Formation and turnover of branched actin networks underlies cell migration and other essential force-driven processes. Type I nucleation-promoting factors (NPFs) such as WASP recruit actin monomers to Arp2/3 complex to stimulate nucleation. In contrast, mechanisms of type II NPFs such as Abp1 (also known as HIP55 and Drebrin-like protein) are less well understood. Here, we use single-molecule analysis to investigate yeast Abp1 effects on Arp2/3 complex, and find that Abp1 strongly enhances Arp2/3-dependent branch nucleation by stabilizing Arp2/3 on sides of mother filaments. Abp1 binds dynamically to filament sides, with sub-second lifetimes, yet associates stably with branch junctions. Further, we uncover a role for Abp1 in protecting filament junctions from GMF-induced debranching by competing with GMF for Arp2/3 binding. These data, combined with EM structures of Abp1 dimers bound to Arp2/3 complex in two different conformations, expand our mechanistic understanding of type II NPFs.
Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch–junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin–actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament–Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.
more » « less- Award ID(s):
- 2011846
- NSF-PAR ID:
- 10506793
- Publisher / Repository:
- National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 29
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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