Browsing by Author "Wagner, Andrew"

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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.
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    Investigating the role of NPFs in the Arp3ΔC phenotype
    Kiemel, Jonathan; Rodnick-Smith, Max; Liu, Su-Ling; Nolen, Brad; Wagner, Andrew
    The Arp2/3 complex is an assembly of seven protein subunits that nucleates branched actin networks involved in cellular functions such as endocytosis. Previous work has determined the complex is intrinsically inactive, and can be turned “on” by activators like ATP or proteins called nucleation promotion factors (NPFs). It has been hypothesized that the complex remains in an auto-inhibited state due to the c-terminus of the Arp3 subunit. Deletion of the c terminus (Arp3ΔC) results in a hyperactive Arp 2/3 complex in a purified in-vitro system. Strikingly, this complex is inhibited by the canonical NPF wiskott-aldrich syndrome protein (wsp1). These contrary phenomena are complicated further by the observation of endocytosis in-vivo. In S. Pombe fission yeast, Arp3ΔC generates endocytic patches that have a reduced internalization percentage compared to wild type cells but assemble at nearly the same abundance. This suggests preferential binding of a single NPF to Arp2/3 that polymerizes actin, but in an incorrect manner for endocytosis. Here, we will investigate involvement of dip1 amongst other NPFs in the Arp3ΔC phenotype. Dip1 has been shown to be involved in the temporal regulation of actin polymerization during endocytosis; deletion of this activator results in both decreased patch density and longer but more stochastic patch lifetimes before internalization. Utilizing S. pombe as a model organism, Arp3ΔC will be combined with NPFΔ constructs to determine which activator is responsible for actin polymerization in the absence of the Arp3 c-terminus. Analysis of crosses is accomplished primarily with spinning disk confocal microscopy.