Nano-scale actin-network characterization of fibroblast cells lacking functional Arp2/3 complex (original) (raw)
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bioRxiv (Cold Spring Harbor Laboratory), 2023
Cells migrate by adapting their leading-edge behaviours to heterogeneous extracellular microenvironments (ECMs) during cancer invasions and immune responses. Yet it remains poorly understood how such complicated dynamic behaviours emerge from millisecond-scale assembling activities of protein molecules, which are hard to probe experimentally. To address this gap, we established a spatiotemporal "resistance-adaptive propulsion" theory based on the protein interactions between Arp2/3 complexes and polymerizing actin filaments, and a multiscale dynamic modelling system spanning from molecular proteins to the cell. Combining spatiotemporal simulations with experiments, we quantitatively find that cells can accurately self-adapt propulsive forces to overcome heterogeneous ECMs via a resistance-triggered positive feedback mechanism, dominated by polymerization-induced actin filament bending and the bending-regulated actin-Arp2/3 binding. However, for high resistance regions, resistance triggered a negative feedback, hindering branched filament assembly, which adapts cellular morphologies to circumnavigate the obstacles. Strikingly, the synergy of the two opposite feedbacks not only empowers cells with both powerful and flexible migratory capabilities to deal with complex ECMs, but also endows cells to use their intracellular proteins efficiently. In addition, we identify that the nature of cell migration velocity depending on ECM history stems from the inherent temporal hysteresis of cytoskeleton remodelling. We also quantitatively show that directional cell migration is dictated by the competition between the local stiffness of ECMs and the local polymerizing rate of actin network caused by chemotactic cues. Our results reveal that it is the polymerization force-regulated actin filament-Arp2/3 complex binding interaction that dominates self-adaptive cell migrations in complex ECMs, and we provide a predictive theory and a spatiotemporal multiscale modelling system at the protein level. .
PLoS Biology, 2005
Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.
Molecular Biology of the Cell, 2013
Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.
Control of Actin Filament Treadmilling in Cell Motility
Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues. 449 Annu. Rev. Biophys. 2010.39:449-470. Downloaded from arjournals.annualreviews.org by 89.133.73.22 on 05/25/10. For personal use only.
Proceedings of The National Academy of Sciences, 1998
The Arp2͞3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2͞3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2͞3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 M. The high affinity of Arp2͞3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2͞3 complex. Arp2͞3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2͞3 bound to sides and pointed ends of actin filaments and examples of Arp2͞3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ؎ 7°. From these in vitro biochemical properties, we propose a model for how Arp2͞3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells. 1 mM MgCl 2 , and 50 mM KCl.
Journal of Biological Chemistry, 2012
Background: The Arp2/3 complex preferentially forms branches from newly polymerized actin filaments, but the mechanism is unknown. Results: Caldesmon bound to polymerizing actin preserves this preferred binding and branching activity of Arp2/3. Conclusion: By stabilizing filaments in their nascent state, caldesmon potentiates actin nucleation and branching by the Arp2/3 complex. Significance: This work describes a novel mechanism regulating actin dynamics. Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.
Actin dynamics in cell migration
Essays In Biochemistry
Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model – also inspired by previous literature – in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascin...
Proceedings of the National Academy of Sciences, 2005
RNA interference silencing of up to 90% of Arp3 protein expression, a major subunit of the Arp2͞3 complex, proportionately decreases the intracellular motility of Listeria monocytogenes and actin nucleation activity ascribable to the Arp2͞3 complex in mouse embryonic fibroblasts. However, the Arp2͞3-deficient cells exhibit unimpaired lamellipodial actin network structure, translational locomotion, spreading, actin assembly, and ruffling responses. In addition, Arp3-silenced cells expressing neural Wiskott-Aldrich syndrome protein-derived peptides that inhibit Arp2͞3 complex function in wild-type cells retained normal PDGF-induced ruffling. The Arp2͞3 complex can be dispensable for leading-edge actin remodeling.