MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes.
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ABSTRACT:
SUBMITTER: Galloni C
PROVIDER: S-EPMC8193582 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.
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Project description:We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.
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Project description:Arp2/3 complex is an actin filament nucleation and branching machinery conserved in all eukaryotes from yeast to human. Arp2/3 complex branched networks generate pushing forces that drive cellular processes ranging from membrane remodeling to cell and organelle motility. Several molecules regulate these processes by directly inhibiting or activating Arp2/3 complex and by stabilizing or disassembling branched networks. Here, we review recent advances in our understanding of Arp2/3 complex regulation, including high-resolution cryoelectron microscopy (cryo-EM) structures that illuminate the mechanisms of Arp2/3 complex activation and branch formation, and novel cellular pathways of branch formation, stabilization, and debranching. We also identify major gaps in our understanding of Arp2/3 complex inhibition and branch stabilization and disassembly.
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Project description:Small GTPases regulate cytoskeletal dynamics, cell motility, and division under precise spatiotemporal control. Different small GTPases exhibit cross talks to exert feedback response or to act in concert during signal transduction. However, whether and how specific cytoskeletal components' feedback to upstream signaling factors remains largely elusive. Here, we report an intriguing finding that disruption of the Arp2/3-branched actin specifically reduces RhoA activity but upregulates its total protein abundance. We further dissect the mechanisms underlying these circumstances and identify the altered cortactin/p190RhoGAP interaction and weakened CCM2/Smurf1 binding to be involved in GTP-RhoA reduction and total RhoA increase, respectively. Moreover, we find that cytokinesis defects induced by Arp2/3 inhibition can be rescued by activating RhoA. Our study reveals an intricate feedback from the actin cytoskeleton to the small GTPase. Our work highlights the role of Arp2/3-branched actin in signal transduction aside from its function in serving as critical cytoskeletal components to maintain cell morphology and motility.
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Project description:Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.
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