University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 An Examination Of The Cytoplasmic Dynein Stepping Mechanism At The Single Molecule Level Lisa Gail Lippert University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Lippert, Lisa Gail, "An Examination Of The Cytoplasmic Dynein Stepping Mechanism At The Single Molecule Level" (2016). Publicly Accessible Penn Dissertations. 2428. https://repository.upenn.edu/edissertations/2428 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2428 For more information, please contact [email protected]. An Examination Of The Cytoplasmic Dynein Stepping Mechanism At The Single Molecule Level Abstract Rotational motions play important roles within biological processes. These motions can drive energy production as with the F1-ATP synthase or accompany domain motions during a conformational change such as the relative rotation of the large and small ribosomal subunits during protein synthesis. Studying these motions can provide insight into the mechanics of enzyme function that cannot be obtained by measuring its localization or chemical output alone. Rotational tracking can be done in the context of single molecule studies to observe enzymatic function at the single particle level. This presents an advantage over bulk solution studies because simultaneously occurring events, such as a solution of enzymes catalyzing a reaction, are not necessarily identical. By measuring the motions of a single molecule, short-lived states and rare events that would otherwise be averaged out can be detected. Here single molecule rotational tracking is utilized to examine the stepping mechanism of the cellular transport motor, cytoplasmic dynein. Cytoplasmic dynein walks along microtubules toward the minus end and is responsible for a wide range of cellular functions including cargo transport and chromosome alignment during cell division. This work employs a position and rotational tracking method, polarized total internal reflection fluorescence (polTIRF) microscopy. This technique requires a polarized fluorescent probe that is rigidly attached to the protein domain of interest and an optical system capable of measuring the orientation of such a probe. A functionalization method was developed to water-solubilize CdSe/CdS semiconductor quantum nanorods, which have polarized fluorescence emission, and coat them with the biotin binding protein, NeutrAvidin, in order to attach them to biotinylation sites within the dynein ring. A method was also developed to quantify the number and density of functional biotin binding sites on the nanorod surface and compare it to that of commercially available streptavidin quantum dots. These nanorods were attached to cytoplasmic dynein via two inserted biotinylation sites in AAA5 and AAA6 of the ring domain and rotational motions of the dynein ring were measured in real time using a home-built optical system capable of measuring both position and orientation simultaneously. These measurements revealed small, frequent ring rotations that occurred more than twice as frequently as steps along the microtubule track. The observed ring rotations are too small to be attributed to a classic powerstroke mechanism in which large-scale tilting produces forward motion, but instead support a flexible stalk model where tension between the two dynein heads, produced by conformational changes of the linker domain, results in bending of the flexible coiled-coil stalk and hinging at the microtubule binding domain. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Yale E. Goldman Keywords Dynein, Fluorescence, Nanoparticles, Polarization, Single molecule Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2428 AN EXAMINATION OF THE CYTOPLASMIC DYNEIN STEPPING MECHANISM AT THE SINGLE MOLECULE LEVEL Lisa G. Lippert A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation ________________________ Yale E. Goldman, M.D., Ph. D. Professor of Physiology Graduate Group Chairperson ________________________ Kim A. Sharp, Ph. D. Associate Professor of Biochemistry and Biophysics Dissertation Committee Michael Ostap, Ph. D. Professor of Physiology Feng Gai, Ph. D. Professor of Chemistry Erika L.F. Holzbaur, Ph. D. Professor of Physiology Michael A. Lampson, Ph. D. Associate Professor of Biology Samara L. Reck-Peterson, Ph. D. Professor of Cellular and Molecular Medicine AN EXAMINATION OF THE CYTOPLASMIC DYNEIN STEPPING MECHANISM AT THE SINGLE MOLECULE LEVEL COPYRIGHT 2016 Lisa G. Lippert This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ ACKNOWLEDGMENT This work would not have been possible without the support of my advisor, Dr. Yale E. Goldman. His dedication to teaching provided me with the tools necessary to complete this project, and striving to reach his high scientific standards has made me a better and more precise researcher. He has been a fantastic mentor, and I consider myself lucky to have had the opportunity to work with him. I would also like to thank the current and former Goldman lab members as well as collaborators who contributed to this work. Tali Dadosh and Jeffrey Hallock helped to develop the methods presented here. My friends and lab-mates Betsy McIntosh and Michael Woody provided insightful scientific and non-scientific discussions. I am honored to have collaborated with Samara Reck-Peterson, Chris Murray, Klaus Schulten and Erika Holzbaur, each of whom made essential contributions and improved both the quality and impact of this research. Finally, I would like to thank my family. My parents, Skip and Laura Rung, instilled in me the importance of an education. And my husband, Andrew Lippert, has patiently supported me throughout this entire process. His willingness to prioritize my career enabled me to follow the opportunities that I have been given. iii ABSTRACT AN EXAMINATION OF THE CYTOPLASMIC DYNEIN STEPPING MECHANISM AT THE SINGLE MOLECULE LEVEL Lisa G. Lippert Yale E. Goldman, M.D., Ph. D. Rotational motions play important roles within biological processes. These motions can drive energy production as with the F1-ATP synthase or accompany domain motions during a conformational change such as the relative rotation of the large and small ribosomal subunits during protein synthesis. Studying these motions can provide insight into the mechanics of enzyme function that cannot be obtained by measuring its localization or chemical output alone. Rotational tracking can be done in the context of single molecule studies to observe enzymatic function at the single particle level. This presents an advantage over bulk solution studies because simultaneously occurring events, such as a solution of enzymes catalyzing a reaction, are not necessarily identical. By measuring the motions of a single molecule, short-lived states and rare events that would otherwise be averaged out can be detected. Here single molecule rotational tracking is utilized to examine the stepping mechanism of the cellular transport motor, cytoplasmic dynein. Cytoplasmic dynein walks along microtubules toward the minus end and is responsible for a wide range of cellular functions including cargo transport and chromosome alignment during cell division. This work employs a position and rotational iv tracking method, polarized total internal reflection fluorescence (polTIRF) microscopy. This technique requires a polarized fluorescent probe that is rigidly attached to the protein domain of interest and an optical system capable of measuring the orientation of such a probe. A functionalization method was developed to water-solubilize CdSe/CdS semiconductor quantum nanorods, which have polarized fluorescence emission, and coat them with the biotin binding protein, NeutrAvidin, in order to attach them to biotinylation sites within the dynein ring. A method was also developed to quantify the number and density of functional biotin binding sites on the nanorod surface and compare it to that of commercially available streptavidin quantum dots. These nanorods were attached to cytoplasmic dynein via two inserted biotinylation sites in AAA5 and AAA6 of the ring domain and rotational motions of the dynein ring were measured in real time using a home- built optical system capable of measuring both position and orientation simultaneously. These measurements revealed small, frequent ring rotations that occurred more than twice as frequently as steps along the microtubule track. The observed ring rotations are too small to be attributed to a classic powerstroke mechanism in which large-scale tilting produces forward motion, but instead support a flexible stalk model where tension between the two dynein heads, produced by conformational changes of the linker domain, results in bending of the flexible coiled-coil stalk and hinging at the microtubule binding domain. v TABLE OF CONTENTS ACKNOWLEDGMENT ...........................................................................