MECHANISMS OF BRANCHED ACTIN NETWORK FORMATION THROUGH COORDINATE ACTIVATION OF ARP2/3 COMPLEX by LUKE ANDREW HELGESON A DISSERTATION Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2014 DISSERTATION APPROVAL PAGE Student: Luke Andrew Helgeson Title: Mechanisms of Branched Actin Network Formation through Coordinate Activation of Arp2/3 Complex This dissertation has been accepted and approved in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Biology by: Bruce Bowerman Chairperson Bradley Nolen Advisor Alice Barkan Core Member J. Andrew Berglund Core Member Kenneth Prehoda Institutional Representative and J. Andrew Berglund Dean of the Graduate School Original approval signatures are on file with the University of Oregon Graduate School. Degree awarded December 2014 ii © 2014 Luke Andrew Helgeson iii DISSERTATION ABSTRACT Luke Andrew Helgeson Doctor of Philosophy Department of Biology December 2014 Title: Mechanisms of Branched Actin Network Formation through Coordinate Activation of Arp2/3 Complex Fundamental cellular processes such as motility and endocytosis rely on the actin cytoskeleton to translate biochemical protein interactions into mechanical forces. Cells utilize an extensive collection of actin binding proteins to comprehensively regulate actin networks during these dynamic cell operations. Branched actin networks, which are geometrically and functionally disparate from linear networks, are required for numerous cellular actions. Actin-related protein 2/3 complex (Arp2/3 complex) nucleates branched actin filaments upon activation by regulatory proteins known as nucleation promoting factors (NPFs). Often, several biochemically distinct NPFs are required for the same cellular structure, leading us to hypothesize that multiple NPFs can coordinately activate Arp2/3 complex to regulate the nucleation, architecture and assembly of branched networks. We identified and dissected the mechanisms of two sets of NPFs which coordinately activate Arp2/3 complex. Overall, these findings provide a better understanding of how Arp2/3 complex is activated and how cells control branched actin networks. In chapters II and III, we investigated the mechanism of synergistic activation of Arp2/3 complex by the NPFs cortactin and WASP family proteins. We found that iv cortactin accelerates the release of WASP family proteins from a branching intermediate, a previously unknown rate limiting step. Further dissection of the mechanism revealed that cortactin is specifically suited to displace WASP family proteins through a unique Arp2/3 complex binding region and target stalled branching intermediates with high affinity. Three different WASP family members were tested for their capacity to synergize with cortactin in Arp2/3 complex activation, establishing a list of cellular structures where cortactin-mediated synergistic activation is likely occurring. In chapter IV, we investigated the ability of Dip1 and Wsp1 to coordinately activate Arp2/3 complex during branched network formation. We established that Dip1 activation of Arp2/3 complex results in the formation of linear filaments which can template Wsp1 mediated branching. Subsequent kinetic data and modeling revealed that Dip1 and Wsp1 likely increase the rate of network formation by simultaneously binding to and co- activating Arp2/3 complex. These findings suggest that, together, Dip1 and Wsp1 regulate the initiation and rate of branched network assembly. This dissertation includes previously published and unpublished co-authored material and videos files. v CURRICULUM VITAE NAME OF AUTHOR: Luke Andrew Helgeson GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene, OR Iowa State University, Ames, IA DEGREES AWARDED: Doctor of Philosophy, Biology, 2014 University of Oregon Bachelor of Science, Biochemistry, 2009, Iowa State University AREAS OF SPECIAL INTEREST: Biochemistry Biophysics Microscopy PROFESSIONAL EXPERIENCE: Graduate Teaching Assistant, Department of Biology, University of Oregon, Eugene, OR, 2009-2010 GRANTS, AWARDS, AND HONORS: Pete von Hippel Scholar Award, Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, OR. 2014. NIH Training Grant: Molecular Biology and Biophysics. Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, OR. 2010-2013 Robert Stupka Memorial Scholarship. Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA. 2009 Dexter Memorial Fund. Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA. 2005/2006. Award for Competitive Excellence Scholarship. Iowa State University, Ames, IA. 2005-2009. vi PUBLICATIONS: Helgeson, Luke A, Julianna G Prendergast, Andrew R Wagner, Max Rodnick-Smith, and Brad J Nolen. 2014. “Interactions with Actin Monomers, Actin Filaments and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks.” The Journal of Biological Chemistry 289 (42) (August 26): 28856–28869. doi:10.1074/jbc.M114.587527. Helgeson, Luke A, and Brad J Nolen. 2013. “Mechanism of Synergistic Activation of Arp2/3 Complex by Cortactin and N-WASP.” eLife 2 (September 3): e00884. doi:10.7554/eLife.00884. Hetrick, Byron, Min Suk Han, Luke A Helgeson, and Brad J Nolen. 2013. “Small Molecules CK-666 and CK-869 Inhibit Actin-Related Protein 2/3 Complex by Blocking an Activating Conformational Change.” Chemistry & Biology 20 (5) (April): 701–12. doi:10.1016/j.chembiol.2013.03.019. Liu, Su-Ling, Jordan R May, Luke A Helgeson, and Brad J Nolen. 2013. “Insertions within the Actin Core of Actin-Related Protein 3 (Arp3) Modulate Branching Nucleation by Arp2/3 Complex.” The Journal of Biological Chemistry 288 (1) (January 4): 487–97. doi:10.1074/jbc.M112.406744. Leung Daisy W, Dominika Borek, Mina Farahbakhsh, Parameshwaran Ramanan, Jay C Nix, Tianjiao Wang, Kathleen C Prins, Zbyszek Otwinowski, Richard B Honzatko, Luke A Helgeson, Christopher F Basler, Gaya K Amarasinghe. 2010. “Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins.” Acta.Crystallogr. Sect. F Struct. Biol.Cryst.Commun. 66(6): 689-92. Leung Daisy W, Kathleen C Prins, Dominika M Borek, Mina Farahbakhsh, JoAnn M Tufariello, Parameshwaran Ramanan, Jay C Nix, Luke A Helgeson, Zbyszek Otwinowski, Richard B Honzatko, Christopher F Basler, Gaya K Amarasinghe. 2010. “Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35”. Nature Structural & Molecular Biology. 17(2): 165-72. vii ACKNOWLEDGMENTS First and foremost, I would like to express my sincere gratitude to my mentor and thesis advisor, Dr. Brad Nolen. Under Brad‟s guidance, I developed a strong set of scientific skills which will be fundamental to my success for years to come. I am thankful to all past and present members of the Nolen lab; without them I would not have been able to accomplish my goals. I would especially like to thank Dr. Byron Hetrick for his mentoring and Dr. Su-Ling Liu who was always helpful and full of knowledge. I would like to thank my dissertation advisor committee for their assistance and words of encouragement throughout the years. Additionally, I would like to thank the University of Oregon, Institute of Molecular Biology community for their various means of support. I am thankful to my undergraduate mentor, Dr. Gaya Amarasinghe, for helping me build a solid foundation for performing rigorous scientific research. Successful science requires a clear and refreshed mind. I am especially grateful for my friends who have allowed me to maintain a refreshed mind and have made my graduate school experience memorable. Specifically, I am forever grateful to those friends who ventured into the wilderness with me to experience and explore all the beauty nature has to offer. I would especially like to thank Dr. Alesia McKeown for her invaluable companionship throughout this challenging endeavor. Alesia‟s love, support and humor constantly sustains me and brings joy to my life. Last but not least, I am forever thankful to my family for their love throughout the years. My parents, Benny and Janice, and sister, Beth, have been constant sources of inspiration, knowledge and encouragement. Without their love and support, I would not be where I am today. viii I dedicate this work to my family, past and present. Thank you for everything. ix TABLE OF CONTENTS Chapter Page I. INTRODUCTION .................................................................................................... 1 II. MECHANISM OF SYNERGISTIC ACTIVATION OF ARP2/3 COMPLEX BY CORTACTIN AND N-WASP .......................................................................... 15 Introduction ............................................................................................................ 15 Results .................................................................................................................... 19 Discussion .............................................................................................................. 43 Material and Methods ............................................................................................ 49 Bridge to Chapter III .............................................................................................. 58 III. INTERACTIONS WITH ACTIN