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«By KIMBERLY A. INTERLIGGI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR ...»

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CHARACTERIZATION OF ACTIN-BASED MOTILITY ON MODIFIED SURFACES FOR

IN VITRO APPLICATIONS IN NANODEVICES

By

KIMBERLY A. INTERLIGGI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007 1 © 2007 Kimberly A. Interliggi 2 To my encouraging parents, Denni and Vicki Interliggi, my siblings, Jen and Tom, and my boyfriend, Matt Crim, for their continued support.

3

ACKNOWLEDGMENTS

I thank my advisor, Dr. Richard B. Dickinson, for his support over the past four years. His guidance and encouragement have helped me succeed in many aspects of my graduate experience. I appreciate Dr. Yiider Tseng for always expressing his interest in my research and career and encouraging me along the way. I thank Dr. Spyros Svoronos for his dedication as a committee member. I acknowledge Dr. Anuj Chauhan for his guidance and helpful advice from the very beginning of my graduate career. I also thank Dr. Jennifer Curtis for her dedication to the graduate students in this department. Finally, I acknowledge the faculty and staff in the Department of Chemical Engineering for continuously supporting my efforts in many aspects of my graduate school career.

I am grateful to have had the opportunity to work with Dr. Daniel Purich in the Department of Biochemistry. Dr. Purich was involved on a daily basis with my lab work and was constantly teaching me new skills and sharing new ideas with me. I also thank Dr. William Zeile, who taught me many experimental and research techniques, and was always willing to listen to my research problems and help me work through them. Dr. Joseph Phillips and Dr. Fangliang Zhang are greatly appreciated for their companionship and knowledge in the lab. I also thank Dr.

Suzanne Ciftan-Hens and Dr. Gary McGuire at International Technology Center in Raleigh, NC for their collaboration on this project.

I thank Dr. Adam Feinberg, who was instrumental in helping me start my project. I thank the members of my group, who were consistently helping me to understand and work through daily problems and always provided good company: Dr. Luzelena Caro, Dr. Colin Sturm, Gaurav Misra, Dr. Jeff Sharp, Dr. Huilian Ma, and Adam Wulkan. I also thank Andre Baran for his assistance and constant support.

4

TABLE OF CONTENTS

page ACKNOWLEDGMENTS

LIST OF FIGURES

LIST OF ABBREVIATIONS

ABSTRACT

CHAPTER 1 INTRODUCTION

1.1 Actin

1.1.1 Filament Growth

1.1.2 Actomyosin

1.1.3 Actin-Associated Proteins

1.2 Experimental Methods for Actin Polymerization on Modified Substrata

1.2.1 Total Internal Reflection Fluorescence Microscopy

1.2.2 Microcontact Printing

1.3 Actin-Based Motility

1.3.1 Listeria and Particle Motility

1.3.2 Mechanisms

1.4 Bionanodevices

1.4.1 Sliding Filament Assay

1.4.2 Immobilized Filament Assay

1.4.3 Material Transport

2 GUIDANCE OF ACTIN FILAMENT ELONGATION ON FILAMENT-BINDING TRACKS

2.1 Introduction

2.2 Materials and Methods

2.2.1 Protein Preparation

2.2.2 Microcontact Printing

2.2.3 Actin Polymerization

2.2.4 TIRF Microscopy and Data Analysis

2.3 Results

2.3.1 Filament Binding to Stamped Surfaces

2.3.2 Alignment of Filaments

2.3.3 NEM-Myosin Concentration

2.3.4 Control of Actin Polymerization

2.4 Discussion

2.4.1 Mechanism for Filament Alignment

2.4.2 Potential and Applications

53 SIMULATING ACTIN FILAMENT ELONGATION ON MODIFIED SURFACES.........63

3.1 Introduction

3.2 Methodology

3.3 Results

3.3.1 Description of Simulated Filament Elongation

3.3.2 Probability of Filament Rebinding

3.3.3 Alignment of Filaments

3.4 Discussion

4 ACTIN-BASED MOTILITY OF LISTERIA AND PARTICLES ON MODIFIED SURFACES

4.1 Introduction

4.2 Materials and Methods

4.2.1 Listeria monocytogenes Growth and Protein Purification

4.2.2 Bead Preparation

4.2.3 Motility Assay

4.2.4 Fabrication of Channel Devices

4.2.5 Microscopy and Analysis

4.3 Results

4.3.1 Confining Particle Propulsion to the Surface

4.3.2 Effectiveness of NEM-Myosin Surfaces

4.3.3 Particle Velocity and Tail Characterization

4.3.4 Guiding Particle Propulsion

4.4 Discussion

4.4.1 Mechanics of Actin Rocket Tails on Surfaces

4.4.2 Biochemical Considerations

4.4.3 Considerations for Bionanotechnology

5 SINGLE FILAMENT ACTIN-BASED MOTILITY OF PARTICLES

5.1 Introduction

5.2 Materials and Methods

5.2.1 Protein Preparations





5.2.2 Bead Functionalization

5.2.3 Motility Assays

5.2.3.1 Attached beads

5.2.3.2 NEM-myosin surfaces

5.2.4 Microscopy and Analysis

5.3 Results

5.3.1 Actin Asters

5.3.2 Single Actin Filaments

5.4 Discussion

66 SUMMARY AND FUTURE WORK

6.1 Single Actin Filaments

6.2 Actin-Based Motility

6.3 Recommendation for Future Work

6.3.1 Filament-Binding Tracks

6.3.2 Three-Dimensional Surfaces for Larger Structures

6.3.2 Use of End-Tracking Motors

APPENDIX: MATLAB CODE

LIST OF REFERENCES

BIOGRAPHICAL SKETCH

–  –  –

1-1 Treadmilling of an actin filament

1-2 Experimental set-up for binding filaments to a surface.

1-3 Schematic of microcontact printing protein on glass

2-1 Images of microcontact-printed myosin tracks on a glass coverslip

2-2 Time-lapse image of actin filaments on a BSA-stamped surface

2-3 Total internal reflection fluorescence microscopy images of elongating actin filaments on NEM-myosin tracks

2-4 Filaments in BSA region undergo large thermal undulations

2-5 Total internal reflection fluorescence microscopy images of undulating ends of elongating filaments

2-6 Filament elongating passed the track edge at small θ.

2-7 Filament alignment as a function of filament density on tracks..

2-8 Effect of track width on filament alignment

2-9 Histogram showing the fraction, f, of filament ends that rebind to tracks after their elongating ends cross track boundaries

2-10 Scatter plot showing each segment alignment with the track edge as a function of the distance from the track edge

2-11 Dependence of filament alignment and elongation rate on the concentration of NEM-myosin

2-12 Scatter plots showing the alignment of individual filament segments

2-13 Filaments accumulate in the BSA region of the stamped surface.

2-14 Incubation time of lyophilized rhodamine actin

2-15 Increasing the concentration of profilin visibly decreases the density of actin filaments on NEM-myosin treated surfaces

2-16 Elongation rate of actin filaments as a function of profilin concentration

8 2-17 Illustration of the likely mechanism for actin filament alignment on NEM-myosin tracks

3-1 The x- and y-positions of elongating filament ends as a function of time

3-2 Effect of change in length (step-size of simulation) on the filament rebinding probability

3-3 Effect of the number of modes on filament rebinding probability

3-4 Effect of total filament length on filament rebinding probability

Effect of binding probability constant, Kp (µm-1×sec-1), on filament rebinding 3-5 probability.

3-6 Effect of persistence length on filament rebinding probability.

3-7 Effect of track width and number of modes on the alignment of filaments

3-8 Effect of track width and binding probability constant on the alignment of filaments.....83 3-9 Effect of binding probability on the alignment of filaments

3-10 Effect of track width and persistence length on the alignment of filaments

4-1 Listeria and 500-nm diameter bead propelled by actin rocket tails

4-2 Rotation of a helical actin rocket tail in solution

4-3 Listeria rocket tails on NEM-myosin and BSA-treated surfaces

4-4 500-nm diameter beads attached to rocket tails bound on NEM-myosin and BSA-treated surfaces

4-5 Fields-of-view with large percentage of tails bound to surface

4-6 Fraction of actin rocket tails bound to NEM-myosin and BSA-treated surfaces............103 4-7 Change in x- and y-position over time for beads on NEM-myosin and BSA surfaces...104 4-8 Helical actin rocket tail confined to NEM-myosin-treated surfaces

4-9 Actin rocket tails in total internal reflection fluorescence microscopy

4-10 Magnified image of actin rocket tail with protruding filaments

4-11 Actin tail elongation on NEM-myosin and BSA surfaces.

4-12 Average tail elongation rates determined from the slope of a best-fit line

–  –  –

4-14 Stamped surfaces with 500-nm diameter beads attached to actin tails

4-15 Actin tails bound to NEM-myosin-treated exposed glass of fabricated device...............111 4-16 Actin rocket tail encounters CYTOP wall

5-1 Growth of actin filaments/bundles on 50-nm diameter beads

5-2 Fluorescent intensity of actin filaments/bundles

5-3 Actin asters recovery after photobleaching

5-4 Enlarged image of asters reappearing after photobleaching

5-5 Fluorescent intensity of photobleached filaments

5-6 Recovery rates and equilibrium intensities of photobleached filaments

5-7 50-nm diameter beads bound to surface with single filaments or bundles

5-8 50-nm diameter beads in solution on NEM-myosin surfaces

5-9 Actin motility assay with and without beads on NEM-myosin surfaces.

–  –  –

ADF Actin depolymerizing factor ADP Adenosine diphosphate AFM Atomic force microscopy APES 3-aminopropyltriethoxysilane Arp2/3 Actin related proteins 2/3 ATP Adenosine triphosphate BHI Brain-heart infusion media BS3 Bis(sulfosuccinimidyl suberate) BSA Bovine serum albumin DMSO Dimethyl sulfoxide DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EGTA Ethylene glycol tetraacetic acid EM Electron microscopy HMM Heavy meromyosin NEM N-ethylmaleimide PDMS Polydimethylsiloxane PMSF Phenylmethanesulphonyl fluoride TIRF Total internal reflection fluorescence VASP Vasodilator-stimulated phosphoprotein

–  –  –

Chair: Richard Dickinson Major: Chemical Engineering The cytoskeletal protein actin generates forces for various processes by polymerizing into filaments. In vivo, actin works with the motor protein myosin to produce muscle contractions and with proteins acting as end-tracking motors responsible for cell and bacterial motility, such as the motility of Listeria monocytogenes. End-tracking proteins bind the polymerizing end of an actin filament to a motile surface, creating persistent attachment during filament elongation.

Both types of motors use the energy from ATP hydrolysis and can be exploited in vitro in bionanodevices, which require forces to transport objects on a micro- or nano-scale, possibly against flow or diffusion gradients.

Our study has focused on the guidance of single-filament elongation and filament bundles (rocket tails) to orient elongation in vitro. Microcontact printing, a technique that stamps protein patterns onto glass surfaces through adsorption, was used to create filament-binding tracks of modified myosin (void of its motor activity), which successfully bound and guided single actin filament elongation in a manner dependent on track width and surface conditions. These results confirm the capability of this method to be used for the motility of objects attached to single actin filaments and for the creation of immobilized tracks of actin filaments for myosin-mediated

–  –  –

make predictions for other types of filaments and systems.

Modified myosin surfaces also confined actin rocket tails attached to particles and bacteria, reducing the Brownian motion of the motile objects. Channels formed through photolithographic techniques on glass surfaces were used to attempt to guide these particles.

Single actin filaments attached to smaller particles were also characterized to determine the potential for single-filament propulsion in nanodevices. We conclude that actin filament-binding proteins can be applied to surfaces using adsorption and microcontact printing and that this technique is effective in binding and guiding filaments in various systems, including single and bundled filaments. We predict this technique can be applied to other systems undergoing actin-based motility, making it a versatile method for bionanotechnology.

–  –  –

Muscle contraction, cell movement, intracellular bacteria motility and cell division are all processes that involve biomolecular motors, a group of proteins that utilize the energy from adenosine triphosphate (ATP) hydrolysis to do work on a system (1). These biomolecular motors can be exploited for the in vitro transport of nano-cargo (e.g. beads, bacteria, DNA) (2-7), as well as in biosensors, microfluidic devices, and micro- and nanoelectromechanical systems (MEMS and NEMS) (8-15). One approach has been to pattern surface regions with a high-affinity for the adsorption of the molecular motors kinesin and myosin to guide transport of pre-assembled microtubules and actin filaments, respectively (8, 15-21). Other methods rely on surface-immobilized microtubules and actin filaments to create tracks for kinesin-mediated (3, 5,

22) and myosin-mediated particle transport (6, 23, 24).



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