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Purpose To present the clinical, imaging, pathological and immunohistochemical features of giant cell angiofibroma (GCA). Case presentation In this paper we report an atypical case of a GCA extending from the parotid to the parapharyngeal space. The lesion was being treated as a vascular malformation for one year prior to surgical removal. We summarize the clinical manifestations, imaging, pathological and molecular features of this rare disease. After complete surgical removal of the tumor, immunohistochemical analysis revealed strong positivity for the mesenchymal markers vimentin, CD34, CD31 and CD99 in neoplastic cells. Tumor proliferation antigen marker Ki67 was partly positive (<5% of cells). Tumor cells were negative for muscle-specific actin, epithelial membrane antigen, smooth muscle actin, cytokeratin pan, S100, desmin, glial fibrillary acidic protein, myogenin, MyoD1 and F8. The morphological and immunohistochemical profile was consistent with the diagnosis of GCA. Conclusion GCA is a rare soft tissue tumor that can easily be misdiagnosed in the clinical preoperative setting. In view of the clinical, pathological and molecular features of the tumor, complete surgical removal is the current optimal treatment option, providing accurate diagnosis and low to minimal recurrence rate. [ABSTRACT FROM AUTHOR]

In HIV-infected people, resting CD4+ T cells are the main reservoir of latent virus and the reason for the failure of drug therapy to cure HIV infection. Still, we do not have a complete understanding of the factors regulating HIV replication in these cells. A recent paper in Cell describes a new trick that the virus uses to infect resting T cells. Interaction between the viral gp120 and cellular HIV coreceptor, CXCR4, during viral entry initiates signaling that activates cofilin, the main regulator of actin polymerization. As a result of this activation, actin is depolymerized, thus destroying the natural barrier to HIV replication. I discuss implications of this study for our understanding of HIV biology and development of novel anti-HIV therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: One of the main roles of the intestinal mucosa is to protect against environmental hazards. Supplementation of xylo-oligosaccharides (XOS) is known to selectively stimulate the growth of beneficial intestinal bacteria and improve gut health and function in chickens. XOS may have an impact on the integrity of the intestinal epithelia where cell turnover is critical to maintain the compatibility between the digestive and barrier functions. The aim of the study was to evaluate the effect of XOS and an arabinoxylan-rich fraction (AXRF) supplementation on gut function and epithelial integrity in broiler chickens. Methods: A total of 128 broiler chickens (Ross 308) were assigned into one of two different dietary treatments for a period of 42 d: 1) control diet consisting of a corn/soybean meal-based diet; or 2) a control diet supplemented with 0.5% XOS and 1% AXRF. Each treatment was randomly distributed across 8 pens (n = 8) with 8 chickens each. Feed intake and body weight were recorded weekly. On d 42, one male chicken per pen was selected based on average weight and euthanized, jejunum samples were collected for proteomics analysis. Results: Dietary XOS/AXRF supplementation improved feed efficiency (P < 0.05) from d 1 to 42 compared to the control group. Proteomic analysis was used to understand the mechanism of improved efficiency uncovering 346 differentially abundant proteins (DAP) (Padj < 0.00001) in supplemented chickens compared to the non-supplemented group. In the jejunum, the DAP translated into decreased ATP production indicating lower energy expenditure by the tissue (e.g., inhibition of glycolysis and tricarboxylic acid cycle pathways). In addition, DAP were associated with decreased epithelial cell differentiation, and migration by reducing the actin polymerization pathway. Putting the two main pathways together, XOS/AXRF supplementation may decrease around 19% the energy required for the maintenance of the gastrointestinal tract. Conclusions: Dietary XOS/AXRF supplementation improved growth efficiency by reducing epithelial cell migration and differentiation (hence, turnover), actin polymerization, and consequently energy requirement for maintenance of the jejunum of broiler chickens. [ABSTRACT FROM AUTHOR]

Background: The actin cytoskeleton is involved in an array of integral structural and developmental processes throughout the cell. One of actin's best-studied binding partners is the small ubiquitously expressed protein, profilin. Arabidopsis thaliana is known to encode a family of five profilin sequence variants: three vegetative (also constitutive) profilins that are predominantly expressed in all vegetative tissues and ovules, and two reproductive profilins that are specifically expressed in pollen. This paper analyzes the roles of the three vegetative profilin members, PRF1, PRF2, and PRF3, in plant cell and organ development. Results: Using a collection of knockout or severe knockdown T-DNA single mutants, we found that defects in each of the three variants gave rise to specific developmental deficiencies. Plants lacking PRF1 or PRF2 had defects in rosette leaf morphology and inflorescence stature, while those lacking PRF3 led to plants with slightly elongated petioles. To further examine these effects, double mutants and double and triple gene-silenced RNAi epialleles were created. These plants displayed significantly compounded developmental defects, as well as distinct lateral root growth morphological phenotypes. Conclusion: These results suggest that having at least one vegetative profilin gene is essential to viability. Evidence is presented that combinations of independent function, quantitative genetic effects, and functional redundancy have preserved the three vegetative profilin genes in the Arabidopsis lineage. [ABSTRACT FROM AUTHOR]

A conference paper on whether or not the pores on sinusoidal endothelial cells (SECs) under three-dimensional perfusion co-culture treatment with Swinholide-A behave like those in primary culture cells is presented. Results reveal that the number of pores on M1 cells in co-culture was increased by the effect of actin inhibitor. Both share stress and cell-to-cell interaction are vital factors of reappearance of fenestrae in immortalized endothelial cells.

Background: Coronins are a family of highly evolutionary conserved proteins reportedly involved in the regulation of actin cytoskeletal dynamics, although only coronin 3 has been shown to be related to cancer cell migration. In glioblastoma cells, the knockdown of coronin 3 inhibits cell proliferation and invasion. Coronin 3 is also associated with the aggression and metastasis of hepatocellular carcinoma. In this paper, we analyze the migration, invasion and metastasis abilities of gastric cancer cells after up- or down-regulation of coronin 3, and explore the mechanism of coronin 3 in the process of gastric cancer metastasis. Results: The expression of coronin 3 was higher in the highly metastatic sub-cell line MKN28-M, which we established in our laboratory. We also demonstrated that the expression of coronin 3 was remarkably higher in lymph lode metastases than in primary gastric cancer tissues, and over-expression of coronin 3 was correlated with the increased clinical stage and lymph lode metastasis. Recombinant lentiviral vectors encoding shRNAs were designed to down-regulate coronin 3 expression in gastric cancer cell lines. Stable knockdown of coronin 3 by this lentiviral vector could efficiently inhibit the migration and invasion of MKN45 gastric cancer cells. In contrast, up-regulation of coronin 3 significantly enhanced migration and invasion of MKN28-NM cells. In addition, knockdown of coronin 3 significantly reduced liver metastasis in mice after tail vein injection of gastric cancer cells. The Human Tumor Metastasis PCR Array was used to screen the metastasis-associated genes identified by the down-regulation of coronin 3, and the results suggested that, following the knockdown of coronin 3, the tumor cell migration and invasion were inhibited by the reduced expression of MMP-9 and cathepsin K. Conclusion: Coronin 3 is highly expressed in gastric cancer metastases and can promote the metastatic behaviors of gastric cancer cells, including their migration and invasion. [ABSTRACT FROM AUTHOR]

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light. [ABSTRACT FROM AUTHOR]

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, responsible for severe economic losses to the swine industry worldwide. The interferon-inducible GTPase guanylate-binding protein 1 (GBP1) exhibits antiviral immunity. Our findings show that there is a robust upregulation in the expression of porcine GBP1 during PRV infection. GBP1 knockout promotes PRV infection, while GBP1 overexpression restricts it. Importantly, we found that GBP1 impeded the normal structure of actin filaments in a GTPase-dependent manner, preventing PRV virions from reaching the nucleus. We also discovered that viral US3 protein bound GBP1 to interfere with its GTPase activity. Finally, the interaction between US3 and GBP1 requires US3 serine/threonine kinase activity sites and the GTPase domain (aa 1 to 308) of GBP1. Taken together, this study offers fresh perspectives on how PRV manipulates the host's antiviral immune system. [ABSTRACT FROM AUTHOR]

The p27kip1 protein, mainly known as a negative regulator of cell proliferation, has also been involved in the control of other cellular processes, including the regulation of cytoskeleton dynamics. Notably, these two functions involve distinct protein domains, residing in the N- and C-terminal halves, respectively. In the last two decades, p27kip1 has been reported to interact with microtubule and acto-myosin cytoskeletons, both in direct and indirect ways, overall drawing a picture in which several factors play their role either in synergy or in contrast one with another. As a result, the role of p27kip1 in cytoskeleton dynamics has been implicated in cell migration, both in physiologic and in neoplastic contexts, modulating cytokinesis, lipid raft trafficking, and neuronal development. Recently, two distinct papers have further reported a central role for p27kip1 in the control of microtubule stability and post-translational modifications, dissecting the interaction between p27kip1 and α-tubulin-acetyl-transferase (α-TAT), an enzyme involved in the stability of microtubules, and protein-regulator of cytokinesis 1 (PRC1), a nuclear regulator of the central spindle during mitosis. In light of these recent evidences, we will comment on the role of p27kip1 on cytoskeleton regulation and its implication for cancer progression. [ABSTRACT FROM AUTHOR]

Background: Tumor metastasis is the primary cause of mortality in cancer patients. Migratory breast cancer cells in lymphatic and blood vessels seek new sites and form metastatic colonies in the lung and bone, and then these cancer cells often wreak considerable havoc. With advances in nanotechnology, nanomaterials and nanotechnologies are widely applied in tumor therapy. In this paper, small size fullerenol nanoparticles, which are separated by isoelectric focusing electrophoresis (IFE) for discrepancy of isoelectric point (pI), are used in the study of tumor metastasis. Results: In this study, the commendable inhibition of tumor metastasis was uncovered by intravenous injection of purified fullerenol fraction with special surface charge and functional groups, which was separated by IFE for discrepancy of pI. By investigating the actin dynamics in several cancer cell lines, we found these small size fullerenol nanoparticles disturbed actin dynamics. Young's modulus detection and cell migration assays revealed that fullerenol lowered stiffness and restrained migration of breast cancer cells. Filopodia, the main supporting structures of actin bundles, are important for cell motility and adhesion. Scanning electron microscopy showed that fullerenol reduced the number and length of filopodia. Simultaneously, the inhibition of integrin to form clusters on filopodias, which was likely induced by reorganizing of actin cytoskeleton, impacted cancer cell adhesion and motility. Conclusions: With intravenous injection of these fullerenol nanoparticles, tumor metastasis is well inhibited in vivo. The underlying mechanism most likely to be attributed to the effect of fullerenol nanoparticles on disturbing actin dynamics. With the disordered actin fiber, cell function is varied, including decreased cell stiffness, reduced filopodia formation, and inactivated integrin. [ABSTRACT FROM AUTHOR]

Background: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. Methods: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. Results: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. Conclusions: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is one of the neurodegenerative diseases, characterized by the accumulation of abnormal protein deposits, which disrupts signal transduction in neurons and other glia cells. The pathological protein in neurodegenerative diseases, Tau and amyloid-β contribute to the disrupted microglial signaling pathways, actin cytoskeleton, and cellular receptor expression. The important secondary messenger lipids i.e., phosphatidylinositols are largely affected by protein deposits of amyloid-β in Alzheimer's disease. Phosphatidylinositols are the product of different phosphatidylinositol kinases and the state of phosphorylation at D3, D4, and D5 positions of inositol ring. Phosphatidylinositol 3,4,5-triphosphate (PI 3, 4, 5-P3) involves in phagocytic cup formation, cell polarization, whereas Phosphatidylinositol 4,5-bisphosphate (PI 4, 5-P2)-mediates the process of phagosomes formation and further its fusion with early endosome.. The necessary activation of actin-binding proteins such as Rac, WAVE complex, and ARP2/3 complex for the actin polymerization in the process of phagocytosis, migration is regulated and maintained by PI 3, 4, 5-P3 and PI 4, 5-P2. The ratio and types of fatty acid intake can influence the intracellular secondary lipid messengers along with the cellular content of phaphatidylcholine and phosphatidylethanolamine. The Amyloid-β deposits and extracellular Tau seeds disrupt phosphatidylinositides level and actin cytoskeletal network that hamper microglial-signaling pathways in AD. We hypothesize that being a lipid species intracellular levels of phosphatidylinositol would be regulated by dietary fatty acids. Further we are interested to understand phosphoinositide-based signaling cascades in phagocytosis and actin remodeling. D93Rndy_JWMVrxE9wNkr5r Video Abstract [ABSTRACT FROM AUTHOR]

Background: Trichoderma spp. are majorly composed of plant-beneficial symbionts widely used in agriculture as bio-control agents. Studying the mechanisms behind Trichoderma-derived plant benefits has yielded tangible bio-industrial products. To better take advantage of this fungal-plant symbiosis it is necessary to obtain detailed knowledge of which genes Trichoderma utilizes during interaction with its plant host. In this study, we explored the transcriptional activity undergone by T. virens during two phases of symbiosis with maize; recognition of roots and after ingress into the root cortex. Results: We present a model of T. virens – maize interaction wherein T. virens experiences global repression of transcription upon recognition of maize roots and then induces expression of a broad spectrum of genes during colonization of maize roots. The genes expressed indicate that, during colonization of maize roots, T. virens modulates biosynthesis of phytohormone-like compounds, secretes a plant-environment specific array of cell wall degrading enzymes and secondary metabolites, remodels both actin-based and cell membrane structures, and shifts metabolic activity. We also highlight transcription factors and signal transduction genes important in future research seeking to unravel the molecular mechanisms of T. virens activity in maize roots. Conclusions: T. virens displays distinctly different transcriptional profiles between recognizing the presence of maize roots and active colonization of these roots. A though understanding of these processes will allow development of T. virens as a bio-control agent. Further, the publication of these datasets will target future research endeavors specifically to genes of interest when considering T. virens – maize symbiosis. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNA (lncRNA) actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) is oriented in an antisense direction to the protein-coding gene AFAP1 in the opposite strand. Previous studies showed that lncRNA AFAP1-AS1 was upregulated and acted as an oncogene in a variety of tumors. However, the expression and biological functions of lncRNA AFAP1-AS1 in tongue squamous cell carcinoma (TSCC) are still unknown. Methods: The expression level of AFAP1-AS1 was measured in 103 pairs of human TSCC tissues and corresponding adjacent normal tongue mucous tissues. The correlation between AFAP1-AS1 and the clinicopathological features was evaluated using the chi-square test. The effects of AFAP1-AS1 on TSCC cells were determined via a CCK-8 assay, clone formation assay, flow cytometry, wound healing assay and transwell assay. Furthermore, the effect of AFAP1-AS1 knockdown on the activation of the Wnt/ß-catenin signaling pathway was investigated. Finally, CAL-27 cells with AFAP1-AS1 knockdown were subcutaneously injected into nude mice to evaluate the effect of AFAP1-AS1 on tumor growth in vivo. Results: In this study, we found that lncRNA AFAP1-AS1 was increased in TSCC tissues and that patients with high AFAP1-AS1 expression had a shorter overall survival. Short hairpin RNA (shRNA)-mediated AFAP1-AS1 knockdown significantly decreased the proliferation of TSCC cells. Furthermore, AFAP1-AS1 silencing partly inhibited cell migration and invasion. Inhibition of AFAP1-AS1 decreased the activity of the Wnt/β-catenin pathway and suppressed the expression of EMT-related genes (SLUG, SNAIL1, VIM, CADN, ZEB1, ZEB2, SMAD2 and TWIST1) in TSCC cells. In addition, CAL-27 cells with AFAP1-AS1 knockdown were injected into nude mice to investigate the effect of AFAP1-AS1 on tumorigenesis in vivo. Downregulation of AFAP1-AS1 suppressed tumor growth and inhibited the expression of EMT-related genes (SLUG, SNIAL1, VIM, ZEB1, NANOG, SMAD2, NESTIN and SOX2) in vivo. Conclusions: Taken together, our findings present a road map for targeting the newly identified lncRNA AFAP1-AS1 to suppress TSCC progression, and these results elucidate a novel potential therapeutic strategy for TSCC. [ABSTRACT FROM AUTHOR]

Background: Cytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters. Results: We developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls. Conclusions: Our new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin mobility in transgenic A. thaliana expressing Lifeact fusion proteins with different fluorophores, highlighting the need for cautious interpretation of experimental results, as well as to reveal hitherto uncharacterized anisotropy of cytoskeletal mobility in elongated plant cells. [ABSTRACT FROM AUTHOR]

Background: The navigation of magnetotactic bacteria relies on specific intracellular organelles, the magnetosomes, which are membrane-enclosed crystals of magnetite aligned into a linear chain. The magnetosome chain acts as a cellular compass, aligning the cells in the geomagnetic field in order to search for suitable environmental conditions in chemically stratified water columns and sediments. During cytokinesis, magnetosome chains have to be properly positioned, cleaved and separated in order to be evenly passed into daughter cells. In Magnetospirillum gryphiswaldense, the assembly of the magnetosome chain is controlled by the actin-like MamK, which polymerizes into cytoskeletal filaments that are connected to magnetosomes through the acidic MamJ protein. MamK filaments were speculated to recruit the magnetosome chain to cellular division sites, thus ensuring equal organelle inheritance. However, the underlying mechanism of magnetic organelle segregation has remained largely unknown. Results: Here, we performed in vivo time-lapse fluorescence imaging to directly track the intracellular movement and dynamics of magnetosome chains as well as photokinetic and ultrastructural analyses of the actin-like cytoskeletal MamK filament. We show that magnetosome chains undergo rapid intracellular repositioning from the new poles towards midcell into the newborn daughter cells, and the driving force for magnetosomes movement is likely provided by the pole-to-midcell treadmilling growth of MamK filaments. We further discovered that splitting and equipartitioning of magnetosome chains occurs with unexpectedly high accuracy, which depends directly on the dynamics of MamK filaments. Conclusion: We propose a novel mechanism for prokaryotic organelle segregation that, similar to the type-II bacterial partitioning system of plasmids, relies on the action of cytomotive actin-like filaments together with specific connectors, which transport the magnetosome cargo in a fashion reminiscent of eukaryotic actin-organelle transport and segregation mechanisms. [ABSTRACT FROM AUTHOR]

Background: Formins are a highly conserved family of cytoskeletal remodeling proteins. A growing body of evidence suggests that formins play key roles in the progression and spread of a variety of cancers. There are 15 human formin proteins and of these the Diaphanous-Related Formins (DRFs) are the best characterized. Included in the DRFs are the Formin-Like proteins, FMNL1, 2 & 3, each of which have been strongly implicated in driving tumorigenesis and metastasis of specific tumors. In particular, increased FMNL2 expression correlates with increased invasiveness of colorectal cancer (CRC) in vivo and for a variety of CRC cell-lines in vitro. FMNL2 expression is also required for invasive cell motility in other cancer cell-lines. There are multiple alternatively spliced isoforms of FMNL2 and it is predicted that the encoded proteins will differ in their regulation, subcellular localization and in their ability to regulate cytoskeletal dynamics. Results: Using RT-PCR we identified four FMNL2 isoforms expressed in CRC and melanoma cell-lines. We find that a previously uncharacterized FMNL2 isoform is predominantly expressed in a variety of melanoma and CRC cell lines; this isoform is also more effective in driving 3D motility. Building on previous reports, we also show that FMNL2 is required for invasion in A375 and WM266.4 melanoma cells. Conclusions: Taken together, these results suggest that FMNL2 is likely to be generally required in melanoma cells for invasion, that a specific isoform of FMNL2 is up-regulated in invasive CRC and melanoma cells and this isoform is the most effective at facilitating invasion. [ABSTRACT FROM AUTHOR]

Background: The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown. Methods: We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells. Results: Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division. Conclusions: Taken together, the data suggest a model in which mitochondria are transported by a microtubulemediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis. [ABSTRACT FROM AUTHOR]

Background: Excessive mechanical overload may be involved in bladder wall remodelling. Since the activity of Rho kinase is known to be upregulated in the obstructed bladder, we investigate the roles of the RhoA/Rho kinase pathway in mechanical overloaded bladder smooth muscle cells.Methods: Human bladder smooth muscle cells were stimulated on silicon culture plates by 15 % elongated uni-axial cyclic stretch at 1 Hz. The activity of c-Jun NH2-terminal kinase was measured by western blotting and actin stress fibers were observed by stained with phallotoxin conjugated with Alexa-Fluor 594.Results: The activity of c-Jun NH2-terminal kinase 1 peaked at 30 min (4.7-fold increase vs. before stretch) and this activity was partially abrogated by the RhoA inhibitor, C3 exoenzoyme or by the Rho kinase inhibitor, Y-27632. Stretch induced the strong formation of actin stress fibers and these fibers re-orientated in a direction that was perpendicular to the stretch direction. The average angle of the fibers from the perpendicular to the direction of stretch was significantly different between before, and 4 h after, stretch. Actin stress fibers reorganization was also suppressed by the C3 exoenzyme or Y-27632.Conclusions: Bladder smooth muscle cells appear to have elaborate mechanisms for sensing mechanical stress and for adapting to mechanical stress overload by cytoskeletal remodeling and by activating cell growth signals such as c-Jun NH2-terminal kinase via RhoA/Rho kinase pathways. [ABSTRACT FROM AUTHOR]

Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation. [ABSTRACT FROM AUTHOR]

Background: Membrane microdomains represent dynamic membrane nano-assemblies enriched in signaling molecules suggesting their active involvement in not only physiological but also pathological molecular processes. The hyperlipidemic stress is a major risk factor of atherosclerosis, but its exact mechanisms of action at the membrane microdomains level remain elusive. The aim of the present study was to determine whether membrane-cytoskeleton proteome in the pulmonary tissue could be modulated by the hyperlipidemic stress, a major risk factor of atherosclerosis. Results: High resolution mass spectrometry based proteomics analysis was performed for detergent resistant membrane microdomains isolated from lung homogenates of control, ApoE deficient and statin treated ApoE deficient mice. The findings of the study allowed the identification with high confidence of 1925 proteins, 291 of which were found significantly altered by the modified genetic background, by the statin treatment or both conditions. Principal component analysis revealed a proximal partitioning of the biological replicates, but also a distinct spatial scattering of the sample groups, highlighting different quantitative profiles. The statistical significant over-representation of Regulation of actin cytoskeleton, Focal adhesion and Adherens junction Kyoto Encyclopedia of Genes and Genomes signaling pathways was demonstrated through bioinformatics analysis. The three inter-relation maps comprised 29 of regulated proteins, proving membrane-cytoskeleton coupling targeting and alteration by hyperlipidemia and/or statin treatment. Conclusions: The findings of the study allowed the identification with high confidence of the main proteins modulated by the hyperlipidemic stress involved in the actin-dependent pathways. Our study provides the basis for future work probing how the protein activities at the membrane-cytoskeleton interface are dependent upon genetic induced hyperlipidemia. [ABSTRACT FROM AUTHOR]

Background: Cyclin D3, which induces progression through the G1 phase of the cell cycle, is a regulator of Cyclin-dependent kinases 4 and 6. Previous studies revealed that abnormal expression of Cyclin D3 was found in many different cancers. However, the role of Cyclin D3 in breast cancer (BC) remains unknown. The aim of this study is to examine the expression pattern of Cyclin D3 in BC and to evaluate its biological role and clinical significance in prognosis prediction. The mechanism involved is also evaluated. Methods: Immunohistochemical staining was used to detect the expression of Cyclin D3. qRT-PCR was used to detect the mRNA level of Cyclin D3 in BC tissues and BC cell lines. Transwell assay was used to examine the role of Cyclin D3 in the migration and invasion of BC cells. Mass Spectrometry was used to search for the interacting protein with Cyclin D3. Co-Immunoprecipitation assay and GST-Pull Down assay were used to validate the interaction of Cyclin D3 and its interaction protein. Results: Through detecting Cyclin D3 expression in 243 breast cancer patients' tissue array, we found Cyclin D3 expression was correlated with ER status (p = 0.000), PR status (p = 0.001), HER2 status (p = 0.002) and tumor differentiation (p = 0.045). The Kaplan-Meier survival curves indicated that the disease free survival (DFS) was significantly poor in high Cyclin D3 expression BC patients (p = 0.004). Furthermore, expression of Cyclin D3 was significantly associated with BC prognosis and was shown to be an independent prognostic marker in breast cancer (p = 0.028). By IHC staining and qPCR detection, Cyclin D3 expression was found to be down-regulated both in BC tissues and in BC cell lines compared with the corresponding normal controls. Further investigation showed Cyclin D3 was involved in the metastasis of BC cells and physically interacted with actin in vivo and in vitro. Conclusion: Our studies revealed that Cyclin D3 was upregulated in breast cancer and represented a novel predictor of BC prognosis. [ABSTRACT FROM AUTHOR]

Background The classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14- 21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system. The predominant membrane-associated isoform of MBP is not simply a structural component of compact myelin but is highly post-translationally modified and multifunctional, having interactions with numerous proteins such as Ca2+-calmodulin, and with actin, tubulin, and proteins with SH3-domains, which it can tether to a lipid membrane in vitro. It co-localizes with such proteins in primary oligodendrocytes (OLGs) and in early developmental N19-OLGs transfected with fluorescently-tagged MBP. Results To provide further evidence for MBP associations with these proteins in vivo, we show here that MBP isoforms are co-immunoprecipitated from detergent extracts of primary OLGs together with actin, tubulin, zonula occludens 1 (ZO-1), cortactin, and Fyn kinase. We also carry out live-cell imaging of N19-OLGs co-transfected with fluorescent MBP and actin, and show that when actin filaments re-assemble after recovery from cytochalasin D treatment, MBP and actin are rapidly enriched and co-localized at certain sites at the plasma membrane and in newly-formed membrane ruffles. The MBP and actin distributions change similarly with time, suggesting a specific and dynamic association. Conclusions These results provide more direct evidence for association of the predominant 18.5-kDa MBP isoform with these proteins in primary OLGs and in live cells than previously could be inferred from co-localization observations. This study supports further a role for classic MBP isoforms in protein-protein interactions during membrane and cytoskeletal extension and remodeling in OLGs. [ABSTRACT FROM AUTHOR]

Background BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signaling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signaling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. Results We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PtdIns(3,4,5)P3 (PIP3) in BMP2- induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2 dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5β to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knockdown of p55γ or LL5β or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. Conclusions Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3- LL5β signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2- induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts. [ABSTRACT FROM AUTHOR]

Background Cryptosporidiosis in dogs has been reported worldwide, involving both asymptomatic and diarrheic dogs. Large-scale surveys of Cryptosporidium infection in dogs have been performed in some countries using differents diagnostic methods. But, few data are available on the infection rate and molecular characteristics of Cryptosporidium spp. in dogs in China. Result In this study, 770 fecal samples from 66 locations in Henan Province were examined. The average Cryptosporidium infection rate was 3.8%, with dogs in kennels having the highest rate of 7.0% (X2 = 14.82, P < 0.01). The infection rate was 8.0% in dogs younger than 90 days, which was significantly higher than that in the other age groups (1.1-3.8%;X2 = 18.82, P < 0.01). No association was noted between the infection rate and the sex of the dogs. Twenty-nine Cryptosporidium-positive samples were amplified at the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin loci using PCR. Sequence analysis of these amplicons identified only Cryptosporidium canis, which showed 100% identity with the published sequences of the SSU rRNA, HSP70, and actin genes. Conclusions Our results confirm that C. canis is popular in the dog population in China, considering the large number of dogs in China and the close contact between dogs and humans, the role of C. canis in the transmission of human cryptosporidiosis warrants attention. [ABSTRACT FROM AUTHOR]

Background: Cytoskeletal proteins are often involved in the virus life cycle, either at early steps during virus entry or at later steps during formation of new virus particles. Though actin filaments have been shown to play a role in the production of measles virus (MV), the importance of actin dynamics for virus assembly and budding steps is not known yet. Aim of this work was thus to analyze the distinctive consequences of F-actin stabilization or disruption for MV protein trafficking, particle assembly and virus release. Results: MV infection studies in the presence of inhibitors differently affecting the actin cytoskeleton revealed that not only actin disruption but also stabilization of actin filaments interfered with MV particle release. While overall viral protein synthesis, surface expression levels of the MV glycoproteins, and cell-associated infectivity was not altered, cell-free virus titers were decreased. Interestingly, the underlying mechanisms of interference with late MV maturation steps differed principally after F-actin disruption by Cytochalasin D (CD) and F-actin stabilization by Jasplakinolide (Jaspla). While intact actin filaments were shown to be required for transport of nucleocapsids and matrix proteins (M-RNPs) from inclusions to the plasma membrane, actin dynamics at the cytocortex that are blocked by Jaspla are necessary for final steps in virus assembly, in particular for the formation of viral buds and the pinching-off at the plasma membrane. Supporting our finding that F-actin disruption blocks M-RNP transport to the plasma membrane, cell-to-cell spread of MV infection was enhanced upon CD treatment. Due to the lack of M-glycoprotein-interactions at the cell surface, M-mediated fusion downregulation was hindered and a more rapid syncytia formation was observed. Conclusion: While stable actin filaments are needed for intracellular trafficking of viral RNPs to the plasma membrane, and consequently for assembly at the cell surface and prevention of an overexerted fusion by the viral surface glycoproteins, actin dynamics are required for the final steps of budding at the plasma membrane. [ABSTRACT FROM AUTHOR]