Actin-related Protein Complex Research Articles

Enhanced Migration of Fuchs Corneal Endothelial Cells by Rho Kinase Inhibition: A Novel Ex Vivo Descemet's Stripping Only Model.

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation

AbstractPlatelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ–dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

The Impact of Arp2/3 Complex Inhibition on Cytoskeleton Dynamics and Mitochondrial Function during Goat Oocyte Meiosis.

F-actin is of critical importance in oocyte meiotic maturation. Actin assembly and its dynamics are mainly regulated by actin nucleation factors. The actin-related protein complex 2/3 (Arp2/3) is responsible for the organization of F-actin filaments. However, the role of Arp2/3 complex in goat oocytes has not been fully elucidated. Our findings demonstrate that Arp2/3 complex activity is necessary for the maturation of goat oocytes. The Arp2/3 complex-specific inhibitor CK666 impairs the maturation of goat oocytes and alters the genes associated with cumulus expansion, both of which suggest that normal meiosis is affected. Arp2, one of the subunits of the Arp2/3 complex, was found to be mainly accumulated at the oocyte cortex and to co-localize with F-actin during goat oocyte maturation in our results. Thus, we further investigated the cytoskeleton dynamics and found that Arp2/3 complex inhibition disrupts the F-actin assembly and spindle organization. Further analysis revealed that, in addition to direct effects on the cytoskeleton, Arp2/3 complex could also induce ROS accumulation and oxidative stress by disrupting mitochondrial distribution and function, ultimately increasing the rate of early apoptosis in goat oocytes. Our study provides evidence that the Arp2/3 complex is a key regulator of goat oocyte maturation through its regulation of the cytoskeleton dynamics and mitochondrial function.

Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses.

Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity.

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections.

Cellular and pathophysiological consequences of Arp2/3 complex inhibition: role of inhibitory proteins and pharmacological compounds.

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

Abstract P105: Gaseous Air Pollutants and DNA Methlyation in a Methylome-wide Association Study of an Ethnically and Environmentally Diverse Population of U.S. Adults

Background: Epigenetic mechanisms underlying associations between coronary artery disease (CAD) and ambient exposures to carbon monoxide (CO), nitrogen oxides (NO 2 /NOx), ozone (O 3 ), and sulfur dioxide (SO 2 ) are incompletely understood. We therefore examined associations between these gaseous air pollutants and DNA methylation (DNAm)—a process that controls gene expression. Methods: We conducted a race/ethnicity- and study-stratified, methylome-wide association study of 12 Women’s Health Initiative and Atherosclerosis Risk in Communities subpopulations (N=8567, mean age 61.3 years, 83% female, 46% African American, 8% Hispanic/Latino American). In each subpopulation, we estimated daily mean ambient pollutant concentrations at geocoded participant addresses using national-scale, lognormal ordinary kriging. We averaged the concentrations over the 2, 28, and 365 days before phlebotomy, the source of leukocyte DNA used in estimating DNAm at 485,000 Cytosine-phosphate-Guanine (CpG) sites on the Illumina Infinium HumanMethylation450 BeadChip. We estimated pollutant-DNAm associations using multi-level, linear mixed-effects models adjusted for socio-demographic, behavioral, and meteorological factors; estimated leukocyte proportions; and technical covariates. Then we combined estimates from the 12 subpopulations in inverse variance-weighted meta-analyses and characterized CpG sites associated with significant associations and little evidence of heterogeneity among subpopulations ( p <1.0x10 -7 ; p Cochran’s Q >0.05). Results: Although we found no significant or suggestive CO-, O 3 -, or SO 2 -DNAm associations, we found a significant 28-day mean NO 2 -DNAm association at cg01885635 near ZNF621 on chr. 3 ( p =5.01x10 -8 ) and suggestive 365-day mean NO 2 -DNAm associations at cg12684684 near ARPC2 on chr. 2 ( p =9.3x10 -7 ), cg19693031 near TXNIP on chr. 1 ( p =7.3x10 -7 ), and cg01885635 near ZNF621 on chr. 3 ( p =6.7x10 -7 ). We also found 2- and 365-day mean NOx-DNAm associations at cg27032760 near KCTD16 on chr. 5 ( p =4.1x10 -7 ) and cg15008743 near ZNF83 on chr. 19 ( p =2.8x10 -7 ), respectively. ZNF621 and ZNF83 encode zinc finger proteins that possess transcription factor activity. ARPC2 encodes an actin-related protein complex subunit that has been associated with mean platelet volume, a known risk factor for myocardial infarction (MI) and post-MI mortality. TXNIP encodes a thioredoxin-binding protein that protects cells from oxidative stress, is highly glucose responsive, and has been associated with triglycerides, blood pressure, diabetes, and CAD. KCTD16 encodes a potassium channel tetramerization domain-containing protein. Conclusions: Pending replication, the preliminary findings suggest that biologically plausible epigenetic mechanisms may underlie associations between nitrogen oxide exposures and CAD in multi-racial/ethnic populations.

P84: COMBINED IMMUNODEFICIENCY DUE TO DEFICIENCY OF ACTINRELATED PROTEIN COMPLEX 1B (ARPC1B)

A number of cytoskeletal defects arising from perturbations in actin polymerisation and resulting in combined primary immunodeficiency (PID) have been described. Defects include Wiskott Aldrich Syndrome (WAS), WAS Interacting Protein (WIP) and Dedicator of Cytokinesis 8 (DOCK-8) deficiencies. A newly reported cytoskeletal defect is Actin-related Protein Complex 1B (ARPC1B) deficiency. Here we report the first Australian, youngest yet diagnosed, and 5th only reported case of ARPC1B deficiency. A female infant born to Nepalese parents presented with bloody diarrhoea and metabolic acidosis at 2 months of age. A diagnosis of cow’s milk enteritis was made and she was commenced on an elemental formula with resolution of the hematochezia and improvement in the diarrhoea. Subsequently she developed an eczematous rash which progressed to an erosive purpuric dermatitis with ulceration, and later took on a psoriasiform appearance. Skin biopsy showed a leucocytoclastic vasculitis. Her progress was complicated by cytomegalovirus infection, recurrent suppurative otitis media, twice by periorbital cellulitis, persistent oral candidiasis, urinary tract infection and failure to thrive. Investigations showed defective neutrophil chemotaxis and random migration, reduced Natural Killer cell cytotoxicity, decreased naïve T-cells, variable thrombocytopenia, eosinophilia, and raised IgA and IgE. Whole exome sequencing showed her to be homozygote for an exon 2 splice site mutation in ARPC1B (c.64+2T>A) which is predicted to cause loss of gene function. Western blot analysis showed low expression of the ARPC1B protein. ARPC1B is a regulatory subunit of the actin-related protein 2/3 complex critical for F-actin polymerisation in haemopoetic cells. Deficiency of ARPC1B results in defective actin polymerisation and a combined immunodeficiency similar but distinct to WAS.

Regulation of intraocular pressure by microRNA cluster miR-143/145

Glaucoma is a major cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), which causes optic nerve damage and retinal ganglion cell death, is the primary risk factor for blindness in glaucoma patients. IOP is controlled by the balance between aqueous humor secretion from the ciliary body (CB) and its drainage through the trabecular meshwork (TM). How microRNAs (miRs) regulate IOP and glaucoma in vivo is largely unknown. Here we show that miR-143 and miR-145 expression is enriched in the smooth muscle and trabecular meshwork in the eye. Targeted deletion of miR-143/145 in mice results in significantly reduced IOP, consistent with an ~2-fold increase in outflow facilities. However, aqueous humor production in the same mice appears to be normal based on a microbeads-induced glaucoma model. Mechanistically, we found that miR-143/145 regulates actin dynamics and the contractility of TM cells, consistent with its regulation of actin-related protein complex (ARPC) subunit 2, 3, and 5, as well as myosin light chain kinase (MLCK) in these cells. Our data establish miR-143/145 as important regulators of IOP, which may have important therapeutic implications in glaucoma.

The role of viral protein Ac34 in nuclear relocation of subunits of the actin-related protein 2/3 complex.

The actin nucleator actin-related protein complex (Arp2/3) is composed of seven subunits: Arp2, Arp3, p40/ARPC1 (P40), p34/ARPC2 (P34), p21/ARPC3 (P21), p20/ARPC4 (P20), and p16/ARPC5 (P16). Arp2/3 plays crucial roles in a variety of cellular activities through regulation of actin polymerization. Autographa californica multiple nucleopolyhedrovirus (AcMNPV), one of the beststudied alphabaculoviruses, induces Arp2/3 nuclear relocation and mediates nuclear actin polymerization to assist in virus replication. We have demonstrated that Ac34, a viral late-gene product, induces translocation of the P40 subunit of Arp2/3 to the nucleus during AcMNPV infection. However, it remains unknown whether Ac34 could relocate other Arp2/3 subunits to the nucleus. In this study, the effects of the viral protein Ac34 on the distribution of these subunits were studied by an immunofluorescence assay. Arp2, P34, P21, and P20 cloned from Spodoptera frugiperda (Sf9) cells showed mainly cytoplasmic localization and were relocated to the nucleus in the presence of Ac34. In addition, Arp3 was localized in the cytoplasm in both the presence and absence of Ac34, and P16 showed whole-cell localization. In contrast to Sf9 cells, all subunits of mammalian Arp2/3 showed no nuclear relocation in the presence of Ac34. Co-immunoprecipitation analysis of the interaction between Ac34 and Arp2/3 subunits revealed that Ac34 bound to P40, P34, and P20 of Sf9 cells. However, none of the subunits of mammalian Arp2/3 interacted with Ac34, indicating that protein-protein interaction is essential for Ac34 to relocate Arp2/3 subunits to the nucleus.

Possibility of “Comet-Like” Transport of Acetylcholine Receptors in Command <i>Helix</i> Neurons in Cellular Analog of Habituation

The presence of "comet-like" radial transport of acetylcholine receptors by actin microfilaments without the participation of myosin motors in the depression of acetylcholine-induced inward chloric current (ACh-current) in command neurons of defensive behavior of the land snail, Helix lucorum, in a cellular analog of habituation was investigated. For that purpose the effects of CK548, CK-636 (inhibitors of actin-related protein complex Arp2/3, whose activation triggers rapid actin polymerization and the formation of the "comet-like" tail on the actinic filament) and wiskostatin (an N-WASP protein inhibitor, activating Arp2/3) on the depression of ACh-current were studied. The attenuation of ACh-current depression was observed upon the addition of CK548. At the same time, CK-636 and wiskostatin irreversibly strengthened the depression of this current and suppressed its spontaneous recovery. The results of CK-548 action and its mathematical modeling allow suggesting the presence of "comet-like" transport of acetylcholine receptors, initiated by Arp2/3 protein complex in receptor endo and exocytosis in command neurons of Helix lucorum in a cellular analog of habituation. Irreversible inhibition of vital metabolic processes of the neuron by wiskostatin and CK-636, which lead to the decrease in the level of ATP, could have caused irreversible effects of these blockers on current depression.

Abstract 5517: MD-164 strongly inhibited various tumor cells and also metastasis of pancreatic tumor cells via modulation of Arp2/3 complexes.

Abstract Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology in Korea Daejon 305-806, Republic of Korea The most difficult problem in the treatment of cancer patients is a metastatic recurrence. We focused to find novel anti-metastatic chemicals without cytotoxicity. In preliminary results, more than 800 commercially available pharmaceuticals and pre-clinical drugs were screened to identify inhibitors of tumor cell migration that are intimately tied to the steps of metastasis. The ARP2/3 complex which induces actin-polymerization and Y-branching is an essential element for lamellipodia, invadopodia and filopodia at the early stages of the metastasis in cells. Two classes of small molecule inhibitors of Arp2/3 were reported. However, inhibitors of ARPC1∼5 subunit have not yet been reported. Regulating the ARPC1∼5 subunits for inhibition of actin polymerization by utilizing small molecules will benefit actin based cell migration research. Furthermore, Inhibitors of specific ARPC1∼5 subunit are highly advantageous for developing anti-metastasis drug without toxicity and side effects. As a result of the screening, MD-164 was found to effectively reduce migration and invasion in various cancer cell lines by targeting an ARP2/3 complex subunit2, a component of actin related protein complex 2/3 (ARP 2/3). When AsPc-1 cells stably expressing luciferase were injected into the tail vein of the mice and the mice were treated with MD164, MD164 inhibited lung metastases about 70%. Citation Format: Byoung-Mog Kwon, Young Min Han. MD-164 strongly inhibited various tumor cells and also metastasis of pancreatic tumor cells via modulation of Arp2/3 complexes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2013-5517 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Differential expression of genes and proteins between electric organ and skeletal muscle in the mormyrid electric fishBrienomyrus brachyistius

Electric organs (EOs) have evolved independently in vertebrates six times from skeletal muscle (SM). The transcriptional changes accompanying this developmental transformation are not presently well understood. Mormyrids and gymnotiforms are two highly convergent groups of weakly electric fish that have independently evolved EOs: while much is known about development and gene expression in gymnotiforms, very little is known about development and gene expression in mormyrids. This lack of data limits prospects for comparative work. We report here on the characterization of 28 differentially expressed genes between SM and EO tissues in the mormyrid Brienomyrus brachyistius, which were identified using suppressive subtractive hybridization (SSH). Forward and reverse SSH was performed on tissue samples of EO and SM resulting in one cDNA library enriched with mRNAs expressed in EO, and a second library representing mRNAs unique to SM. Nineteen expressed sequence tags (ESTs) were identified in EO and nine were identified in SM using BLAST searching of Danio rerio sequences available in NCBI databases. We confirmed differential expression of all 28 ESTs using RT-PCR. In EO, these ESTs represent four classes of proteins: (1) ion pumps, including the α- and β-subunits of Na(+)/K(+)-ATPase, and a plasma membrane Ca(2+)-ATPase; (2) Ca(2+)-binding protein S100, several parvalbumin paralogs, calcyclin-binding protein and neurogranin; (3) sarcomeric proteins troponin I, myosin heavy chain and actin-related protein complex subunit 3 (Arcp3); and (4) the transcription factors enhancer of rudimentary homolog (ERH) and myocyte enhancer factor 2A (MEF2A). Immunohistochemistry and western blotting were used to demonstrate the translation of seven proteins (myosin heavy chain, Na(+)/K(+)-ATPase, plasma membrane Ca(2+)-ATPase, MEF2, troponin and parvalbumin) and their cellular localization in EO and SM. Our findings suggest that mormyrids express several paralogs of muscle-specific genes and the proteins they encode in EOs, unlike gymnotiforms, which may post-transcriptionally repress several sarcomeric proteins. In spite of the similarity in the physiology and function of EOs in mormyrids and gymnotiforms, this study indicates that the mechanisms of development in the two groups may be considerably different.

Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages

Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP.WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients.

SRY-box containing gene 11 (Sox11) transcription factor is required for neuron survival and neurite growth

The transcription factor Sox11 is expressed at high levels in developing sensory neurons and injured adult neurons but little is known about its transcriptional targets and function. In this study we examined the role of Sox11 using Neuro2a neuroblastoma cells and cultured mouse dorsal root ganglia (DRG) neurons. Results show Sox11 has an essential role in regulation of neuron survival and neurite outgrowth in Neuro2a cells and primary sensory neurons. Neuro2a cells increase expression of Sox11 as they differentiate in culture. Following addition of 20 μM retinoic acid (RA), a stimulus for differentiation that enhances neurite growth and differentiation, Sox11 level rises. RNAi-mediated knockdown of Sox11 in RA-differentiated Neuro2a cells caused a decrease in neurite growth and an increase in the percent of apoptotic cells. RNA expression analysis showed that Sox11 knockdown modulated the level of mRNAs encoding several genes related to cell survival and death. Further validation in the Neuro2a model showed Sox11 knockdown increased expression of the pro-apoptotic gene BNIP3 (BclII interacting protein 1 NIP3) and decreased expression of the anti-apoptotic gene TANK (TNF receptor-associated factor family member-associated NFκB activator). Cultured primary DRG neurons also express Sox11 and treatment with Sox11 small interfering RNA (siRNA) caused a significant decrease in neurite growth and branching and a decrease in mRNA encoding actin-related protein complex 3 (Arpc3), an actin organizing protein that may be involved in axon growth. The percent of apoptotic neurons also increased in cultures of DRG neurons treated with Sox11 siRNA. Similar to Neuro2a cells, a decrease in TANK gene expression occurred, suggesting at least some overlap in Sox11 transcriptional targets in Neuro2a and DRG neurons. These data are consistent with a central role for Sox11 in regulating events that promote neurite growth and neuron survival.

Insights into Allosteric Control of Vinculin Function from Its Large Scale Conformational Dynamics

Vinculin is an important constituent of both cell-cell and cell-matrix junctions, where it plays crucial roles in the regulation of cell adhesion and migration. When activated, it mediates the linkage between cadherins (cell-cell) or integrins (cell-matrix) and the actin cytoskeleton through interactions with various proteins. The activation of vinculin requires structural conversions from an autoinhibited conformation to the "open" conformations in which the occluded binding sites of its different ligands become exposed, while the structural dynamics underlying the vinculin activation remains largely unknown. Here we report the first computational study of large scale conformational dynamics of full-length vinculin. We find that the "holding" and "releasing" motions between vinculin tail and pincer-like structure formed by first three domains of vinculin are the dominant motions near the native state of vinculin, indicating that an inherent flexibility of vinculin has a large influence on its allostery. We also find a cooperative dissociation between the head and tail domains of vinculin with increasing temperature in both thermodynamic and kinetic simulations, implying that vinculin may function as an allosteric switch in response to external signals. We show that the kinetics of vinculin unfolding exhibits specific sequential patterns, suggesting that a sophisticated interplay between domains may synergistically contribute to vinculin activation. We further find that the interaction between vinculin-binding site peptide from talin and vinculin significantly destabilizes the intramolecular head-tail interactions, suggesting a direct role of talin binding in vinculin activation.

Sleeping Beauty Transposon-Based Phenotypic Analysis of Mice: Lack of Arpc3 Results in Defective Trophoblast Outgrowth

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.

Using small molecules to overcome drug resistance induced by a viral oncogene

We used small molecule screening to discover compounds and mechanisms for overcoming E6 oncogene-mediated drug resistance. Using high-throughput screening in isogenic cell lines, we identified compounds that potentiate doxorubicin's lethality in E6-expressing colon cancer cells. Such compounds included quaternary ammonium salts, protein synthesis inhibitors, 11-deoxyprostaglandins, and two additional classes of compounds-analogs of 1,3-bis(4-morpholinylmethyl)-2-imidazolidinethione (a thiourea) and acylated secondary amines that we named indoxins. Indoxins upregulated topoisomerase IIalpha, the target of doxorubicin, thereby increasing doxorubicin lethality. We developed a photolabeling strategy to identify targets of indoxin and discovered a nuclear actin-related protein complex as a candidate indoxin target.