Characterization of two classes of small molecule inhibitors of Arp2/3 complex

Nolen, Brad J.; Tomašević, Nenad; Russell, Alan J.; Pierce, David W.; Jia, Zhiheng; McCormick, Chad D.; Hartman, James J.; Sakowicz, Roman; Pollard, Thomas D.

doi:10.1038/nature08231

Cited by 492 publications

(502 citation statements)

References 24 publications

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“…However, CK-666 did not inhibit actin cable assembly from GST-Bni1 FH1-COOH-coated beads in HUarrested extracts (Fig. S4 F and G) (72). Furthermore, although Bni1-derived actin filaments also form the contractile actomyosin ring in vivo (73), we did not detect the type II myosin Myo1 or the IQGAP protein Iqg1 by MS or by detection of fluorescently tagged proteins ( Fig.…”

Section: Ms Reveals the Protein Composition Of Bni1-nucleated Actin Cmentioning

confidence: 87%

Cell-cycle regulation of formin-mediated actin cable assembly

Miao

Wong

Mennella

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cellcycle regulation is conserved in vertebrates. The use of the cablereconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Cdk1 | three-dimensional structured-illumination microscopy | actin nucleation E ukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5-14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16)(17)(18)(19) or cellular extracts (20-25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified pr...

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Section: Ms Reveals the Protein Composition Of Bni1-nucleated Actin Cmentioning

confidence: 87%

Cell-cycle regulation of formin-mediated actin cable assembly

Miao

Wong

Mennella

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Small molecule inhibitors were added to inhibit formins (20 μM SMIFH2) and Arp2/3 (100 μM CK666) activity. These concentrations have been shown previously to disrupt formin and Arp2/3 activities in MDCK cells without inducing cytotoxic or off-target effects (14,(43)(44)(45). Importantly, the effects of these inhibitor concentrations are reversible so that recovery of normal function can be monitored.…”

Section: Resultsmentioning

confidence: 99%

Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of hightraction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42-and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cellcell adhesion.C ell adhesion is essential for tissue structure and function. Cells use specialized types of adhesions to interact with the surrounding environment, including integrin-based focal adhesions at cell-extracellular matrix (cell-ECM) contacts and cadherin-based adhesions at cell-cell contacts (1). Integrins bind to the ECM and intracellular proteins that link to the actin cytoskeleton and important signaling pathways (2). Similarly, cadherins regulate cellcell recognition and adhesion (3) and, through cytoplasmic adaptor proteins (catenins, vinculin) (4, 5), also link to the actin cytoskeleton and other proteins with signaling and scaffolding functions (6).Initiation of cell-cell adhesion requires significant reorganization of the actin cytoskeleton and is tightly controlled by the activities of actin nucleating proteins and Rho GTPases. Adhesion is initiated when filopodia from opposing cells come into contact with one another (7,8), and this process is regulated by Cdc42 activity (9, 10) and formin-dependent actin polymerization (11)(12)(13)(14). Intermediate stages of cell-cell contact formation involve lateral expansion of the contact by Rac1-induced and Arp2/3-dependent lamellipodial activity (15, 16). Finally, com...

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“…CK666 appears to block movement of the Arp2/3 complex into its active structure (Nolen et al 2009). Following treatment with 500 μM CK666 at the 1-cell stage, most embryos failed to develop to the 8-cell stage.…”

Section: Inhibition Of the Arp2/3 Complex Impairs Early Porcine Embrymentioning

confidence: 99%

“…In the current study, we inhibited Arp2/3 activity using CK666, a small molecule Arp2/3-specific inhibitor (Nolen et al 2009). Inhibition of the Arp2/3 complex by CK666 involved downregulation of selected transcription factors, including Pou5f1, Sox2, and Nanog at the 4-cell stage, and prevented blastocyst formation.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the Arp2/3 complex impairs early embryo development of porcine parthenotes

Lin

et al. 2016

Animal Cells and Systems

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The Arp2/3 complex, which nucleates actin filaments, comprises a stable assembly of seven-protein subunits including two actin-related proteins (Arp2 and Arp3). Previous work showed that Arp2/3 binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. In the present study, we show that the Arp2/3 complex is critical for cytokinesis during early embryonic development in porcine parthenotes. The Arp2/3 complex is concentrated at the cortex of each cell at the 1-, 2-, and 4-cell stages, and at the periphery at the morula stage. The amount of Arp2/3 significantly decreased at the blastocyst stage in parthenogenetically activated porcine embryos. Inhibition of the Arp2/3 complex in the pig embryos by the Arp2/3-specific inhibitor CK666 resulted in abnormal cell division, a decrease in developmental rate and total cell numbers, and an increase in the ratio of trophectoderm cell number to inner cell mass number in blastocyst-stage embryos. In addition, 4-cell stage embryos subjected to CK666 treatment exhibited significantly decreased expression of ZGA genes (Pou5f1, Sox2, and Nanog), suggesting that the Arp2/3 complex plays an important role in early porcine embryo development. Thus, our data demonstrate that the Arp2/3 complex is required for early embryonic development in pigs and appears to regulate the expression of pluripotency genes. ARTICLE HISTORY

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Characterization of two classes of small molecule inhibitors of Arp2/3 complex

Cited by 492 publications

References 24 publications

Cell-cycle regulation of formin-mediated actin cable assembly

Cell-cycle regulation of formin-mediated actin cable assembly

Changes in E-cadherin rigidity sensing regulate cell adhesion

Inhibition of the Arp2/3 complex impairs early embryo development of porcine parthenotes

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