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Dissertation Characterizing the Diffusional Behavior DISSERTATION CHARACTERIZING THE DIFFUSIONAL BEHAVIOR AND TRAFFICKING PATHWAYS OF KV2 . 1 USING SINGLE PARTICLE TRACKING IN LIVE CELLS Submitted by Aubrey Weigel Graduate Degree Program in Bioengineering In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2013 Doctoral Committee: Advisor: Diego Krapf Michael Tamkun James Bamburg Randy Bartels ABSTRACT CHARACTERIZING THE DIFFUSIONAL BEHAVIOR AND TRAFFICKING PATHWAYS OF KV2 . 1 USING SINGLE PARTICLE TRACKING IN LIVE CELLS Studying the diffusion pattern of membrane components yields valuable information regarding membrane structure, organization, and dynamics. Single particle tracking serves as an excellent tool to probe these events. We are investigating of the dynamics of the voltage gated potassium channel, Kv2.1. Kv2.1 uniquely localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. The work reported here examines the diffusion pattern of Kv2.1 and determines alternate functional roles of surface clusters by investigating recycling pathways using single particle tracking in live cells. The movement of Kv2.1 on the cell surface is found to be best modeled by the combination of a stationary and non-stationary process, namely a continuous time random walk in a fractal geometry. Kv2.1 surface structures are shown to be specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells. Both Kv2.1 and Kv1.4, a non-clustering membrane protein, are inserted and retrieved from the plasma membrane at the perimeter of Kv2.1 clusters. From the distribution of cluster sizes, using a Fokker-Planck formalism, we find there is no evidence of a feedback mechanism controlling Kv2.1 domain size on the cell surface. Interestingly, the sizes of Kv2.1 clusters are rather governed by fluctuations in the endocytic and exocytic machinery. Lastly, we pinpoint the mechanism responsible for inducing Kv2.1 non-ergodic dynamics: the capture of Kv2.1 into growing clathrin- coated pits via transient binding to pit proteins. ii ACKNOWLEDGEMENTS There are many people I would like to thank. Without the support from friends, family and colleagues I wouldn’t be where I am today. I owe a great deal of gratitude to my graduate com- mittee. I thank you all very much for the time and effort you have invested. Your encouragment and mentorship is truely appreciated. Diego, when I started the lab was brand new, and my how it has changed since then! We were both younger then, and experiencing a lot of things for the first time. It was a bit of a bumpy road at times but in the end a great time. It was an experience for the both of us growing and building the lab to what it is today. An experience for which I will forever be grateful. I have learned so much working with you. I have witnessed what it truly means to be passionate about science, how to juggle this passion and life with a bit of grace, and how to slam a ’clinker’ on unsuspecting foosball competitors. Thank you for all of your guidance and your patience. Mike, you have been an amazing mentor. While you’d like to have the world see you as a grumpy, ruthless pessimist, I feel priveledged to have known your truer side: a wise, merciful pessimist. You have an amazing talent of looking at the larger picture and answering the bigger questions. An attribute that is very admirable. Not only has it been very fortunate to collaborate with you and your lab but it has been truly instrumental and foundational to the body of work that I have been able to put together for my PhD. (You should also be commended for your ability to consistently brew excellent beer) To my lab-mates past and present, you guys rock! Much of what I have learned has come from interactions with everyone. Kristen, you reminded me there is more to life than just science. Jenny, you have been an amazing officemate - I wouldn’t trade our time together for anything, I think that we have learned so much from eachother. Sanaz, you are just starting to get your feet wet in this lab and already you have done so much! I know that you are going to great things! Bryce, you have great experiments ahead of you, and all the best to you in the future. A huge deal of thanks goes to Kari Ecklund as well. So much time doing tedious data analysis was saved with your efforts! The work in Chapter 4 determing the size of Kv2.1 clusters was made possible with your efforts. Also, thank you Mike Reid for taking the time to help our lab with your help in analyzing the seemingly infinite arrays of data that we put together in Chapter 4.ToEdwin Chong iii and Shankar Ragi, I also owe a great deal of thanks. Your contribution of simlutions and analytical expertise helped us form a beautiful story in Chapter 4. To everyone, thank you for all of your support, patience, and friendship. Tamkunites, where do I even begin. Every ounce of cellular and molecular biology I know, I owe to you. I can not thank each and every one of you enough for taking the time to ’show me the ropes’. I know it had to be painful at first - a physicist doing molecular biology! We have grown so far beyond just being collaborators and I thank you all for your endeared friendship. Liz, I can not send you enough gratitude for taking the time to work with me and teach me the ins and outs of plasmid cloning and mutagenesis. Phil, it has been a great privelege to get to know you. To the both of you, it has been awesome working with you! I have learned a great deal from our improntu brain storm sessions, throwing our edeas at eachother, though vague, often not well thought through, usually not to the point, they have been extremly instructional and have shaped the thinking of us all. Bears, beers, and science should be combined in a frequent fashion. I would specifically like to acknowledge the work that is presented in Chapter 4 which was largely done by the hands of Emily Deutsch, Liz Akin, Phil Fox, Rob Loftus and Chris Haberkorn. All of the experiments in neurons, mirroring what we observed in HEK cells was performed by this group, as well as the elegant experiments showing the insertion of nascent Kv2.1 channels. I am so fortunate to be surrounded by such a grand support group of friends and family. Thank you, for your love, your support, your guidance, and your patience. iv DEDICATION Dedicated to the loving memory of my father, Michael Rankin, 1961–2010 v TABLE OF CONTENTS 1 CHAPTER 1: INTRODUCTION ................................................................................................. 1 1.1 The cell membrane ........................................................................................................................ 1 1.2 Microscopy tools ........................................................................................................................... 2 1.3 Diffusion: Theoretical models ..................................................................................................... 8 1.4 Review of experimental observations in live cells ................................................................. 21 1.5 The voltage-gated potassium channel, Kv2.1 ......................................................................... 26 1.6 Overview of this dissertation .................................................................................................... 27 2 CHAPTER 2: ERGODIC AND NONERGODIC PROCESSES COEXIST IN THE PLASMA MEMBRANE ............................................................................................................................... 30 2.1 Introduction ................................................................................................................................. 30 2.2 Methods ........................................................................................................................................ 32 2.3 Results ........................................................................................................................................... 35 2.4 Discussion .................................................................................................................................... 50 2.5 Acknowledgements and contributions .................................................................................... 51 3 CHAPTER 3: OBSTRUCTED DIFFUSION PROPAGATOR ANALYSIS FOR SINGLE PARTICLE TRACKING ............................................................................................................. 52 3.1 Introduction ................................................................................................................................. 52 3.2 Materials and methods ............................................................................................................... 56 3.3 Results ........................................................................................................................................... 57 3.4 Discussion .................................................................................................................................... 68 3.5 Conclusions .................................................................................................................................. 69 3.6 Acknowledgements and contributions .................................................................................... 70 4 CHAPTER 4: KV2.1 CELL SURFACE CLUSTERS ARE INSERTION PLATFORMS FOR ION CHANNEL DELIVERY TO THE PLASMA
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