Force and phosphate release from Arp2/3 complex promote dissociation of actin filament branches

Abstract

<jats:p>Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to &lt;1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name “young/strong” and “old/weak.” The “young/strong” state 1 has adenosine 5′-diphosphate (ADP)−P<jats:sub><jats:italic>i</jats:italic></jats:sub>bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the “old/weak” state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP−Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP−P<jats:sub><jats:italic>i</jats:italic></jats:sub>−Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling “old” actin filament branches in cells.</jats:p>

Myosin 1b flattens and prunes branched actin filaments

“…Bieling and colleagues ( Bieling et al, 2016 ) suggested that compressive forces on a branched actin gel alter the internal architecture of the network, increasing its density. Recent work using microfluidics ( Pandit et al, 2020 ) confirms that Arp2/3-dependent branches can break when forces in the pN range are applied. In our experiments on stabilized and dynamic F-actin, the mother filament and the branches are propelled by myosins in divergent directions owing to the geometry of the Arp2/3-generated branch, which induces friction forces on the filaments ( Fig.…”

The cell pushes back: The Arp2/3 complex is a key orchestrator of cellular responses to environmental forces

“…Not only the formation, but also dissociation of actin filament branches can depend on Arp2/3 activity. Two distinct mechanical states of the Arp2/3 complex have been described with different stability to force, with a released phosphate state to favour debranching upon force [ 19 ]. In addition to allowing the cell to respond with variable force in lamellipodia protrusions, these features give branched actin networks a versatility allowing unique roles in cells and tissues.…”

E-cadherin is a structuring component of invadopodia in pancreatic cancer

“…Arp2/3 complex is an heptameric protein complex that polymerizes actin filaments as branches from existing filaments generating a highly branched actin filaments network. Arp2/3 complex powers various cell processes including cell motility, endocytosis, vesicle trafficking and adherens junction stability (45, 46). Its impact on actin polymerisation is critical for invadopodia-based invasion program by driving invasive cell protrusions through the matrix and maintaining tight apposition of surface-exposed MT1-MMP with ECM (47).…”