Chemistry Theses and Dissertations

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https://hdl.handle.net/1794/2046

This collection contains some of the theses and dissertations produced by students in the University of Oregon Chemistry Graduate Program. Paper copies of these and other dissertations and theses are available through the UO Libraries.

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Browsing Chemistry Theses and Dissertations by Subject "Actin"

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    Conformational Changes of Arp2/3 Complex in the Branched Actin Nucleation Pathway
    (University of Oregon, 2016-10-27) Rodnick-Smith, Max; Nolen, Bradley
    Branched actin networks play an important role in cellular processes ranging from cell motility, endocytosis, and adhesion. The Actin-related protein 2/3 (Arp2/3) complex nucleates actin branches from the sides of existing actin filaments. Arp2/3 complex is highly regulated and requires association with ATP, actin monomers, actin filaments and a class of proteins called nucleation promoting factors (NPFs) to undergo an activating conformational change where the actin-related subunits, Arp2 and Arp3, arrange into a filament-like conformation that templates a new actin branch. While some progress has been made, the individual roles of each of these factors on the activating conformational change is poorly understood. In addition, it is still unclear how Arp2/3 complex is held in its inactive state, which is vital for understanding how activation occurs. In this dissertation, we dissect key interfaces in Arp2/3 complex that are responsible for holding it in an inactive state, and specifically evaluate the roles of ATP and WASP, the canonical NPF, in the activating conformational change of Arp2/3 complex. In chapter II, we investigated the contacts made between the Arp2 and Arp3 subunits in their inactive state, and the role of ATP in stimulating the active conformation. We found that two key interfaces, the αE/αF loop in Arp2 and the C-terminus of Arp3, a conserved extension not present in actin, are vital for holding Arp2/3 complex in its autoinhibited state. Evaluation of the role of ATP demonstrated that binding of ATP is required for the activating conformational change and displaces the Arp3 C-terminus, an important step in destabilization of the inactive state. In chapter III, we investigated the mechanism of WASP-induced conformational changes using an engineered crosslinking assay that only forms crosslinks when Arp2/3 is in its active conformation. We discovered that many WASP-related proteins are capable of stimulating this conformational change through a mechanism that involves displacement of the Arp3 C-terminus. Interestingly, purified Arp2/3 complex crosslinked in the active conformation was hyperactive compared to WASP-mediated activation, demonstrating that WASP activation limits nucleation and that actin monomer delivery is not required for nucleation. This dissertation contains unpublished co-authored material.
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    Discovery and Characterization of WISH/DIP/SPIN90 Proteins as a Class of ARP2/3 Complex Activators that Function to Seed Branched Actin Networks
    (University of Oregon, 2018-04-10) Wagner, Andrew; Stevens, Tom
    Assembly of branched actin filaments produces dynamic structures required during membrane associated processes including cell motility and endocytosis. The Actin Related Protein 2/3 (Arp2/3) complex is the only known regulator capable of nucleating actin branches. To specify the sub cellular localization and timing of actin assembly the complex is tightly regulated. Canonical activation of the Arp2/3 complex by Wiskott-Aldrich Syndrome proteins (WASP), requires preformed actin filaments, ensuring the complex nucleates new actin filaments off the sides of preformed filaments. WASP proteins can therefore propagate branch formation but cannot initiate a Y-branch without performed filaments. A key question, then, is what is the source of preformed filaments that seed branched actin network formation in cells? It is unclear how activation of Arp2/3 by multiple regulators is balanced to specify actin filament architectures that are productive in vivo. In this dissertation, we identified WISH/DIP1/SPIN90 (WDS) family proteins as activators of the Arp2/3 complex that do not require preformed filaments, and evaluated whether WDS proteins seed branching nucleation. In chapter II, we dissected the biochemical properties of WDS proteins and found they activate the Arp2/3 complex using a non-WASP like mechanism. Importantly, we discovered WDS-mediated Arp2/3 activation produces linear, unbranched filaments, and this activity is conversed from yeast to mammals. These observations highlight that WDS proteins have the biochemical capacity to seed actin branches. In chapter III, we observed WDS-generated linear filaments can seed WASP-mediated branching directly using single molecule microscopy with fluorescently labeled Dip1. We find that WDS-mediated nucleation co-opts features of branching nucleation. In chapter IV, we investigated how WDS activity is balanced with WASP. We discovered WDS proteins use a single turnover mechanism to activate Arp2/3 and this is conserved during endocytosis. In contrast, WASP-mediated activation is multi-turnover, highlighting a crucial difference between WDS proteins and WASP. Our observations explain how Arp2/3 may limit linear filament production to initiate networks and favor branches during network propagation. Finally, we use fission yeast to show that increasing Dip1 is sufficient to cause defects in actin assembly and the timing of actin patches at sites of endocytosis.