Actin Accumulation Research Articles

P.9.12 Exploring the pathological mechanism of Actin myopathies in zebrafish

Congenital myopathies are characterized by progressive muscle weakness and low muscle tone. Mutations in ACTA1 are responsible for up to 20% of all congenital myopathies ranging in severity from long-term survival with minor muscle weakness to lethality prior to or shortly after birth. To date over 200 different ACTA1 mutations have been described. At the cellular level ACTA1 mutations result in multiple pathologies including; nemaline bodies, intranuclear rods, actin aggregates, congenital fibre type disproportion, or cores, however, the exact pathophysiology of muscle weakness remains unknown. To investigate the mechanism by which mutations results in the distinct pathologies we have generated stable transgenic zebrafish strains expressing human ACTA1 tagged with GFP. We show, that expression of ACTA1 variants in zebrafish recapitulates the histo-pathological hallmarks of their respective diseases. Using the advantages of the zebrafish model system we followed the progression of disease in vivo and identified distinct mechanisms of muscle weakness. We show that in ACTA1D286G-GFP fish, nemaline bodies initiate as punctate actin aggregations at the muscle attachment sites before elongating to form the characteristic rod shape. In addition to its inclusion in rods, ACTA1D286G-GFP is incorporated into sarcomeres, which is the likely cause of muscle weakness. By contrast the formation of actin aggregates in ACTA1D154N myopathy occurs in the cytoplasm without a clearly defined site of origin and aggregates remain punctate, never elongating to form rods. The accumulation of aggregates triggers cell death leading to muscle weakness. The generation of zebrafish models and identification of distinct mechanisms of disease provides an experimental platform for the development, testing and specific targeting of potential therapies to combat Actin myopathies.

Wounded cells drive rapid epidermal repair in the earlyDrosophilaembryo

Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures--a medial actomyosin network and an actomyosin purse string--contributes to the increased efficiency of wound repair in the early embryo.

P.9.11 Transgenic zebrafish expressing mutant skeletal muscle actin, acta1a, model human nemaline myopathy

The nemaline myopathies (NMs) are a heterogeneous group of congenital myopathies defined by moderate to severe muscle weakness and accumulation of rod-like structures (nemaline bodies) in myofibers. Heterozygous dominant mutations of the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases. Despite our current understanding of normal actin function, the mechanisms underlying muscle pathology remain unclear and there are no curative therapies available for patients. In order to elucidate the molecular defects behind ACTA1-related NM, we are generating a panel of transgenic zebrafish lines that express a series of disease-linked dominant mutations on the zebrafish acta1a transcript, the predominant actin expressed in fast myofibers. We initially tested whether three of these variants, acta1a H42Y, M134V and V165M would lead to a muscle phenotype by injecting the mutant mRNA into zebrafish embryos. The fish overexpressing mutant actins displayed myopathic phenotypes characterized by delayed hatching from the chorion and curved bodies to variable degrees that corresponded with the severity of disease in patients with these mutations. We next generated an acta1a V165M transgenic line that stably expresses mutant actin in all fast myofibers and characterized its neuromuscular phenotypes by morphological and histological analysis at different time points. The acta1a V165M fish display muscle weakness as evidenced by their thin bodies and curved tails at 2 days-post-fertilization (dpf) and reduced motility in touch-evoked escape response assay at 5 dpf. Whole-mount phalloidin staining at 2 dpf revealed actin aggregates in the affected fish muscle, while electron microscopy demonstrated Z-line thickening and severe myofibrillar disorganization. These results provide proof of concept that zebrafish models of actin mutations recapitulate the human disease and have robust myopathic phenotypes that may be amenable for high-throughput chemical screening.

Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on slingshot homolog 1-mediated cofilin hyperactivation

Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation.

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

T-cell apoptosis induced by intratumoral activated hepatic stellate cells is associated with lung metastasis in hepatocellular carcinoma

Profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro has recently been described in hepatocellular carcinoma (HCC). In the present study, we investigated the immune inhibitory activity of HSCs in vivo in an orthotopic rat HCC model with lung metastasis. Rats (n=24) were randomly sacrificed on days 7, 14, 21 and 28 (n=4 each). Lung tissues were stained with hematoxylin and eosin. Liver sections were stained for immunofluorescence analysis. T-cell apoptosis was detected using double staining for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Staining revealed marked and continuous accumulation of α-smooth muscle actin with tumor progression after orthotopic tumor implantation in rat liver. T lymphocyte numbers gradually increased following tumor progression, and subset analysis revealed an increase in the distribution of liver CD8+ and CD4+ T cells. Double staining for CD3 and TUNEL demonstrated T-cell apoptosis. Apoptotic T cells were more frequent in the HCC livers compared to the normal livers, and were spatially associated with intratumoral activated HSCs (tHSCs), suggesting a direct interaction. T-cell apoptosis was more frequently induced in the co-cultures of activated splenic T cells(aT)/tHSCs compared to aT/quiescent (q) HSCs or qT/tHSCs. tHSCs were positively correlated with T-cell apoptosis, and the percentage of T-cells undergoing apoptosis was positively correlated with the number of lung metastasis nodules. T-cell apoptosis may be promoted via an interaction with tHSCs, suggesting that tHSCs regulate T cells and contribute to lung metastasis in HCC.

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

Periostin downregulation is an early marker of inhibited neonatal murine lung alveolar septation

Extreme preterm birth exposes the saccular lung to multiple teratogens, which ultimately retard alveolar development. Specifically, therapeutic high level oxygen supplementation adversely affects the premature lungs and results in blunted alveolarization. Prolonged hyperoxic lung injury has previously been shown to upregulate the matricellular protein Periostin (Postn) and stimulate ectopic accumulation of alpha smooth muscle actin (αSMA) myofibroblasts. Therapies that promote lung septation are lacking largely due to a lack of reliable early biomarkers of injury. Thus, we determined if Postn expression correlated with the initial appearance of myofibroblasts in the saccular lung and was required for early alveolar development. Lung development in C57BL/6J mice following room-air (RA, 21%-O₂) or continuous hyperoxia (85%-O₂) from birth (P0) through postnatal day P14 was correlated with Postn and αSMA expression. Alveolarization in Postn knockout mice exposed to room-air, 60%-, and 85%-O₂ was also examined. Postn was widely expressed in distal lung septa through P2 to P4 and peak expression coincided with accumulation of saccular myofibroblasts. Initially, 85%-O₂ prematurely downregulated Postn and αSMA expression and suppressed proliferation before the first evidence of distal lung simplification at P4. By P14, chronic 85%-O₂ resulted in secondary upregulation of Postn and αSMA in blunted septa. Myofibroblast differentiation and alveolar development was unaffected in Postn null mice and acute 85%-O₂ exposure equally inhibited septal formation in Postn null and wild-type littermates. Postn expression is tightly correlated with the presence of αSMA-myofibroblasts and is a novel early biomarker of acutely inhibited alveolar septation during a crucial window of lung development.

Sleeping Giants: Emerging Roles for the Fat Cadherins in Health and Disease

The vertebrate Fat cadherins comprise a small gene family of four members, Fat1-Fat4, all closely related in structure to Drosophila ft and ft2. Over the past decade, knock-out mouse studies, genetic manipulation, and large sequencing projects has aided our understanding of the function of vertebrate Fat cadherins in tissue development and disease. The majority of studies of this family have focused on Fat1, with evidence now showing it can bind enable (ENA)/Vasodilator-stimulated phosphoprotein (VASP), β-catenin and Atrophin proteins to influence cell polarity and motility; HOMER-1 and HOMER-3 proteins to regulate actin accumulation in neuronal synapses; and scribble to influence the Hippo signaling pathway. Fat2 and Fat3 can regulate cell migration in a tissue specific manner and Fat4 appears to influence both planar cell polarity and Hippo signaling recapitulating the activity of Drosophila ft. Knowledge about the exact downstream signaling pathways activated by each family member remains in its infancy, but it is becoming clearer that they have tissue specific and redundant roles in development and may be lost or gained in cancer. In this review, we summarize the recent progress on understanding the role of the Fat cadherin family, integrating the current knowledge of molecular interactions and tissue distributions, together with the accumulating evidence of their changed expression in human disease. The latter is now beginning to promote interest in these molecules as both biomarkers and new targets for therapeutic intervention.

F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in theCaenorhabditis elegansembryo

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.

Attenuation of LDHA expression in cancer cells leads to redox-dependent alterations in cytoskeletal structure and cell migration

Aerobic glycolysis, the preferential use of glycolysis even in the presence of oxygen to meet cellular metabolic demands, is a near universal feature of cancer. This unique type of metabolism is thought to protect cancer cells from damaging reactive oxygen species (ROS) produced in the mitochondria. Using the cancer cell line MDA-MB-435 it is shown that shRNA mediated knockdown of lactate dehydrogenase A (LDHA), a key mediator of aerobic glycolysis, results in elevated mitochondrial ROS production and a concomitant decrease in cell proliferation and motility. Redox-sensitive proteins affected by oxidative stress associated with LDHA knockdown were identified by Redox 2D-PAGE and mass spectrometry. In particular, tropomyosin (Tm) isoforms Tm4, Tm5NM1 and Tm5NM5, proteins involved in cell migration and cytoskeletal dynamics, exhibited changes in disulfide bonding and co-localized with peri-nuclear actin aggregates in LDHA knockdown cells. In contrast, treatment with the thiol-based antioxidant N-acetylcysteine promoted the relocalization of Tms to cortical actin microfilaments and partially rescued the migration defects associated with attenuated LDHA expression. These results suggest that aerobic glycolysis and reduced mitochondrial ROS production create an environment conducive to cytoskeletal remodeling; key events linked to the high cell motility associated with cancer.

Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage

Actin, one of the most evolutionarily conservative proteins in eukaryotes, is distributed both in the cytoplasm and the nucleus, and its dynamics plays important roles in numerous cellular processes. Previous evidence has shown that actin interacts with p53 and this interaction increases in the process of p53 responding to DNA damage, but the physiological significance of their interaction remains elusive. Here, we show that DNA damage induces both actin polymerization and p53 accumulation. To further understand the implication of actin polymerization in p53 function, cells were treated with actin aggregation agent. We find that the protein level of p53 decrease. The change in p53 is a consequence of the polymeric actin anchoring p53 in the cytoplasm, thus impairing p53 nuclear import. Analysis of phosphorylation and ubiquitination of p53 reveals that actin polymerization promotes the p53 phosphorylation at Ser315 and reduces the stabilization of p53 by recruiting Aurora kinase A. Taken together, our results suggest that the actin polymerization serves as a negative modulator leading to the impairment of nuclear import and destabilization of p53. On the basis of our results, we propose that actin polymerization might be a factor participating in the process of orchestrating p53 function in response to DNA damage.

Optical fiber light source directs neurite growth

Guiding the growth of a neurite by directing ~800 nm laser light to the leading edge of the neurite's growing region can be accomplished by controlling the position and direction in three dimensional space of a tapered optical fiber through which the light is projected. We control the position, angle and power of the laser beam to direct the growth of actin accumulations in neurites which affects their mobility.

Cucurbitacin IIa induces caspase-3-dependent apoptosis and enhances autophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages

Cucurbitacin IIa (CuIIa), a member of cucurbitacin family, is isolated from the root of Hemsleya amabilis which has been used as an ancient remedy for bacillary dysentery and gastroenteritis. The anti-inflammatory properties of CuIIa have long been recognized but the underlying mechanism is largely unknown. In this study, we investigated the anti-inflammatory effect of CuIIa on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that CuIIa inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependent manner. Whereas CuIIa did not cause apoptosis in unstimulated RAW 264.7 cells, it did induce a significant apoptosis in LPS-stimulated cells, which was caspase-3-dependent and associated with downregulation of survivin. Furthermore, LPS induced autophagy in RAW 264.7 cells and this effect was further enhanced by CuIIa as evidenced by increased levels of LC3-II conjugates and formation of LC3 puncta. In addition, CuIIa disrupted actin cytoskeleton via inducing actin aggregation. However, neither the synthesis of tumor necrosis factor-α, nor the activation of the mitogen-activated protein kinases and NF-κB pathways in LPS-stimulated cells was suppressed by CuIIa treatment. Collectively, these results suggested that induction of apoptosis and enhancement of autophagy contributed to the anti-inflammatory activity of CuIIa against inflammation-related diseases.

Regulation of Pollen Tube Growth by Transglutaminase.

In pollen tubes, cytoskeleton proteins are involved in many aspects of pollen germination and growth, from the transport of sperm cells to the asymmetrical distribution of organelles to the deposition of cell wall material. These activities are based on the dynamics of the cytoskeleton. Changes to both actin filaments and microtubules are triggered by specific proteins, resulting in different organization levels suitable for the different functions of the cytoskeleton. Transglutaminases are enzymes ubiquitous in all plant organs and cell compartments. They catalyze the post-translational conjugation of polyamines to different protein targets, such as the cytoskeleton. Transglutaminases are suggested to have a general role in the interaction between pollen tubes and the extracellular matrix during fertilization and a specific role during the self-incompatibility response. In such processes, the activity of transglutaminases is enhanced, leading to the formation of cross-linked products (including aggregates of tubulin and actin). Consequently, transglutaminases are suggested to act as regulators of cytoskeleton dynamics. The distribution of transglutaminases in pollen tubes is affected by both membrane dynamics and the cytoskeleton. Transglutaminases are also secreted in the extracellular matrix, where they may take part in the assembly and/or strengthening of the pollen tube cell wall.

A2.23 Impaired Natural Killer Cell Function in DOCK8 Deficiency

Introduction DOCK8 mutations are responsible for a rare autosomal recessive immunodeficiency syndrome associated with severe cutaneous viral infections, elevated IgE levels, environmental allergies, autoimmunity, and malignancy. DOCK8 activates CDC42, which is important for cell signalling and actin reorganisation. Natural killer cells play a vital role in tumour surveillance and defence against virally infected cells. NK cell function relies on the accumulation of actin at the NK cell immunologic synapse formed with target cells. Although abnormalities in T and B cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is poorly understood. Objectives/Aims Given the susceptibility to severe cutaneous viral infection and malignancy, we hypothesised there was a substantive defect in NK cell function in patients with DOCK8 deficiency. Methods 10 patients with genetically confirmed DOCK8 deficiency as well as NK cell lines with stably reduced DOCK8 expression were evaluated experimentally using in vitro NK cell cytotoxicity, F-actin content, and confocal immunofluorescence microscopy assays. Results DOCK8-deficient patients and cell lines all had decreased NK cell cytotoxicity and function could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency did not affect NK cell F-actin content, but impaired F-actin accumulation at the lytic immunological synapse. Conclusions DOCK8 deficiency results in severely deficient NK cell function owing to an inability to form a mature lytic immunological synapse via focal F-actin accumulation. This defect may underlie important and previously perplexing attributes of the DOCK8 deficiency clinical syndrome including the unusual susceptibility to viral infection.

Differential interactions of Streptococcus gordonii and Staphylococcus aureus with cultured osteoblasts

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S.gordonii cells had typical features of phagocytosis, similar to S.aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S.gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S.gordonii were enhanced by expression of S.aureus fibronectin-binding protein A (FnBPA) on the S.gordonii cell surface. Lysosomal-associated membrane protein-1 phagosomal membrane marker accumulated with intracellular S.aureus and S.gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12h, unless expressing FnBPA, whereas S.aureus showed extended survival times (>48h). Both S.aureus and S.gordonii DL-1 elicited a rapid interleukin-8 response by osteoblasts, whereas S.gordonii FnBPA was slower. Only S.aureus elicited an interleukin-6 response. Hence, S.gordonii invades osteoblasts by a mechanism similar to that exhibited by S.aureus, and elicits a proinflammatory response that may promote bone resorption.

Defective actin accumulation impairs human natural killer cell function in patients with dedicator of cytokinesis 8 deficiency

Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.

Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-κB nuclear translocation

Cucurbitacin E (CuE), a triterpenoid compound isolated from Cucurbitaceae plants, possesses a wide range of biological activities including anti-inflammatory properties. The present study aimed to investigate the anti-inflammatory effect of CuE and the underlying mechanism of action. The anti-inflammatory effect of CuE was evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Cell proliferation was assessed using a modified MTT assay. Cell cycle distribution was analyzed by propidium iodide staining. The actin cytoskeleton was examined by immunofluorescent staining. The expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β was determined by intracellular cytokine staining. G-actin level and nuclear factor (NF)-κB nuclear translocation were detected by immunoblotting. CuE inhibited cell proliferation and induced cell cycle arrest at G2/M phase in RAW 264.7 cells. CuE also suppressed LPS-induced cell spreading and pseudopodia formation. These effects were associated with decreased G-actin level and severe actin aggregation. Moreover, CuE significantly inhibited both TNF-α and IL-1β production in LPS-stimulated RAW 264.7 cells. This was likely mediated by suppressing LPS-induced nuclear translocation of NF-κB, a critical transcription factor responsible for pro-inflammatory cytokine expression. CuE displayed anti-inflammatory effects through suppression of NF-κB nuclear translocation leading to a decreased expression of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells.

Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis

Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.