Return to search

Discovery and Characterization of WISH/DIP/SPIN90 Proteins as a Class of ARP2/3 Complex Activators that Function to Seed Branched Actin Networks

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.

Identiferoai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/23137
Date10 April 2018
CreatorsWagner, Andrew
ContributorsStevens, Tom
PublisherUniversity of Oregon
Source SetsUniversity of Oregon
Languageen_US
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
RightsAll Rights Reserved.

Page generated in 0.0018 seconds