Self-organization of the actin cytoskeleton /

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Bibliographic Details
Author / Creator:Winkelman, Jonathan David, author.
Ann Arbor : ProQuest Dissertations & Theses, 2015
Description:1 electronic resource (165 pages)
Format: E-Resource Dissertations
Local Note:School code: 0330
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Other authors / contributors:University of Chicago. degree granting institution.
Notes:Advisors: David R. Kovar Committee members: Richard Fehon; Margaret Gardel; Michael Glotzer; Edwin Munro.
Dissertation Abstracts International, Volume: 77-05(E), Section: B.
Summary:Actin self-organizes into many distinct polymer networks within a common cytoplasm. These filament networks drive a number of processes that are essential for life, such as cell division, motility, the establishment and maintenance of cell polarity and endocytosis. Polymer networks have dynamic and structural characteristics that tailor them to carry out their specific functions. Therefore, it is critical that actin networks assemble at the correct time and place and with the appropriate properties.
Actin networks acquire their properties due to the interaction of actin with a milieu of actin binding proteins. In vitro, purified actin will assemble into simple filaments. However, cells contain hundreds of different types of actin binding proteins each with unique biochemical properties that work in concert to regulate actin network dynamics and architecture. The interaction of actin binding proteins with actin as well as each other organizes actin filaments into networks that display the appropriate properties.
ABPs fall into several broad functional categories. Some actin binding proteins, such as Ena/VASPs, formins and capping proteins, bind to the barbed end to control the rate at which the filament polymerizes. Many actin binding proteins bind to the sides of filaments to bundle, regulate filament stability, or even nucleate new filaments. Other side-binders, such as the myosin family, are active ATPase motors that function to transport cargo along actin tracks or slide antiparallel filaments past each other to generate contractile forces. However, the precise function of many actin binding proteins and how they interact with actin and each other to affect network organization is still poorly understood.
Actin networks are associated with specific subsets of actin binding proteins, which give each network its unique functional identity. Determining how actin binding proteins become associated with and endow these networks with their properties is a major open question. In this work, TIRF microscopy and other biochemical assay were used to observe purified fluorescently-labeled proteins interacting with each other and the actin network at single molecule resolution and on millisecond timescales. We observed how proteins could segregate from an initially homogeneous state, in turn directing sorting of other actin binding proteins. We also unraveled the activity of different actin binding proteins and observed how they can modulate each other's activity to tune the organization of actin networks. Specifically, the Ena/VASP family member Enabled (Ena) processively tracks the growing ends of actin filaments. Ena's activity is enhanced by an interaction with the actin bundling protein fascin. The result is that Ena drives polymerization of filament ends that are within the bundle more so than ends that have escaped, resulting in a coherent organization of bundles. We discovered that fascin and ?-actinin contain intrinsic properties that drive their segregation to different networks, Other actin binding proteins that are found in the same networks as fascin in cell, specifically recognize fascin-bundled actin in vitro. The filament spacing within these bundles appears to be a key architectural feature that drives sorting of actin binding proteins in this system. Finally, Ena and the formin Diaphanous (Dia) were discovered to colocalize to the same structures through a direct interaction, and Ena negatively regulated Dia's activity both in vitro and in vivo as direct consequence of this interaction.