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.
The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.
more » « less- NSF-PAR ID:
- 10090578
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. 201901176
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex. This complex generates branched filaments, thereby changing the overall organization of the network. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying the branching-induced reorganization caused by Arp2/3 were studied using a computational model (mechanochemical dynamics of active networks [MEDYAN]); this model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, “avalanches,” that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically linked filaments displayed by branched networks. These far-from-equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the “cytoquakes” recently seen in experiments.
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Abstract Actin networks are highly dynamic cytoskeletal structures that continuously undergo structural remodeling. One prominent way to probe these processes is via Fluorescence Recovery After Photobleaching (FRAP), which can be used to estimate the rate of turnover for filamentous actin monomers. It is thought that head‐to‐tail treadmilling and de novo filament nucleation constitute two primary mechanisms underlying turnover kinetics. More generally, these self‐assembly activities are responsible for many important cellular functions such as force generation, cellular shape dynamics, and cellular motility. In what relative proportions filament treadmilling and de novo filament nucleation contribute to actin network turnover is still not fully understood. We used an advanced stochastic reaction–diffusion model in three dimensions, MEDYAN, to study turnover dynamics of actin networks containing Arp2/3, formin and capping protein at experimentally meaningful length‐ and time‐scales. Our results reveal that, most commonly, treadmilling of older filaments is the main contributor to actin network turnover. On the other hand, although turnover and treadmilling are often used interchangeably, we show clear instances where this assumption would not be justified, for example, finding that rapid turnover is accompanied by slow treadmilling in highly dendritic Arp2/3 networks.
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