G and an antigen binding fragment 12 Creation of aptamers . . . . . . . . . . . . . . . . . . . . . . . . . 14 Microtubules transporting cargo on a kinesin1 coated surface . . 15 The biotinstreptavidin bond . . . . . . . . . . . . . . . . . . . . . 16 Amino and Sulfhydrylspecific chemistry . . . . . . . . . . . . . . 18 Examples of control over microtubules . . . . . . . . . . . . . . . 20

Single molecule roadblock assay . . . . . . . . . . . . . . . . . . . Kinesin1 on streptavidin coated microtubules . . . . . . . . . . . Nanometer tracking . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of obstacles on dwell time and run length of single kinesin1 molecules in BRB20 . . . . . . . . . . . . . . . . . . . . The effect of obstacles on dwell time and run length of single kinesin1 molecules in BRB80 . . . . . . . . . . . . . . . . . . . . Direct measurement of stopping times in BRB20 . . . . . . . . . . Microtubule gliding assay . . . . . . . . . . . . . . . . . . . . . . Streptavidin coating slows down gliding microtubules . . . . . . . Microtubule speed depends on streptavidin coating density . . . . Dependency of k s on the dwell time and run length in the presence of roadblocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii

24 25 27

29

32 34 36 37 38

42

LIST OF FIGURES

3.1 3.2 3.3 3.4

3.5 3.6

4.1 4.2 4.3 4.4 4.5 4.6 4.7

6.1 6.2 6.3 6.4

Microtubule based detection assay . . . . . . . . . . . . . . . . . . 50 Simultaneous detection of streptavidin and antirhodamine antibodies 51 Testing the versatility of the proposed detection . . . . . . . . . . 52 Detecting antistreptavidin antibodies using streptavidincoated microtubules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Covalently linking aptamers to microtubules . . . . . . . . . . . . 54 Aptamers covalently linked to microtubules . . . . . . . . . . . . . 55

Thermoresponsive polymers on microtubules . . . . . . . . . . . . Motility switching of PNIPAMcoated microtubules . . . . . . . . Schematic drawing of a photoswitchable kinesin1 . . . . . . . . . Alexa Fluor 647 ATP . . . . . . . . . . . . . . . . . . . . . . . . . Homology modelling . . . . . . . . . . . . . . . . . . . . . . . . . Tether design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular dynamics calculations . . . . . . . . . . . . . . . . . . .

Photographs of the temperature stage . . . . . . . . . . . . . . . . Temperature stage allowing fast temperature control for imaging . Peltier calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . Measuring bleaching times . . . . . . . . . . . . . . . . . . . . . .

iv

60 61 65 66 67 70 71

89 90 91 95

List

1.1

2.1 2.2 2.3 2.4

4.1

6.1

of

Tables

Covalent modifications of proteins . . . . . . . . . . . . . . . . . .

17

Measured and calculated kinetic parameters of kinesin1 at roadblocks 31 Kinetic parameters of kinesin1 in BRB20 at roadblocks . . . . . . 31 Kinetic parameters of kinesin1 in BRB80 at roadblocks . . . . . . 33 Comparison of kinetic parameters of kinesin1 at roadblocks in different salt concentrations . . . . . . . . . . . . . . . . . . . . . 39

Sequence alignment . . . . . . . . . . . . . . . . . . . . . . . . . .

List of parts for an automated polarity switch . . . . . . . . . . .

v

68

89