MECHANISMS OF ARP2/3 COMPLEX INHIBITION, SYNERGISTIC ACTIVATION, AND BRANCH REGULATION

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Degree type
Doctor of Philosophy (PhD)
Graduate group
Biochemistry and Molecular Biophysics
Discipline
Biochemistry, Biophysics, and Structural Biology
Biochemistry, Biophysics, and Structural Biology
Biology
Subject
Actin
Biochemistry
Biophysics
Cancer Metastasis
Structural Biology
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Copyright date
01/01/2024
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Author
Fregoso, Fred, Erick
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Abstract

Cells utilize the actin cytoskeleton to create membrane protrusions that drive cell migration and other vital cellular processes. Comprising these protrusions are dynamic networks of branched actin filaments, nucleated by the Arp2/3 complex. Regulation of Arp2/3 complex covers activation, inhibition, and (once integrated at the branch junction) branch stability. The literature on activation by WASP-family nucleation promoting factors (NPFs) is vast, converging on the C-terminal central (C) and acidic (A) domains of NPFs binding at two distinct sites on the complex, one spanning Arp2-ArpC1 and the other on Arp3. When isolated with the native WH2 domain as WCA, this peptide activates Arp2/3 complex in polymerization assays and promotes the active state of the complex. In contrast, the amount of literature regarding of inhibition and branch regulation are insufficient to precisely address the molecular mechanisms underpinning these activities. We used cryo-electron microscopy to solve the structures of Arp2/3 complex with bound inhibitor (Arpin), branch stabilizer (Cortactin), or branch destabilizer (Coronin 7), and used an array of biochemical and biophysical experiments to test their unique properties. Arpin and Coronin 7 harbor an A domain like that of NPFs and Cortactin where here we found both also contain a helical C-domain, thus revealing novel C-terminal CA domains of Arpin and Coronin 7. Both CA domains bind to subunit Arp3 only, unlike NPFs that additionally bind Arp2-ArpC1. The C-domain binding of NPF, Arpin and Coronin 7 is all localized to the barbed end of Arp3 with the A domain loosely spanning toward subdomain 4. We show that the direct interaction between Arp2/3 complex and Arpin control migratory persistence of cells, where the underlying inhibitory mechanism is indicated to be both concentrated to a key tryptophan residue (W195) in the C-domain that stabilizes the inactive state, and direct competition with NPF-actin on Arp3. The rapid time scale of dynamic membrane protrusions contrasts greatly with that of in vitro rates suggesting additional systems to potentiate rapid nucleation and disassembly of branched filaments. Cortactin is localized to the cell cortex where it’s shown to stabilize filaments nucleated by Arp2/3 complex and NPFs. Cortactin synergizes with WASP-family NPFs in activating Arp2/3 complex in vitro, hinting at a possible mechanism for rapid nucleation of branched filaments. We demonstrate that Cortactin role in achieving this synergy involves two models: 1) the N-terminal A domain of Cortactin binds Arp2/3 complex and recruits an existing filament via its C-terminal Cortactin repeat (CRs) domain to mediate mother filament binding and 2) increasing the displacement rate of NPFs from the branch after nucleation of the daughter filament to enter another round of activation. Lastly, we found that Coronin 7 CA may inhibit Arp2/3 complex via competition with NPFs to Arp3. The work described address the gaps of knowledge that exists on the molecular mechanism of inhibition, synergistic activation, and branch regulation of Arp2/3 complex.

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Dominguez, Roberto
Date of degree
2024
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