Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

Anti-F-Actin Antibody Positivity on Indirect Immunofluorescence Assay Following Chinese and Alternative Medicine Therapy: A Case Report.

Immunofluorescence on human epithelial type 2 cells is the standard screening assay for the detection of antinuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding. However, the less commonly reported ones include the cytoplasmic fibrillar patterns on indirect immunofluorescence technique (IIFT). The cytoplasmic fibrillar patterns include the cytoplasmic linear (AC-15), cytoplasmic filamentous (AC-16), and cytoplasmic segmental (AC-17). We report a case of cytoplasmic linear (F-actin) detected through IIFT during ANA screening in a 77-year-old man and later reconfirmed on liver mosaic biochip through IIFT on vascular smooth muscle substrate (VSM-47) without features suggestive of anti-smooth muscle antibody involvement post-complementary and alternative medicine therapy initiation.

Unreported cytochalasins from an acid-mediated transformation of cytochalasin J isolated from Diaporthe cf. ueckeri.

Chemical investigation of an endophytic fungus herein identified as Diaporthe cf. ueckeri yielded four known compounds, named cytochalasins H and J and dicerandrols A and B. Reports of acid sensitivity within the cytochalasan family inspired an attempt of acid-mediated conversion of cytochalasins H and J, resulting in the acquisition of five polycyclic cytochalasins featuring 5/6/5/8-fused tetracyclic and 5/6/6/7/5-fused pentacyclic skeletons. Two of the obtained polycyclic cytochalasins constituted unprecedented analogues, for which the trivial names cytochalasins J4 and J5 were proposed, whereas the others were identified as the known phomopchalasin A, phomopchalasin D and 21-acetoxycytochalasin J3. The structures of the compounds were determined by extensive spectral analysis, namely HR-ESIMS, ESIMS and 1D/2D NMR. The stereochemistry of cytochalasins J4 and J5 was proposed using their ROESY data, biosynthetic and mechanistic considerations and by comparison of their ECD spectra with those of related congeners. All compounds except for cytochalasins H and J were tested for antimicrobial and cytotoxic activity. Cytochalasins J4 and J5 showed neither antimicrobial nor cytotoxic activity in the tested concentrations, with only weak antiproliferative activity observable against KB3.1 cells. The actin disruptive properties of all cytochalasins obtained in this study and of the previously reported cytochalasins RKS-1778 and phomopchalasin N were examined, and monitored by fluorescence microscopy using human osteo-sarcoma (U2-OS) cells. Compared to their precursor molecules (cytochalasins H and J), phomopchalasins A and D, 21-acetoxycytochalasin J3, cytochalasins J4 and J5 revealed a strongly reduced activity on the F-actin network, highlighting that the macrocyclic ring is crucial for bioactivity.

Photobiomodulation Therapy Affects the Elastic Modulus, Cytoskeletal Rearrangement and Migration Capability of Human Osteosarcoma Cells.

Photobiomodulation (PBM) therapy utilizes low-power lasers to modulate the viability of living human cells and leads to changes in proliferation, differentiation, adhesion and gene expression, even though the rearrangement of cytoskeleton was not previously studied. The present study aims to evaluate the photobiological effects on the elastic behavior of human osteosarcoma cells (MG-63) and their morphological changes. Fluorescence staining, confocal imaging and atomic force microscopy (AFM) topography were performed to study the effects of PBM therapy with the exposure of 532 nm-25mW, 650 nm-3mW, 650 nm-150mW and 780 nm-70mW beams following the 5-min continuous irradiation. The area of each beam was 3.14cm2 with a source-surface distance of 20 cm. Besides the cell proliferation assessment, the migratory potential of MG-63 was determined with the wound healing technique. The results indicated an increase in stiffness and shape index of radiation-induced cells 24 h after exposure along with the obvious F-actins changes. But, cell stiffening was not observed 72 h after 532 nm laser irradiation. Also, a decrease in the migration rate was seen in all of the groups after 72 h of irradiation except cells treated with 532 nm wavelength. However, 532 nm laser beams increase the migratory potential 24 h after exposure. Within 72 h after irradiation, the cell proliferation was only affected by applying 532 nm and 650 nm-150mW laser beams. It was concluded that applying photobiomodulation with wavelengths of 650 nm (at both utilized powers) and 780 nm alters the migration capability and provides a quantitative description of cytoskeletal changes. Moreover, membrane stiffening can be considered as the biological marker of PBM treatments.

YiQiFuMai lyophilized injection attenuates cerebral ischemic injury with inhibition of neuronal autophagy through intervention in the NMMHC IIA-actin-ATG9A interaction.

BACKGROUND: YiQiFuMai lyophilized injection (YQFM) is derived from a traditional Chinese medicine prescription termed Shengmai San.YQFM is clinically applied to the treatment of cardiovascular and cerebrovascular diseases. It has been found that critical components of YQFM affect non-muscle myosin heavy chain IIA (NMMHC IIA), but its regulation in the excessive autophagy and the underlying mechanism has yet to be clarified. PURPOSE: To evaluate whether YQFM has neuroprotective effects on cerebral ischemia/reperfusion-induced injury by inhibiting NMMHC IIA-actin-ATG9A interaction for autophagosome formation. METHODS: The neuroprotective effects of YQFM were investigated in vivo in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) (n = 6) by detecting neurological deficits, infarct volume, and histopathological changes. The NMMHC IIA-actin-ATG9A interaction was determined using immunofluorescence co-localization, co-immunoprecipitation, and proximity ligation assay. Rat pheochromocytoma (PC12) cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic neurons in in vitro experiments. RESULTS: In MCAO/R model mice, YQFM (1.342 g/kg) attenuated brain ischemia/reperfusion-induced injury by regulating NMMHC IIA-actin-mediated ATG9A trafficking. YQFM (400 µg/ml) also exerted similar effects on OGD/R-induced PC12 cells. Furthermore, RNAi of NMMHC IIA weakened the NMMHC IIA-F-actin-dependent ATG9A trafficking and, therefore, attenuated the neuroprotective activities of YQFM in vitro. CONCLUSION: These findings demonstrated that YQFM exerted neuroprotective effects by regulating the NMMHC IIA-actin-ATG9A interaction for autophagosome formation. This evidence sheds new light on the potential mechanism of YQFM in the treatment of cerebral ischemia/reperfusion.

[Main components in butyl alcohol extract of Baitouweng Decoction inhibited neutrophil chemotaxis].

The present study aims to investigate the effects of the main components(aesculin, berberine hydrochloride, and anemoside B4) in the butyl alcohol extract of Baitouweng Decoction(BAEB) on the chemotaxis of neutrophils induced by dimethyl sulfoxide(DMSO). HL60 cells were cultivated in RPMI-1640 complete medium, and transferred into a 6-well plate(2 × 10~5 per mL) with 4 mL in each well, followed by incubation with DMSO at 1.3% for five days. The morphologic changes of cells were observed under an inverted microscope. The CD11 b expression after DMSO induction was analyzed by flow cytometry. The effects of aesculin, berberine hydrochloride, and anemoside B4 on the cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The effects of the main components on the production and polarization of F-actin protein were also examined by flow cytometry and laser confocal microscopy. PI3 K/Akt signaling pathway was checked by Western blot. As revealed by the results, neutrophil-like HL60 cells were observed after DMSO induction. The CD11 b expression in these cells increased significantly as indicated by the flow cytometry. Additionally, 100 µg·mL~(-1) aesculin, 8 µg·mL~(-1) berberine hydrochloride, and 80 µg·mL~(-1) anemoside B4 were potent in inhibiting the migration of neutrophils and reducing F-actin expression. Berberine hydrochloride was verified to be capable of diminishing phosphorylated PI3 K/Akt protein expression. The findings indicate that aesculin, anemoside B4, and especially berberine hydrochloride in the BAEB can inhibit the chemotaxis of neutrophils, which is possibly achieved by the inhibition of F-actin and PI3 K/Akt signaling pathway.

Main components in butyl alcohol extract of Baitouweng Decoction inhibited neutrophil chemotaxis / 中国中药杂志

The present study aims to investigate the effects of the main components(aesculin, berberine hydrochloride, and anemoside B4) in the butyl alcohol extract of Baitouweng Decoction(BAEB) on the chemotaxis of neutrophils induced by dimethyl sulfoxide(DMSO). HL60 cells were cultivated in RPMI-1640 complete medium, and transferred into a 6-well plate(2 × 10~5 per mL) with 4 mL in each well, followed by incubation with DMSO at 1.3% for five days. The morphologic changes of cells were observed under an inverted microscope. The CD11 b expression after DMSO induction was analyzed by flow cytometry. The effects of aesculin, berberine hydrochloride, and anemoside B4 on the cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The effects of the main components on the production and polarization of F-actin protein were also examined by flow cytometry and laser confocal microscopy. PI3 K/Akt signaling pathway was checked by Western blot. As revealed by the results, neutrophil-like HL60 cells were observed after DMSO induction. The CD11 b expression in these cells increased significantly as indicated by the flow cytometry. Additionally, 100 μg·mL~(-1) aesculin, 8 μg·mL~(-1) berberine hydrochloride, and 80 μg·mL~(-1) anemoside B4 were potent in inhibiting the migration of neutrophils and reducing F-actin expression. Berberine hydrochloride was verified to be capable of diminishing phosphorylated PI3 K/Akt protein expression. The findings indicate that aesculin, anemoside B4, and especially berberine hydrochloride in the BAEB can inhibit the chemotaxis of neutrophils, which is possibly achieved by the inhibition of F-actin and PI3 K/Akt signaling pathway.

ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants.

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore.

Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing.

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 µg·mL-1) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg-1) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Melittin from Apis florea Venom as a Promising Therapeutic Agent for Skin Cancer Treatment.

Melittin, a major component found in bee venom, is produced by the Apis species of the honey bee. In this study, the effect of melittin derived from Apis florea (Mel-AF), which is a wild honey bee species that is indigenous to Thailand, was investigated against human malignant melanoma (A375) cells. In this study, Mel-AF exhibited considerable potential in the anti-proliferative action of A375 cells. Subsequently, the cellular mechanism of Mel-AF that induced cell death was investigated in terms of apoptosis. As a result, gene and protein expression levels, which indicated the activation of cytochrome-c release and caspase-9 expression, eventually triggered the release of the caspase-3 executioner upon Mel-AF. We then determined that apoptosis-mediated cell death was carried out through the intrinsic mitochondrial pathway. Moreover, advanced abilities, including cell motility and invasion, were significantly suppressed. Mel-AF manipulated the actin arrangement via the trapping of stress fibers that were found underneath the membrane, which resulted in the defective actin cytoskeleton organization. Consequently, the expression of EGFR, a binding protein to F-actin, was also found to be suppressed. This outcome strongly supports the effects of Mel-AF in the inhibition of progressive malignant activity through the disruption of actin cytoskeleton-EGFR interaction and the EGFR signaling system. Thus, the findings of our current study indicate the potential usefulness of Mel-AF in cancer treatments as an apoptosis inducer and a potential actin-targeting agent.

Salmonella Effector SpvB Disrupts Intestinal Epithelial Barrier Integrity for Bacterial Translocation.

Salmonella are common enteric bacterial pathogens that infect both humans and animals. Intestinal epithelial barrier, formed by a single layer of epithelial cells and apical junctional complex (AJC), plays a crucial role in host defense against enteric pathogens to prevent bacterial translocation. However, the underlying mechanisms of intestinal epithelial barrier dysfunction caused by Salmonella are poorly understood. It is found that a locus termed Salmonella plasmid virulence (spv) gene exists extensively in clinically important Salmonella serovars. SpvB is a key effector encoded within this locus, and closely related to Salmonella pathogenicity such as interfering with autophagy and iron homeostasis. To investigate the interaction between SpvB and intestinal epithelial barrier and elucidate the underlying molecular mechanism, we used the typical foodborne disease agent Salmonella enterica serovar Typhimurium (Salmonella typhimurium) carrying spvB or not to construct infection models in vivo and in vitro. C57BL/6 mice were orally challenged with S. typhimurium wild-type strain SL1344 or spvB-deficient mutant strain SL1344-ΔspvB. Caco-2 cell monolayer model, as a widely used model to mimic the human intestinal epithelium in vitro, was infected with SL1344, SL1344-ΔspvB, or spvB complementary strain SL1344-c-ΔspvB, respectively. The results showed that SpvB enhanced bacterial pathogenicity during S. typhimurium infection in vivo, and contributed to intestinal epithelial barrier dysfunction in both infection systems. This SpvB-mediated barrier dysfunction was attributed to the cellular redistribution of Claudin-1, Occludin, and E-cadherin junctional proteins. Moreover, by using pharmacological inhibitors, we found that F-actin rearrangement and suppression of protein kinase C (PKC) signaling pathway were involved in SpvB-mediated barrier dysfunction. In conclusion, the study reveals the contribution of Salmonella effector SpvB to the dysfunction of intestinal epithelial barrier integrity, which facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. Our findings broaden the understanding of host-pathogen interactions in salmonellosis, and provide new strategies for the therapy in limiting bacterial dissemination during infection.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing / 中国天然药物

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 μg·mL) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Vitamin C Inhibits Triple-Negative Breast Cancer Metastasis by Affecting the Expression of YAP1 and Synaptopodin 2.

Vitamin C supplementation has been shown to decrease triple-negative breast cancer (TNBC) metastasis. However, the molecular mechanism whereby vitamin C inhibits metastasis remains elusive. It has been postulated that vitamin C reduces the levels of HIF-1α, the master regulator of metastasis, by promoting its hydroxylation and degradation. Here, we show that vitamin C at 100 µM, a concentration achievable in the plasma in vivo by oral administration, blocks TNBC cell migration and invasion in vitro. The protein level of HIF-1α remains largely unchanged in cultured TNBC cells and xenografts, partially due to its upregulated transcription by vitamin C, suggesting that HIF-1α unlikely mediates the action of vitamin C on metastasis. Vitamin C treatment upregulates the expression of synaptopodin 2 and downregulates the expression of the transcription coactivator YAP1, both genes in the Hippo pathway. The changes in SYNPO2 and YAP1 expression were subsequently validated at mRNA and protein levels in cultured TNBC cells and xenografts. Further experiments showed that vitamin C treatment inhibits F-actin assembly and lamellipodia formation, which correlates with the changes in SYNPO2 and YAP1 expression. Overall, these results suggest that vitamin C inhibits TNBC metastasis by affecting the expression of SYNPO2 and YAP1. Vitamin C may thus have a potential role in the prevention and treatment of TNBC metastasis.

Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes.

BACKGROUND: Three-dimensional (3D) floating culture clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. Previous studies have demonstrated that C-MSCs can be transplanted into bony lesions without an artificial scaffold to induce bone regeneration. Moreover, osteoinductive medium (OIM)-treated C-MSCs (OIM-C-MSCs) have shown rapid and increased new bone formation in vivo. To apply OIM-C-MSCs for novel bone regenerative cell therapy, their cellular properties at the molecular level must be elucidated. The transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have been recognized as key players in the mechanotransduction cascade, controlling cell lineage commitment in MSCs. It is plausible that 3D C-MSCs/OIM-C-MSCs cultured in floating conditions could provide distinct microenvironments compared to conventional 2D culture systems and thereby induce unique mechanotransduction cascades. Therefore, this study investigated the YAP/TAZ activity in 3D-cultured C-MSCs/OIM-C-MSCs in floating conditions. METHODS: Human bone marrow-derived MSCs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make round clumps of cells. Then, YAP/TAZ activity, filamentous actin (F-actin) integrity, collagen type I (COL1) production, and the differentiation potency in 3D floating culture C-MSCs/OIM-C-MSCs were analyzed. RESULTS: C-MSCs cultured in floating conditions lost their actin cytoskeleton to downregulate YAP/TAZ activity, which directed cells to undergo adipogenesis/chondrogenesis. OIM treatment induced abundant COL1 deposition, which facilitated Intß1-dependent actin fiber formation and YAP/TAZ activity to elevate the expression levels of osteogenic master transcriptional factor runt-related transcription factor 2 (RUNX2) mRNA in C-MSCs. Importantly, elevation of YAP/TAZ activity via OIM was associated with COL1 deposition and F-actin integrity, suggesting a positive feedback loop in OIM-C-MSCs. CONCLUSION: These findings suggest that OIM-C-MSCs, which form a unique microenvironment that maintains high YAP/TAZ activity, can serve as better candidates for bone regenerative cell therapy than C-MSCs.

Enterolactone alters FAK-Src signaling and suppresses migration and invasion of lung cancer cell lines.

BACKGROUND: Systemic toxicity of chemotherapeutic agents and the challenges associated with targeting metastatic tumors are limiting factors for current lung cancer therapeutic approaches. To address these issues, plant-derived bioactive components have been investigated for their anti-cancer properties because many of these agents are non-toxic to healthy tissues. Enterolactone (EL) is a flaxseed-derived mammalian lignan that has demonstrated anti-migratory properties for various cancers, but EL has not been investigated in the context of lung cancer, and its anticancer mechanisms are ill-defined. We hypothesized that EL could inhibit lung cancer cell motility by affecting the FAK-Src signaling pathway. METHODS: Non-toxic concentrations of EL were identified for A549 and H460 human lung cancer cells by conducting 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Dephenyltetrazolium Bromide (MTT) assays. The anti-migratory and anti-invasive potential of EL for lung cancer cell lines was determined by scratch wound healing and Matrigel® invasion assays. Changes in filamentous actin (F-actin) fiber density and length in EL-treated cells were determined using phalloidin-conjugated rhodamine dye and fluorescent microscopy. Vinculin expression in focal adhesions upon EL treatment was determined by immunocytochemistry. Gene and protein expression levels of FAK-Src signaling molecules in EL-treated lung cancer cells were determined using PCR arrays, qRT-PCR, and western blotting. RESULTS: Non-toxic concentrations of EL inhibited lung cancer cell migration and invasion in a concentration- and time-dependent manner. EL treatment reduced the density and number of F-actin fibers in lung cancer cell lines, and reduced the number and size of focal adhesions. EL decreased phosphorylation of FAK and its downstream targets, Src, paxillin, and decreased mRNA expression of cell motility-related genes, RhoA, Rac1, and Cdc42 in lung cancer cells. CONCLUSIONS: Our data suggest that EL suppresses lung cancer cell motility and invasion by altering FAK activity and subsequent activation of downstream proteins needed for focal adhesion formation and cytoskeletal rearrangement. Therefore, administration of EL may serve as a safe and complementary approach for inhibiting lung tumor cell motility, invasion, and metastasis.

Isolation of Actin and Actin-Binding Proteins.

Actin-binding proteins mediate and regulate the dynamics of actin and the organization of highly ordered structures of F-actin. Villin is generally expressed in plant cells and is associated with G-actin or F-actin dependent on Ca2+ concentrations. Using a DNase I affinity column chromatography approach, the villin and the G-actin can be isolated from plant material. An outline of this method including the preparation of crude protein extract from plant material, its application on the affinity column, and the successive elution of villin with a solution containing EGTA and then of G-actin with denatured reagents is presented.

Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.

Alcoholic Extract of Ashwagandha Leaves Protects Against Amnesia by Regulation of Arc Function.

Our earlier report on scopolamine-induced amnesia and its improvement by pre-treatment with i-Extract (alcoholic extract of Ashwagandha leaf) suggested that the i-Extract mediated nootropic effect may involve neuronal immediate early gene, Arc. With a hypothesis that the i-Extract induced expression of Arc protein may cause augmentation in Arc function, we examined the effect of i-extract on a major function of Arc protein, i.e. F-actin expansion, using Arc antisense oligodeoxynucleotides (ODN). Stereotaxic infusion of Arc antisense ODN in the CA1 region of hippocampus decreased the level of Arc protein as demonstrated by immunoblotting. However, this decrease was attenuated when treated with i-Extract prior to infusion of Arc antisense ODN. We noted a significant decrease in the polymerization of F-actin during scopolamine-induced amnesia as well as Arc antisense ODN infusion that was restored rather enhanced when pre-treated with i-Extract in both the cases. We also compared the corresponding changes between CA1 (the infusion site) and CA3 (neighbouring site of infusion) regions of hippocampus, and found more pronounced effects in CA1 than in the CA3 region. The extent of F-actin polymerization, as revealed by changes in the dendritic spine architecture through Golgi staining, showed that both scopolamine as well as Arc antisense ODN disrupted the spine density and mushroom-shaped morphology that was again regained if pre-treated with i-Extract. In conclusion, the findings reveal that the Arc helps in polymerization of F-actin and subsequent changes in the morphology of dendritic spines after pre-treatment with i-Extract in scopolamine-induced amnesic mice, suggesting an important role of Arc in scopolamine-induced amnesia and its recovery by i-Extract.

Structure of the 34†kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Šresolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.