The role of Arabidopsis Actin-Related Protein 3 in amyloplast sedimentation and polar auxin transport in root gravitropism.

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.

Mis-localization of Arp2 mRNA impairs persistence of directional cell migration.

Arp2/3 complex is an actin polymerization nucleator and localized in the leading protrusions of migrating cells. It has been unclear how this complex is targeted to the protrusions and whether its localization is functionally important. We previously demonstrated that mRNAs encoding for the subunits of the complex were localized in the protrusions of fibroblasts, suggesting a mechanism to target the complex to the protrusions. We here present data demonstrating the importance of Arp2/3 complex mRNA localization in directional cell migration. Using a novel mechanism by which Dia1 mRNA is targeted to the perinuclear endoplasmic reticulum, we redirected the mRNA encoding Arp2, a subunit of the Arp2/3 complex, to the perinuclear region in fibroblasts. Knockdown of Arp2 alone caused dramatic reduction of the complex and resulted in narrow protrusions, increased random cell migration speed and loss of directionality. Rescue with a protrusion-localizing Arp2 mRNA restored normal cell migration behavior, whereas rescue with a mis-localizing Arp2 mRNA failed to restore speed and directionality. These results demonstrate that localization of Arp2/3 complex mRNAs in the leading protrusions is functionally important for directional cell migration.

The nuclear actin-related protein Act3p/Arp4 influences yeast cell shape and bulk chromatin organization.

ACT3/ARP4 is an essential gene, coding for the actin-related protein Act3p/Arp4 of Saccharomyces cerevisiae located within the nucleus. Act3p/Arp4 is a stoichiometric component of the NuA4, INO80, and SWR1 chromatin modulating complexes, and recruits these complexes onto chromatin for their proper chromatin functions. Mutated Act3p/Arp4 leads to impairment of the functions of these complexes and affects transcription of specific genes. Our results revealed significant disorder in the cell size and shape of act3/arp4 mutant cells, when grown at permissive temperature. act3/arp4 mutants have also demonstrated an increase in their nuclear diameters, thus suggesting that Act3p/Arp4 is a key regulator in the maintenance of cellular shape and nuclear organization. Furthermore, the use of Chromatin Yeast Comet Assay (ChYCA) for assessment of single-cell bulk chromatin organization in act3/arp4 mutant cells allowed us to detect an elevated sensitivity toward nuclease action, denoting differences in higher-order chromatin structure of the mutants.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

Arp3 is required during preimplantation development of the mouse embryo.

The role of Arp3 in mouse development was investigated utilizing a gene trap mutation in the Arp3 gene. Heterozygous Arp3(WT/GT) mice are normal, however, homozygous Arp3(GT/GT) embryos die at blastocyst stage. Earlier embryonic stages appear unaffected by the mutation, probably due to maternal Arp3 protein. Mutant blastocysts isolated at E3.5 fail to continue development in vitro, lack outgrowth of trophoblast-like cells in culture and express reduced levels of the trophoblast marker Cdx2, while markers for inner cell mass continue to be present. The recessive embryonic lethal phenotype indicates that Arp3 plays a vital role for early mouse development, possibly when trophoblast cells become critical for implantation.

The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia.

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.

Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis.

During polarized growth and tissue morphogenesis, cells must reorganize their cytoplasm and change shape in response to growth signals. Dynamic polymerization of actin filaments is one cellular component of polarized growth, and the actin-related protein 2/3 (ARP2/3) complex is an important actin filament nucleator in plants. ARP2/3 alone is inactive, and the Arabidopsis thaliana WAVE complex translates Rho-family small GTPase signals into an ARP2/3 activation response. The SCAR subunit of the WAVE complex is the primary activator of ARP2/3, and plant and vertebrate SCARs are encoded by a small gene family. However, it is unclear if SCAR isoforms function interchangeably or if they have unique properties that customize WAVE complex functions. We used the Arabidopsis distorted group mutants and an integrated analysis of SCAR gene and protein functions to address this question directly. Genetic results indicate that each of the four SCARs functions in the context of the WAVE-ARP2/3 pathway and together they define the lone mechanism for ARP2/3 activation. Genetic interactions among the scar mutants and transgene complementation studies show that the activators function interchangeably to meet the threshold for ARP2/3 activation in the cell. Interestingly, double, triple, and quadruple mutant analyses indicate that individual SCAR genes vary in their relative importance depending on the cell type, tissue, or organ that is analyzed. Differences among SCARs in mRNA levels and the biochemical efficiency of ARP2/3 activation may explain the functional contributions of individual genes.

Helicobacter pylori CagA induces AGS cell elongation through a cell retraction defect that is independent of Cdc42, Rac1, and Arp2/3.

Helicobacter pylori, which infects over one-half the world's population, is a significant risk factor in a spectrum of gastric diseases, including peptic ulcers and gastric cancer. Strains of H. pylori that deliver the effector molecule CagA into host cells via a type IV secretion system are associated with more severe disease outcomes. In a tissue culture model of infection, CagA delivery results in a dramatic cellular elongation referred to as the "hummingbird" phenotype, which is characterized by long, thin cellular extensions. These actin-based cytoskeletal rearrangements are reminiscent of structures that are regulated by Rho GTPases and the Arp2/3 complex. We tested whether these signaling pathways were important in the H. pylori-induced cell elongation phenotype. Contrary to our expectations, we found that these molecules are dispensable for cell elongation. Instead, time-lapse video microscopy revealed that cells infected by cagA(+) H. pylori become elongated because they fail to release their back ends during cell locomotion. Consistent with a model in which CagA causes cell elongation by inhibiting the disassembly of adhesive cell contacts at migrating cells' lagging ends, immunohistochemical analysis revealed that focal adhesion complexes persist at the distal tips of elongated cell projections. Thus, our data implicate a set of signaling molecules in the hummingbird phenotype that are different than the molecules previously suspected.

Correlation between liver metastasis of the colocalization of actin-related protein 2 and 3 complex and WAVE2 in colorectal carcinoma.

Directed movement of normal cells occurs when actin-related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma.