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The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100-184 or 100-200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.
BACKGROUND The aim of this study was to analyze the prognostic value of ARPC5 in patients with multiple myeloma (MM). MATERIAL AND METHODS MM gene expression studies GSE6477, GSE31162, GSE24080, and GSE19784 were obtained and analyzed. The expression of ARPC5 was assessed in normal plasma cells, baseline MM cells, and relapsed MM cells. Univariate and multivariable analyses were used to determine the relationship between ARPC5 expression and clinical characteristics and survivals of MM patients. Quantitative PCR was used to detect the expression ARPC5 in bone marrow mononuclear cells of MM patients and normal controls. GSEA was conducted to identify associated mechanisms. RESULTS ARPC5 expression was significantly increased in baseline MM cells compared to normal plasma cells (P=0.0414). Meanwhile, ARPC5 was significantly increased in relapsed MM cells compared to baseline MM cells (P<0.0001). ARPC5 expression was significantly associated with β2-microglobin (P=0.047), serum lactate dehydrogenase (P=0.007), and rates of aspirate plasma cells (P=0.007). Meanwhile, patients in the ARPC5 high expression group were associated with poor overall survival (P=0.0027) and event-free survival (P=0.0102) compared to those in the ARPC5 low expression group. Multivariable analysis indicated that ARPC5 was an independent prognostic factor for MM patients. Quantitative PCR demonstrated that ARPC5 was significantly increased in MM patients. GSEA results indicated that ARPC5 might affect cellular growth of myeloma cells through mammalian target of rapamycin (mTOR)C1 signaling pathway. CONCLUSIONS ARPC5 could be treated as an independent biomarker for patients with MM.
T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.
Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus.
Actin-related protein 2/3 complex subunit 5 (ARPC5) is one of the members of actin-related protein 2/3 complex and plays an important role in cell migration and invasion. However, little is known about the expression pattern, prognosis value, and biological function of ARPC5 in pan-cancer. Thus, we focus on ARPC5 as cut point to explore a novel prognostic and immunological biomarker for cancers.
Actin-related protein 2/3 complex subunit 4 (ARPC4) acts as an actin nucleator in actin cytoskeleton branching and contributes to cell migration. ARPC4 has previously been demonstrated to be abnormally expressed in various colorectal carcinoma cell lines, particularly SW620 cells. The present study explored the biological action and the possible mechanisms underlying the function of ARPC4 in the progression of carcinoma. The proliferation and migration of SW620 cells transfected with ARPC4-specific short interfering (si)RNAs were assessed using western blot, cell counting, flow cytometry and transwell assays. SW620 cells exhibited the highest ARPC4 expression of the cell lines investigated, and siRNA538 was the most effective of the siRNAs considered. The results of the present study demonstrated that ARPC4-silencing exhibited a significant effect on the capacity of cells for migration, but did not affect their proliferative ability. ARPC4-silencing inhibited human SW620 cell migration, but not proliferation, in vitro, suggesting that ARPC4 may be a putative therapeutic target for colorectal carcinoma.
A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 µM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.
Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.
Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis.
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.
The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical "or gate" capable integrating diverse upstream signals.
Gravitropism is vital for shaping directional plant growth in response to the forces of gravity. Signals perceived in the gravity-sensing cells can be converted into biochemical signals and transmitted. Sedimentation of amyloplasts in the columella cells triggers asymmetric auxin redistribution in root tips, leading to downward root growth. The actin cytoskeleton is thought to play an important role in root gravitropism, although the molecular mechanism has not been resolved. DISTORTED1 (DIS1) encodes the ARP3 subunit of the Arabidopsis Actin-Related Protein 2/3 (ARP2/3) complex, and the ARP3/DIS1 mutant dis1-1 showed delayed root curvature after gravity stimulation. Microrheological analysis revealed that the high apparent viscosity within dis1-1 central columella cells is closely associated with abnormal movement trajectories of amyloplasts. Analysis using a sensitive auxin input reporter DII-VENUS showed that asymmetric auxin redistribution was reduced in the root tips of dis1-1, and the actin-disrupting drug Latrunculin B increased the asymmetric auxin redistribution. An uptake assay using the membrane-selective dye FM4-64 indicated that endocytosis was decelerated in dis1-1 root epidermal cells. Treatment and wash-out with Brefeldin A, which inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, showed that cycling of the auxin-transporter PIN-FORMED (PIN) proteins to the plasma membrane was also suppressed in dis1-1 roots. The results reveal that ARP3/DIS1 acts in root gravitropism by affecting amyloplast sedimentation and PIN-mediated polar auxin transport through regulation of PIN protein trafficking.
The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.
Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.
Methamphetamine (METH) is a drug of abuse with neurotoxic and neuroinflammatory effects, which include disruption of the blood-brain barrier (BBB) and alterations of tight junction protein expression. This study focused on the actin cytoskeletal rearrangement as a modulator of METH-induced redistribution of tight junction protein occludin in brain endothelial cells. Exposure to METH resulted in a shift of occludin localization from plasma membranes to endosomes. These changes were accompanied by activation of the actin-related protein 2/3 (Arp2/3) complex, which stimulates actin polymerization by promoting actin nucleation. In addition, METH-induced coronin-1b phosphorylation diminishes the inhibitory effect of nonphosphorylated coronin-1b on actin nucleation. Blocking actin nucleation with CK-666, a specific inhibitor of the Arp2/3 complex, protected against METH-induced occludin internalization and increased transendothelial monocyte migration. Importantly, treatment with CK-666 attenuated a decrease in occludin levels in brain microvessels and BBB permeability of METH-injected mice. These findings indicate that actin cytoskeletal dynamics is detrimental to METH-induced BBB dysfunction by increasing internalization of occludin.
WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).
Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6-9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.
Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of ∼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.
Oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury including cytoskeleton reorganization, which is closely related to actin-related protein 2/3 (Arp2/3) complex. The aim of this study was to investigate the role of Arp2/3 complex in ox-LDL-induced endothelial dysfunction. In this study, we found that Arp2 and Arp3 expression was increased under atherosclerotic conditions both in ApoE-/- mice and in ox-LDL-stimulated human coronary artery endothelial cells (HCAECs). Arp2/3 complex inhibitor CK666 significantly reduced ox-LDL-induced ROS generation and cytoskeleton reorganization, and increased NO release in HCAECs. Pretreatment with LOX-1- but not CD36-blocking antibody markedly decreased ox-LDL-induced Arp2 and Arp3 expression. Moreover, Rac-1 siRNA remarkably suppressed ox-LDL-stimulated Arp2 and Arp3 expression. Additionally, CK666 reduced endothelial nitric oxide synthase (eNOS) expression and atherosclerotic lesions in ApoE-/- mice. Collectively, ox-LDL induces endothelial dysfunction by activating LOX-1/Rac-1 signaling and upregulating Arp2/3 complex expression.
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