RECK-Mediated Inhibition of Glioma Migration and Invasion

RECK is an anti-tumoral gene whose activity has been associated with its inhibitory effects regulating MMP-2, MMP-9, and MT1-MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP-2, MMP-9, and MT1-MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin-myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylatecl focal adhesion kinase (P-FAK) in mature focal adhesions associated with stress fibers; whereas P-FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor-suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52-61, 2010. (C) 2010 Wiley-Liss. Inc.

Myosin Va phosphorylated on ser(1650) is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of geneexpression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phosphoser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

Quercetin Reduces Neutrophil Recruitment Induced by CXCL8, LTB(4), and fMLP: Inhibition of Actin Polymerization

Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Further-more, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.

Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury

The mechanism of isoproterenol-induced myocardial damage is unknown, but a mismatch of oxygen supply vs. demand following coronary hypotension and myocardial hyperactivity is the best explanation for the complex morphological alterations observed. Severe alterations in the structural integrity of the sarcolemma of cardiomyocytes have been demonstrated to be caused by isoproterenol. Taking into account that the sarcolemmal integrity is stabilized by the dystrophin-glycoprotein complex (DGC) that connects actin and laminin in contractile machinery and extracellular matrix and by integrins, this study tests the hypothesis that isoproterenol affects sarcolemmal stability through changes in the DGC and integrins. We found different sensitivity of the DGC and integrin to isoproterenol subcutaneous administration. Immunofluorescent staining revealed that dystrophin is the most sensitive among the structures connecting the actin in the cardiomyocyte cytoskeleton and the extracellular matrix. The sarcomeric actin dissolution occurred after the reduction or loss of dystrophin. Subsequently, after lysis of myofilaments, gamma-sarcoglycan, beta-dystroglycan, beta 1-integrin, and laminin alpha-2 expressions were reduced followed by their breakdown, as epiphenomena of the myocytolytic process. In conclusion, administration of isoproterenol to rats results in primary loss of dystrophin, the most sensitive among the structural proteins that form the DGC that connects the extracellular matrix and the cytoskeleton in cardiomyocyte. These changes, related to ischaemic injury, explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol.

T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats

Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30 min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-1 mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

Rho Signaling Pathway and Apical Constriction in the Early Lens Placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49: 368-379, 2011. (C) 2011 Wiley-Liss, Inc.

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1 beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.

Three-dimensional Reconstruction of Ovaries of Leaf-cutting Ant (Atta sexdens rubropilosa) Queens (Hymenoptera: Formicidae)

In this study, the ovary morphology of newly emerged ant queens of Atta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Leukotrienes Target F-actin/Cofilin-1 to Enhance Alveolar Macrophage Anti-fungal Activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-delta (PKC delta) and PI3K but not PKC alpha and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

T3 rapidly modulates TSH beta mRNA stability and translational rate in the pituitary of hypothyroid rats

Whereas it is well known that T3 inhibits TSH beta gene transcription, its effects on TSH beta mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSH beta transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSH beta transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSH beta mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSH beta mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSH beta transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Mapping the cytoskeletal prestress

Park CY, Tambe D, Alencar AM, Trepat X, Zhou EH, Millet E, Butler JP, Fredberg JJ. Mapping the cytoskeletal prestress. Am J Physiol Cell Physiol 298: C1245-C1252, 2010. First published February 17, 2010; doi: 10.1152/ajpcell.00417.2009.-Cell mechanical properties on a whole cell basis have been widely studied, whereas local intracellular variations have been less well characterized and are poorly understood. To fill this gap, here we provide detailed intracellular maps of regional cytoskeleton (CSK) stiffness, loss tangent, and rate of structural rearrangements, as well as their relationships to the underlying regional F-actin density and the local cytoskeletal prestress. In the human airway smooth muscle cell, we used micropatterning to minimize geometric variation. We measured the local cell stiffness and loss tangent with optical magnetic twisting cytometry and the local rate of CSK remodeling with spontaneous displacements of a CSK-bound bead. We also measured traction distributions with traction microscopy and cell geometry with atomic force microscopy. On the basis of these experimental observations, we used finite element methods to map for the first time the regional distribution of intracellular prestress. Compared with the cell center or edges, cell corners were systematically stiffer and more fluidlike and supported higher traction forces, and at the same time had slower remodeling dynamics. Local remodeling dynamics had a close inverse relationship with local cell stiffness. The principal finding, however, is that systematic regional variations of CSK stiffness correlated only poorly with regional F-actin density but strongly and linearly with the regional prestress. Taken together, these findings in the intact cell comprise the most comprehensive characterization to date of regional variations of cytoskeletal mechanical properties and their determinants.

Percolation in a network with long-range connections: Implications for cytoskeletal structure and function

Cell shape, signaling, and integrity depend on cytoskeletal organization. In this study we describe the cytoskeleton as a simple network of filamentary proteins (links) anchored by complex protein structures (nodes). The structure of this network is regulated by a distance-dependent probability of link formation as P = p/d(s), where p regulates the network density and s controls how fast the probability for link formation decays with node distance (d). It was previously shown that the regulation of the link lengths is crucial for the mechanical behavior of the cells. Here we examined the ability of the two-dimensional network to percolate (i.e. to have end-to-end connectivity), and found that the percolation threshold depends strongly on s. The system undergoes a transition around s = 2. The percolation threshold of networks with s < 2 decreases with increasing system size L, while the percolation threshold for networks with s > 2 converges to a finite value. We speculate that s < 2 may represent a condition in which cells can accommodate deformation while still preserving their mechanical integrity. Additionally, we measured the length distribution of F-actin filaments from publicly available images of a variety of cell types. In agreement with model predictions, cells originating from more deformable tissues show longer F-actin cytoskeletal filaments. (C) 2008 Elsevier B.V. All rights reserved.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.