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Single-Molecule Biophysics of Kinesin Family Motor Proteins a Dissertation Submitted to the Department of Physics and the Commit

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Single-Molecule Biophysics of Kinesin Family Motor Proteins a Dissertation Submitted to the Department of Physics and the Commit SINGLE-MOLECULE BIOPHYSICS OF KINESIN FAMILY MOTOR PROTEINS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHYSICS AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Johan Oscar Lennart Andreasson August 2013 © 2013 by Johan Oscar Lennart Andreasson. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/fy688zk4161 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Steven Block, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Sebastian Doniach I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Alexander Dunn Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii iv Abstract Kinesin family proteins are nanoscale motors involved in many essential biological processes, such as intracellular transport and cell division. The biological function of most kinesin motors is to use the energy from ATP hydrolysis to move cargo through a crowded cellular environment, quickly taking 8-nm steps along cytoskeletal microtubules. By maintaining its two motor domains (heads) out of phase, kinesin can complete hundreds of steps per encounter with the microtubule, and can do so against pN-scale loads. The physiological role of kinesin is directly related to its movement and in this dissertation I present several single-molecule studies where the force- dependent motion of individual kinesin motors was studied using optical trapping techniques. In humans, the kinesin superfamily includes over forty genes encoding different kinesin proteins, classified into 15 families, and motors from several families were studied in this work. Optical traps use lasers to detect the position of biological molecules, at nm- scale resolution, and to directly manipulate them by applying pN-scale forces. In this dissertation, I present two novel optical traps. The first uses highly linear electro-optic deflection of the laser light to create an instrument with fast feedback that is optimized for work with kinesin motors. The second instrument, an “Optical Torque Wrench”, is a trap that can apply both forces and torques on birefringent particles. By controlling the light polarization in the sample plane, the rotation of nanofabricated quartz cylinders can be controlled in real time while the applied torque is measured directly. The functionalized particles can be used to twist DNA or other biological molecules. The kinesin motor domains are coordinated during stepping and the inter-head communication is believed to be conferred by the neck linker, a 14-amino acid structural element connecting the head to the common coiled-coil stalk. By extending this segment, we could examine its role in gating the mechanochemical cycle. A six- amino acid insert in the neck linker of a cysteine-light human kinesin construct led to unexpected ATP-dependent backstepping under load. These observations could be explained by a branched pathway where both ATP unbinding and hydrolysis were v gated by the direction of the neck linker. Lengthening the neck linker also led to futile hydrolysis. Further experiments on the effects of neck linker length were done with a series of Drosophila Kinesin-1 mutants, with one to six extra residues in the neck linker. The rate of force-dependent rear head release and the internal strain developed during stepping was determined from force-dependent velocities and we also found that the mechanism of detachment from the microtubule depends on the direction of load. The heterotrimeric Kinesin-2 motors are unique in that they are the only kinesin family motors that consist of two different catalytic domains. Here, the mammalian Kinesin-2, KIF3A/B, was studied in detail by performing optical trapping experiments with both the wild-type dimer and with homodimers (KIF3A/A and KIF3B/B). A pathway that incorporates the individual catalytic cycles for KIF3A and KIF3B could explain all force- and ATP-dependent kinetics and surprisingly we found that the run lengths for KIF3A/B were significantly shorter than for Kinesin-1. Furthermore, motors with the weakly force-dependent KIF3A head “slipped” and exhibited short run lengths that were rescued under no load, indicating that KIF3A/B combines a Kinesin-1-like motor domain (KIF3B) with a unique and “weak” one (KIF3A). Finally, I present motility experiments where force-dependent kinetics were explored for several other kinesin family motors. KIF17 (Kinesin-2) and CENP-E (Kinesin-7) are robust, processive motors whereas KIF4A, a Kinesin-4 motor, is fast but unable to sustain significant loads. These results, together with those for Kinesin- 1, KIF3A/B (Kinesin-2), and other motors, show that forces are needed fully reveal the motor characteristics and differences between various kinesin proteins. They also illustrate the remarkable diversity within the kinesin superfamily. vi Acknowledgements There are many people I would like to thank for making my graduate career a very rewarding experience. First of all, I would like to thank Steve, my advisor, who has been an inspiring mentor throughout my years in the lab. I knew very little about kinesin or optical traps when I first attended a seminar by Steve, but his impressive work and excitement for science inspired me to pursue a career in biophysics. Steve is not only extremely bright but also has a great sense of humor. And I attribute the Block lab’s positive atmosphere to his straightforward and witty personality, as well as his aspiration to give people the freedom to pursue their own ideas. He instills in his students a rigorous approach to science and not to mention a very careful attention to presentation skills. By attracting many talented scientists. he has built an exceptional lab that allows people to grow in a friendly and collaborative environment. I have been fortunate to work daily with several of these scientists who have also become good friends over the years. It was the original Team Kinesin who helped me get started in the lab. I worked on my first project with Megan who taught me a lot about kinesin and set me on the path for my future work in this area. Together with her, Nick, Adrian and Braulio, I also had my first hands-on experience in building traps, and the knowledge they passed on to me was put to good use as Braulio and I continued the building. Braulio was not only a master of optics, but also of electronics, cheap effective solutions, and enjoying life. As the original team Kinesin moved on to new challenges, Bason took over as the senior kinesin team leader, who I was fortunate to not only have the opportunity to work with but also to become close friends with. And in the most recent years, I have enjoyed continuing on the kinesin legacy in the Block lab together with Kevin. I would like to give a special acknowledgement to the the undergraduates and high school students whom I’ve had the privilege of mentoring. Bojan, who worked with me since his first year of college, has never failed to impress me. Over the years, he’s become a good friend, and I look forward to working with him further as we both continue our careers at Stanford. Benamy was a very bright undergraduate who I vii enjoyed working with for two summers given his positive attitude and great sense of humor. Alice was the first high school student to volunteer for me in the lab, and her dedicated work set the path for bringing in addititional students. Taddy volunteered twice and even brought in his friend Ronak for the second summer, and both brought with them a lot of excitement and energy to the lab. Finally, I’d like to extend my thanks to all the rotation students who came through the lab at least partly under my guidance: Sonny, Andrew, Koning, Joel, Jacob and Quintin. In the Block lab, you not only interact with your direct collaborators but also with all of the other lab members as well who have really made the lab an enjoyable place to come to work everyday. After rotating in a position where I worked alone in a concrete office, the welcoming atmosphere of the Block lab was such a stark contrast that I immediately felt at home. Kristina and Will were the senior RNAP team members at the time who provided a lot of inspiration. I also have enjoyed the company of Matt G., Matt L., Peter, and Cuauh, whether in the city, at the gym, or on bikes in the mountains. I’ve shared my longest time in lab with Kirsten, Jing, and Christian, who joined around the time as I did, and with Christian I also enjoyed all the travails and good memories of the physics program. After my fellow European, Volker, left the lab, the remaining Block lab now consists of the following team: Dan, Furqan, Van, Arthur, Andrew, Irena and Anirban. I have great expectations for the future work that will come out of the basement of the Herrin Lab building and will look back fondly on the Block lab, including all the good times, hard work, group meetings, conferences, parties, procrastination, and humorous discussions.
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