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.
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.
more » « less- Award ID(s):
- 1800418
- NSF-PAR ID:
- 10120810
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytoskeleton
- Volume:
- 76
- Issue:
- 11-12
- ISSN:
- 1949-3584
- Page Range / eLocation ID:
- p. 562-570
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
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.
-
In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling. The actin cortex is highly polymorphic, adopting diverse forms such as the ring-like structures found in podosomes, axonal rings, and immune synapses. The biophysical principles that underlie the formation of actin rings and cortices remain unknown. Using a molecular simulation platform called MEDYAN, we discovered that varying the filament treadmilling rate and myosin concentration induces a finite size phase transition in actomyosin network structures. We found that actomyosin networks condense into clusters at low treadmilling rates or high myosin concentrations but form ring-like or cortex-like structures at high treadmilling rates and low myosin concentrations. This mechanism is supported by our corroborating experiments on live T cells, which exhibit ring-like actin networks upon activation by stimulatory antibody. Upon disruption of filament treadmilling or enhancement of myosin activity, the pre-existing actin rings are disrupted into actin clusters or collapse towards the network center respectively. Our analyses suggest that the ring-like actin structure is a preferred state of low mechanical energy, which is, importantly, only reachable at sufficiently high treadmilling rates.more » « less
-
more » « less
After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in
Arabidopsis thaliana (Arabidopsis ) andNicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in theArabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants. -
more » « less
Abstract Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of Fc
γ RIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling.
