The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration

Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 04/2012; 197(2):239-51. DOI: 10.1083/jcb.201112113
Source: PubMed


The Arp2/3 complex nucleates the formation of the dendritic actin network at the leading edge of motile cells, but it is still unclear if the Arp2/3 complex plays a critical role in lamellipodia protrusion and cell motility. Here, we differentiated motile fibroblast cells from isogenic mouse embryonic stem cells with or without disruption of the ARPC3 gene, which encodes the p21 subunit of the Arp2/3 complex. ARPC3(-/-) fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions, with formin proteins (mDia1 and mDia2) concentrated near their tips. The speed of cell migration, as well as the rates of leading edge protrusion and retraction, were comparable between genotypes; however, ARPC3(-/-) cells exhibited a strong defect in persistent directional migration. This deficiency correlated with a lack of coordination of the protrusive activities at the leading edge of ARPC3(-/-) fibroblasts. These results provide insights into the Arp2/3 complex's critical role in lamellipodia extension and directional fibroblast migration.

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    • "As filamentous actin is an essential co-factor in the Arp2/3-complex activation mechanism, actin polymerization by the Arp2/3 complex is an auto-catalytic reaction using newly assembled branched actin filaments as template for further rounds of branching (Beli et al., 2008; Goley and Welch, 2006; Innocenti et al., 2004; Le Clainche and Carlier, 2008; Pollard, 2007; Pollard and Borisy, 2003; Rotty et al., 2013; Suraneni et al., 2012; Yamazaki et al., 2003; Yan et al., 2003). Although these seminal studies have illuminated the mechanism that sustains expansion of lamellipodia and ruffles, the initiation thereof has remained so far enigmatic because origin and availability of the pre-existing actin filaments mediating initial activation of the Arp2/3 complex were mysterious. "
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    ABSTRACT: Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by Arp2/3 complex. Although regulation of Arp2/3-complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an EGF-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments activating the Arp2/3 complex and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium/ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.
    Full-text · Article · Sep 2015 · Journal of Cell Science
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    • "The only machinery found to generate such branched actin networks is the Arp2/3 complex [Pollard, 2007]. It is well established that the Arp2/3 complex is required to form lamellipodia [Steffen et al., 2006; Nicholson-Dykstra and Higgs, 2008; Suraneni et al., 2012; Wu et al., 2012]. The Arp2/3 complex is activated by the WAVE complex at the lamellipodial edge [Innocenti et al., 2004; Steffen et al. 2004; Derivery and Gautreau, 2010]. "
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    ABSTRACT: Branched actin networks generated by the Arp2/3 complex provide the driving force for leading edge protrusion in migrating cells. We recently identified Arpin, a protein that inhibits the Arp2/3 complex in lamellipodia. Arpin is activated by the small GTPase Rac, which triggers lamellipodium formation, and thus Arpin renders protrusions unstable. A conserved role of Arpin is to induce migrating cells to turn in different migration models. Here we investigated the mechanism by which Arpin controls directional persistence. For this analysis, we segmented migration trajectories into alternating phases of active migration and pauses, based on a speed threshold. Regardless of the threshold value, Arpin induced more frequent pausing, during which the cell was more likely to change the direction of its migration. Arpin simultaneously acts on cell speed and directional persistence, which are strongly coupled parameters. Induction of frequent pausing by Arpin is consistent with Arpin circuitry: by inhibiting the Arp2/3 complex as a response to Rac activation, Arpin antagonizes a positive feedback loop that sustains protrusions at the leading edge and maintains active migration. We propose the 'duration of active migration' as a useful proxy to measure feedbacks associated with cell migration. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · Aug 2015 · Cytoskeleton
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    • "However, the development of computational algorithms to analyze the vast amounts of data produced is lagging behind (Myers, 2012). The application of automated, unbiased , computational methods for morphodynamic quantification is rare, with the use of kymographs, for example, still popular (Suraneni et al., 2012; Ura et al., 2012; Wiggan et al., 2012; Dang et al., 2013). Such analyses are time consuming, subject to individual bias, and extract relatively low levels of information . "
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    ABSTRACT: Cell migration is frequently accompanied by changes in cell morphology (morphodynamics) on a range of spatial and temporal scales. Despite recent advances in imaging techniques, the application of unbiased computational image analysis methods for morphodynamic quantification is rare. For example, manual analysis using kymographs is still commonplace, often caused by lack of access to user-friendly, automated tools. We now describe software designed for the automated quantification of cell migration and morphodynamics. Implemented as a plug-in for the open-source platform, ImageJ, ADAPT is capable of rapid, automated analysis of migration and membrane protrusions, together with associated fluorescently labeled proteins, across multiple cells. We demonstrate the ability of the software by quantifying variations in cell population migration rates on different extracellular matrices. We also show that ADAPT can detect and morphologically profile filopodia. Finally, we have used ADAPT to compile an unbiased description of a "typical" bleb formed at the plasma membrane and quantify the effect of Arp2/3 complex inhibition on bleb retraction. © 2015 Barry et al.
    Full-text · Article · Apr 2015 · The Journal of Cell Biology
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