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 Author "Balzer, Connor"

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    The Effects of WISH/DIP/SPIN90 and WASp Family Proteins Regulation of Arp2/3 Complex on Actin Network Architecture and Dynamics
    (University of Oregon, 2020-09-24) Balzer, Connor; Nolen, Brad
    Actin-related protein (Arp) 2/3 complex is required to nucleate branched actin networks necessary for numerous cellular processes including cell motility and endocytosis. To nucleate branched actin filaments, Arp2/3 complex must be activated by nucleation promoting factors (NPFs). The best understood NPFs are from the Wiskott-Aldrich Syndrome protein (WASp) family. To activate nucleation, WASp must recruit actin monomers to the complex and the Arp2/3-WASp-actin monomer assembly must bind a preformed actin filament. The requirement for a preformed actin filament ensures that Arp2/3 complex specifically nucleates branched instead of linear actin filaments but creates a paradox. If an actin filament is required for nucleation of new branches, what is the source of the initial filament? We show that a novel family of Arp2/3 complex NPFs, WISH/DIP/SPIN90 (WDS) proteins, allow Arp2/3 complex to bypass the requirement for a preformed actin filament. The Schizosaccharomyces pombe WDS protein, Dip1, binds Arp2/3 complex and co-opts features of branching nucleation to create linear actin filaments that are suitable substrates for WASp-mediated Arp2/3 complex branching nucleation. In vitro, linear filament nucleation by Dip1-bound Arp2/3 complex dominates over WASp-mediated branching activity. This results in formation of disconnected arrays of actin filaments unlike the highly branched actin architectures observed in vivo. Importantly, unlike WASp, Dip1 remains bound to the end of polymerizing actin filaments, establishing Dip1 as a single-turnover activator of Arp2/3 complex. This mechanistic difference limits the activity of Dip1 relative to WASp, providing a way for cells to ensure that most actin filaments nucleated by Arp2/3 complex are branched, rather than linear, in endocytic actin networks and at other sites where both NPFs are present. Surprisingly, we find that at endocytic sites, WASp plays a role in initiation of actin networks and not just the propagation of networks through branching. In the presence of Dip1, WASp coordinately activates Arp2/3 complex to promote linear filament nucleation. This is accomplished, in part, through the delivery of actin monomers to Dip1-activated Arp2/3 complex. The biochemical properties of Dip1 are conserved in other WDS family proteins suggesting that they are responsible for the initiation of branched actin network assembly in higher eukaryotes. This dissertation contains previously published and unpublished co-authored material.