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Sample records for actin sma expression

  1. Effect of all-trans retinoic acids (ATRA) on the expression of α-smooth muscle actin (α-SMA) in the lung tissues of rats with pulmonary arterial hypertension (PAH).

    PubMed

    Xin, Y; Lv, J-Q; Wang, Y-Z; Zhang, J; Zhang, X

    2015-11-13

    The effect of all-trans retinoic acid (ATRA) on the expression of α-smooth muscle actin (α-SMA) in rats with pulmonary arterial hypertension (PAH) was studied, and the mechanism of the effect of ATRA on PAH was proposed. Thirty male SD rats were randomly divided into normal control, monocrotaline (MCT) model, and ATRA [30 mg/(kg.day)]intervention groups (N = 10 each). The mean pulmonary arterial pressure was recorded. Right ventricular hypertrophy index (RVHI) was calculated (weight of right ventricle: total weight of left ventricle and interventricular septum). The percentages of wall thickness of pulmonary arteriole (WT) to external diameter of artery (WT%) and vascular wall area (WA) to total vascular area (WA%) were determined. Real-time fluorescence-based quantitative PCR and western blot analyses were employed to detect the α-SMA mRNA and protein expressions. The mean pulmonary arterial pressure, RVHI, WT%, and WA% were all obviously higher in the model group than in the control and intervention groups. The values of these indicators in the intervention group were also higher than those in the control group (P < 0.01). The mRNA and protein expression levels of α-SMA were significantly higher in the lung tissue of model rats than those in the control and intervention groups. However, the intervention group showed no statistically significant differences in α-SMA mRNA and protein expression levels compared to the control (P < 0.05). ATRA inhibited the α-SMA mRNA and protein expressionin the lung tissues of rats with MCT-induced PAH, and could be used to treat PAH.

  2. Scleraxis modulates bone morphogenetic protein 4 (BMP4)-Smad1 protein-smooth muscle α-actin (SMA) signal transduction in diabetic nephropathy.

    PubMed

    Abe, Hideharu; Tominaga, Tatsuya; Matsubara, Takeshi; Abe, Naoko; Kishi, Seiji; Nagai, Kojiro; Murakami, Taichi; Araoka, Toshikazu; Doi, Toshio

    2012-06-08

    Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induced SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized scleraxis (Scx) as a new phenotype modulator in advanced glycation end product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morphogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides the TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in diabetic nephropathy.

  3. [Effect of two different acellular lung matrices on α-SMA expression in A549 cells].

    PubMed

    Chen, C; Wang, Z Y; Weng, J; Wang, Z B; Mei, J; Du, X H; Wang, L

    2017-01-24

    Objective: To explore the effect of acellular normal and fibrotic lung matrices on alpha smooth muscle actin (α-SMA) expression in human lung adenocarcinoma cell line A549. Methods: Twenty adult SD rats were randomly divided into normal group and idiopathic pulmonary fibrosis(IPF)group (n=10 each). The pulmonary fibrosis was induced by Bleomycin. Normal and fibrotic decellularized lungs were made, then sections with 500 μm thick were cut by a standard Vibratome. None scaffold was set as control group. A549 cells were seeded dropwise into different slices (normal and fibrotic scaffolds), and cultured for one week in vitro. The expression of α-SMA was measured by immunofluorescence staining and quantitative real time polymerase chain reaction (qRT-PCR). Results: In control group, the expression of α-SMA protein was positive in A549 cells by immunofluorescence staining. However, it expressed weakly both in normal and fibrotic scaffold group, and the fluorescence intensity in fibrotic scaffold group was significant lower than that in normal group (P<0.05). The relative expression amount of α-SMA mRNA in normal and fibrotic scaffold group were (0.70±0.11) and (0.55±0.12), which were significant lower than that of control group (1.28±0.21) (P<0.05). Moreover, the relative expression of α-SMA mRNA in fibrotic scaffold group was decreased compared to that in normal scaffold group (P<0.05). Conclusions: Acellular normal and fibrotic lung scaffold can downregulate the expression of α-SMA in human lung adenocarcinoma cell line A549. It may inhibit the movement of A549 cells in acellular normal and fibrotic lung matrices, especially in acellular fibrotic lung scaffold.

  4. Effect of ultrasound irradiation on α-SMA and TGF-β1 expression in human dermal fibroblasts.

    PubMed

    Maeshige, Noriaki; Terashi, Hiroto; Aoyama, Michiko; Torii, Kazuhiro; Sugimoto, Masaharu; Usami, Makoto

    2011-05-11

    Ultrasound therapy is used to promote pressure ulcer healing as an adjunctive therapy. However, the efficacy and the scientific basis of this treatment are unclear. We investigated the effect of ultrasound irradiation on alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1) expression in human dermal fibroblasts. These are important factors for acceleration of wound closure. We used pulsed ultrasound of 0, 0.1, 0.5, and 1.0 W/cm2. TGF-β1 and α-SMA mRNA was measured by quantitative real-time polymerase chain reaction, α-SMA protein was examined by western blot, and localization of α-SMA was evaluated by immunofluorescence staining. Expression of α-SMA and TGF-β1 mRNA was increased at 24 h but not at 48 h after ultrasound irradiation. There were significant differences between controls of 0 W/cm² and 0.1 W/cm² with a 1.34 ± 0.26 fold increase in α-SMA (P < 0.05) and a 1.78 ± 0.57 fold increase in TGF-β1 (P < 0.05). Protein levels of α-SMA were also increased and detected in ultrasound irradiated fibroblasts at 24 h. Ultrasound irradiation promotes α-SMA expression in human dermal fibroblasts and this suggests the biological mechanism of ultrasound efficacy on chronic wound treatment.

  5. Temporal requirement for high SMN expression in SMA mice

    PubMed Central

    Le, Thanh T.; McGovern, Vicki L.; Alwine, Isaac E.; Wang, Xueyong; Massoni-Laporte, Aurelie; Rich, Mark M.; Burghes, Arthur H.M.

    2011-01-01

    Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron 1 gene (SMN1) and retention of the SMN2 gene, resulting in reduced SMN. SMA mice can be rescued with high expression of SMN in neurons, but when is this high expression required? We have developed a SMA mouse with inducible expression of SMN to address the temporal requirement for high SMN expression. Both embryonic and early postnatal induction of SMN resulted in a dramatic increase in survival with some mice living greater than 200 days. The mice had no marked motor deficits and neuromuscular junction (NMJ) function was near normal thus it appears that induction of SMN in postnatal SMA mice rescues motor function. Early postnatal SMN induction, followed by a 1-month removal of induction at 28 days of age, resulted in no morphological or electrophysiological abnormalities at the NMJ and no overt motor phenotype. Upon removal of SMN induction, five mice survived for just over 1 month and two female mice have survived past 8 months of age. We suggest that there is a postnatal period of time when high SMN levels are required. Furthermore, two copies of SMN2 provide the minimal amount of SMN necessary to maintain survival during adulthood. Finally, in the course of SMA, early induction of SMN is most efficacious. PMID:21672919

  6. The expression of α-SMA in the painful traumatic neuroma: potential role in the pathobiology of neuropathic pain.

    PubMed

    Yan, Hede; Gao, Weiyang; Pan, Zhijun; Zhang, Feng; Fan, Cunyi

    2012-12-10

    The exact mechanism of neuroma-associated pain is not yet fully understood, thus contributing to the substantial challenge faced in managing patients with painful neuromas. We aimed to observe the expression of alpha smooth muscle actin (α-SMA) in the painful traumatic neuroma and to investigate its possible roles in the cause of neuroma-associated pain. Its expression is considered to be a useful phenotypic marker for myofibroblast, and may contribute to its increased contractile activity. We collected peripheral neuroma specimens prospectively and subsequently divided them into two groups: painful (n=21) and non-painful (n=27) based on blinded preoperative visual analogue scale (VAS) pain scores. We also harvested normal nerve specimens from the discarded limbs as a control group (n=8). We performed immunohistological studies to observe the expression of α-SMA in each group, and calculated the expression level by a high-resolution pathological image analysis system. There was no positive staining of α-SMA observed in the control group, slight positive staining in the non-painful group, and obviously positive staining in the painful group. Pearson correlation analysis demonstrated that VAS scores were significantly associated with the expression intensity of α-SMA (R=0.831; p<0.001). Linear regression analysis indicated that the expression intensity of α-SMA was positively related to the scale of VAS (R(2)=0.691, p<0.001). These findings suggest that: 1) expression of α-SMA may play certain roles in painful traumatic neuroma, either as a direct cause of neuroma-associated pain or as an indirect marker of local mechanical stimuli, and 2) the presence of α-SMA in the painful group may provide rationale for transpositional procedures in the management of traumatic neuroma. The persistent existence of α-SMA in the painful group and the correlation with VAS scores may provide insight into the development of new therapeutic strategies.

  7. Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells.

    PubMed

    Hung, Shih-Chieh; Kuo, Pei-Yin; Chang, Ching-Fang; Chen, Tain-Hsiung; Ho, Larry Low-Tone

    2006-06-01

    The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

  8. Assay to evaluate BAL Fluid regulation of Fibroblast α-SMA Expression

    PubMed Central

    Larson-Casey, Jennifer L.; Carter, A. Brent

    2016-01-01

    Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol allows evaluating the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA). Background Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins. PMID:28239621

  9. Recombinant T2 RNase protein of Schistosoma japonicum inhibits expression of α-SMA in LX-2 cells.

    PubMed

    Wang, Jianxin; Peng, Wenxia; Feng, Jinrong; Zhu, Dandan; Chen, Jinling; Sun, Xiaolei; Lyu, Lei; Ju, Shaoqing; Duan, Yinong

    2016-10-01

    Recombinant T2 RNase glycoprotein, which showed a certain degree of homology to Omega-1 from Schistosoma mansoni eggs, was expressed in adult worms of Schistosoma japonicum, but not in eggs of S. japonicum. The direct biological role of the recombinant T2 RNase protein in activation of hepatic stellate cells (HSCs) remains unknown. In the present study, the immortalized human HSC line (LX-2 cells) was treated with the recombinant T2 RNase protein at indicated concentrations for various time points in vitro. The expression levels of α-smooth muscle actin (α-SMA) and Smad4 were detected by Western blot. The results showed that the recombinant T2 RNase protein significantly diminished the expression levels of α-SMA and Smad4 in LX-2 cells. The upregulated expression levels of α-SMA and Smad4 by TGF-β1 in LX-2 cells were both suppressed by the recombinant T2 RNase protein. These data suggest that the recombinant T2 RNase protein may be a potential target of therapeutic strategy for the treatment of hepatic fibrosis.

  10. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a ...

  11. Plastin 3 Expression Does Not Modify Spinal Muscular Atrophy Severity in the ∆7 SMA Mouse.

    PubMed

    McGovern, Vicki L; Massoni-Laporte, Aurélie; Wang, Xueyong; Le, Thanh T; Le, Hao T; Beattie, Christine E; Rich, Mark M; Burghes, Arthur H M

    2015-01-01

    Spinal muscular atrophy is caused by loss of the SMN1 gene and retention of SMN2. The SMN2 copy number inversely correlates with phenotypic severity and is a modifier of disease outcome. The SMN2 gene essentially differs from SMN1 by a single nucleotide in exon 7 that modulates the incorporation of exon 7 into the final SMN transcript. The majority of the SMN2 transcripts lack exon 7 and this leads to a SMN protein that does not effectively oligomerize and is rapidly degraded. However the SMN2 gene does produce some full-length SMN and the SMN2 copy number along with how much full-length SMN the SMN2 gene makes correlates with severity of the SMA phenotype. However there are a number of discordant SMA siblings that have identical haplotypes and SMN2 copy number yet one has a milder form of SMA. It has been suggested that Plastin3 (PLS3) acts as a sex specific phenotypic modifier where increased expression of PLS3 modifies the SMA phenotype in females. To test the effect of PLS3 overexpression we have over expressed full-length PLS3 in SMA mice. To ensure no disruption of functionality or post-translational processing of PLS3 we did not place a tag on the protein. PLS3 protein was expressed under the Prion promoter as we have shown previously that SMN expression under this promoter can rescue SMA mice. High levels of PLS3 mRNA were expressed in motor neurons along with an increased level of PLS3 protein in total spinal cord, yet there was no significant beneficial effect on the phenotype of SMA mice. Specifically, neither survival nor the fundamental electrophysiological aspects of the neuromuscular junction were improved upon overexpression of PLS3 in neurons.

  12. Plastin 3 Expression Does Not Modify Spinal Muscular Atrophy Severity in the ∆7 SMA Mouse

    PubMed Central

    Wang, Xueyong; Le, Thanh T.; Le, Hao T.; Beattie, Christine E.; Rich, Mark M.; Burghes, Arthur H. M.

    2015-01-01

    Spinal muscular atrophy is caused by loss of the SMN1 gene and retention of SMN2. The SMN2 copy number inversely correlates with phenotypic severity and is a modifier of disease outcome. The SMN2 gene essentially differs from SMN1 by a single nucleotide in exon 7 that modulates the incorporation of exon 7 into the final SMN transcript. The majority of the SMN2 transcripts lack exon 7 and this leads to a SMN protein that does not effectively oligomerize and is rapidly degraded. However the SMN2 gene does produce some full-length SMN and the SMN2 copy number along with how much full-length SMN the SMN2 gene makes correlates with severity of the SMA phenotype. However there are a number of discordant SMA siblings that have identical haplotypes and SMN2 copy number yet one has a milder form of SMA. It has been suggested that Plastin3 (PLS3) acts as a sex specific phenotypic modifier where increased expression of PLS3 modifies the SMA phenotype in females. To test the effect of PLS3 overexpression we have over expressed full-length PLS3 in SMA mice. To ensure no disruption of functionality or post-translational processing of PLS3 we did not place a tag on the protein. PLS3 protein was expressed under the Prion promoter as we have shown previously that SMN expression under this promoter can rescue SMA mice. High levels of PLS3 mRNA were expressed in motor neurons along with an increased level of PLS3 protein in total spinal cord, yet there was no significant beneficial effect on the phenotype of SMA mice. Specifically, neither survival nor the fundamental electrophysiological aspects of the neuromuscular junction were improved upon overexpression of PLS3 in neurons. PMID:26134627

  13. The role of bFGF in down-regulating α-SMA expression of chondrogenically induced BMSCs and preventing the shrinkage of BMSC engineered cartilage.

    PubMed

    Li, Qiong; Liu, Tianyi; Zhang, Lu; Liu, Yu; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

    2011-07-01

    Bone marrow stromal cells (BMSCs) have proved to be an ideal cell source for cartilage regeneration. Our previous studies demonstrated that a three-dimensional (3D) cartilage could be constructed successfully in vitro using BMSCs and biodegradable scaffolds. However, an obvious shrinkage and deformation was observed during in vitro chondrogenic induction. According to the literatures, it can be speculated that the up-regulation of smooth muscle actin-alpha (α-SMA) caused by transforming growth factor beta (TGFβ) is one of the leading reasons and that basic fibroblast growth factor (bFGF) could antagonize the role of TGFβ to down-regulate α-SMA expression and prevent the shrinkage of BMSC engineered cartilage. This study testified these speculations by adding bFGF to chondrogenic media. According to the current results, chondrogenic induction significantly up-regulated α-SMA expression of BMSCs at both cell and tissue levels, and the engineered tissue only retained 12.4% of original size after 6 weeks of chondrogenic induction. However, the supplement of bFGF in chondrogenic media efficiently down-regulated α-SMA expression and the engineered tissue still retained over 60% of original size after 6 weeks of culture. Moreover, bFGF showed a beneficial influence on 3D cartilage formation of BMSCs in terms of gene expression and deposition of cartilage specific matrices. All these results suggested that bFGF could repress α-SMA expression caused by chondrogenic induction, efficiently prevent shrinkage of BMSC engineered tissue, and have a positive influence on cartilage formation, which provides a clue for both shape control and quality improvement of BMSC engineered 3D cartilage.

  14. Modulation of MCP-1, TGF-β1, and α-SMA Expressions in Granulation Tissue of Cutaneous Wounds Treated with Local Vitamin B Complex: An Experimental Study

    PubMed Central

    Martinelli-Kläy, Carla P.; Lunardi, Laurelucia O.; Martinelli, Celso Ricardo; Lombardi, Tommaso; Soares, Edson G.; Martinelli, Celso

    2014-01-01

    Vitamin B complex can modulate the inflammatory response and activate wound healing. However, the action mechanisms involved in this process are still unclear. The aim of this study was to evaluate the effects of vitamin B complex on the modulation of monocyte chemotactic protein (MCP)-1, transforming growth factor (TGF)-β1, and α-smooth muscle actin (α-SMA) in granulation tissue growth. Cutaneous ulcers on Wistar rats were topically treated with vitamin B complex. MCP-1, TGF-β1, and α-SMA expressions were evaluated 24, 72, and 168 h after the treatment. Inflammatory cells were counted and collagen fibril staining was performed. After 24 h, more mononuclear cells (p ≤ 0.01) and a higher MCP-1 (p ≤ 0.05) and TGF-β1 (p ≤ 0.01) expression were observed. After 72 h, the number of fibroblasts and mononuclear cells (p ≤ 0.05) was elevated. After 168 h, an increased number of fibroblasts, myofibroblasts, and blood vessels (p ≤ 0.01) as well as a strong intensity of collagen fibril staining were seen. At that point, the cells presented a higher TGF-β1 expression (p ≤ 0.05), and the size of the ulcer area was decreased (p ≤ 0.01). We can conclude that vitamin B complex may stimulate a positive modulation of MCP-1, TGF-β1, and α-SMA expressions in granulation tissue of cutaneous ulcers. PMID:27047929

  15. Modulation of MCP-1, TGF-β1, and α-SMA Expressions in Granulation Tissue of Cutaneous Wounds Treated with Local Vitamin B Complex: An Experimental Study.

    PubMed

    Martinelli-Kläy, Carla P; Lunardi, Laurelucia O; Martinelli, Celso Ricardo; Lombardi, Tommaso; Soares, Edson G; Martinelli, Celso

    2014-01-01

    Vitamin B complex can modulate the inflammatory response and activate wound healing. However, the action mechanisms involved in this process are still unclear. The aim of this study was to evaluate the effects of vitamin B complex on the modulation of monocyte chemotactic protein (MCP)-1, transforming growth factor (TGF)-β1, and α-smooth muscle actin (α-SMA) in granulation tissue growth. Cutaneous ulcers on Wistar rats were topically treated with vitamin B complex. MCP-1, TGF-β1, and α-SMA expressions were evaluated 24, 72, and 168 h after the treatment. Inflammatory cells were counted and collagen fibril staining was performed. After 24 h, more mononuclear cells (p ≤ 0.01) and a higher MCP-1 (p ≤ 0.05) and TGF-β1 (p ≤ 0.01) expression were observed. After 72 h, the number of fibroblasts and mononuclear cells (p ≤ 0.05) was elevated. After 168 h, an increased number of fibroblasts, myofibroblasts, and blood vessels (p ≤ 0.01) as well as a strong intensity of collagen fibril staining were seen. At that point, the cells presented a higher TGF-β1 expression (p ≤ 0.05), and the size of the ulcer area was decreased (p ≤ 0.01). We can conclude that vitamin B complex may stimulate a positive modulation of MCP-1, TGF-β1, and α-SMA expressions in granulation tissue of cutaneous ulcers.

  16. Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle

    PubMed Central

    Chaponnier, Christine; Gabbiani, Giulio

    2016-01-01

    Higher vertebrates (mammals and birds) express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (α-SMA) was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986) . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes. PMID:27335638

  17. [Effects of hydroxycamptothecin on TGFb1, a-SMA and collagen I expression in rat hepatic satellite cells].

    PubMed

    Hu, Guo-xin; Wan, Zan-yan; Shao, Jia-liang; Zhang, Yi; Zhang, Lun-li; Gong, Zuo-jiong

    2012-06-01

    To investigate the molecular mechanism of hydroxycamptothecin (HCPT)-mediated anti-hepatic fibrosis by evaluting its effects on expression of tumor growth factor-beta 1 (TGFb1), alpha-smooth muscle actin (a-SMA) and collagen I (Col I) in hepatic satellite cells (HSCs). Cultured HSCs were treated with different concentrations of HCPT: low-dose group, 0.25 mg/L; middle-dose group, 0.5 mg/L; high-dose group, 0.75 mg/L; and control group, 0 mg/L. Cell proliferation was assessed by the MTT assay. The mRNA expressions of TGFb1, a-SMA and Col I were determined by reverse transcription-polymerase chain reaction. The protein expressions of TGFb1 and a-SMA were detected by Western blot. The content of Col I in the cultured HSCs' supernatant was measured by enzyme-linked immunosorbent assay. The MTT absorbance values of the low-dose group (0.631+/-0.074), middle-dose group (0.469+/- 0.012) and high-dose group (0.204+/- 0.001) were significantly lower than that of the control group (0.793+/-0.098; F = 82.86, P less than 0.01). Compared with the control group, the HCPT-treated groups showed significantly down-regulated gene expressions of TGFb1 (control: 0.716+/-0.064 vs. low: 0.611+/-0.040, middle: 0.510+/-0.014, high: 0.403+/-0.026), a-SMA (control: 0.696+/-0.075 vs. low: 0.579+/-0.037, middle: 0.470+/-0.024, high: 0.299+/-0.017), and Col I (control: 1.019+/-0.056 vs. low: 0.835+/-0.022, middle: 0.696+/-0.055, high: 0.322+/-0.104) (all, P less than 0.01). Meanwhile, HCPT-treated HSCs showed significantly reduced protein expressions of TGFb1 (control: 0.872+/-0.053 vs. low: 0.654+/-0.047, middle: 0.545+/-0.042, high: 0.436+/-0.039) and a-SMA (control: 0.858+/-0.050 vs. low: 0.620+/-0.045, middle: 0.525+/-0.042, high: 0.434+/-0.052) (all, P less than 0.01). The Col I levels secreted by HSCs were significantly lower in the HCPT-treated groups (low: 168.367+/-16.453 ng/ml; middle: 141.284+/-11.731 ng/ml; high: 132.910+/-10.048 ng/ml) than in the control group (188.733 +/-18

  18. Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

    Purpose To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Methods Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actinSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Results Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Conclusions Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes. PMID:26313302

  19. Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.

    PubMed

    Barnes, V L; Musa, J; Mitchell, R J; Barnes, J L

    1999-06-01

    In this study we examined if an association exists between expression of an alternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and alpha-smooth muscle actin (alpha-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and alpha-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and alpha-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and alpha-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and alpha-SMA in anti-GBM disease and suggest a functional link for these two proteins.

  20. Small gastrointestinal stromal tumor in the stomach: identification of precursor for clinical gastrointestinal stromal tumor using c-kit and α-smooth muscle actin expression.

    PubMed

    Mikami, Tetuo; Nemoto, Yuta; Numata, Yoshiko; Hana, Kiyomi; Nakada, Norihiro; Ichinoe, Masaaki; Murakumo, Yoshiki; Okayasu, Isao

    2013-12-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. To find precursors for clinical GISTs of the stomach, small gastric stromal tumors of less than 3 cm were collected and examined immunohistochemically with analysis of the KIT mutation. Sixty-eight of 74 lesions were classified into 4 representative groups according to the expression of c-kit and α-smooth muscle actinSMA): group A, c-kit diffusely positive and αSMA negative (18 cases); group B, c-kit diffusely positive and αSMA focally positive (13); group C, c-kit focally positive and αSMA diffusely positive (27); and group D, c-kit negative and αSMA diffusely positive (10). Of the 4 groups, groups A and B of c-kit diffuse expression showed higher cellularity and labeling indices of p27(Kip1) and Ki-67 than did groups C and D of diffuse αSMA expression. Incidence of KIT exon 11 mutation in groups A and B was 86% (25/29), whereas that in groups C and D was 0% (0/20). Small gastric stromal tumors with c-kit diffuse expression were considered precursors for clinical GIST because they were significantly different from c-kit focally positive or negative tumors. The mutation of KIT is considered as an early event in tumorigenesis of GIST.

  1. Expression of cardiac alpha-actin spares extraocular muscles in skeletal muscle alpha-actin diseases--quantification of striated alpha-actins by MRM-mass spectrometry.

    PubMed

    Ravenscroft, Gianina; Colley, Stephen M J; Walker, Kendall R; Clement, Sophie; Bringans, Scott; Lipscombe, Richard; Fabian, Victoria A; Laing, Nigel G; Nowak, Kristen J

    2008-12-01

    As with many skeletal muscle diseases, the extraocular muscles (EOMs) are spared in skeletal muscle alpha-actin diseases, with no ophthalmoplegia even in severely affected patients. We hypothesised that the extraocular muscles sparing in these patients was due to significant expression of cardiac alpha-actin, the alpha-actin isoform expressed in heart and foetal skeletal muscle. We have shown by immunochemistry, Western blotting and a novel MRM-mass spectrometry technique, comparable levels of cardiac alpha-actin in the extraocular muscles of human, pig and sheep to those in the heart. The sparing of extraocular muscles in skeletal muscle alpha-actin disease is thus probably due to greater levels of cardiac alpha-actin, than the negligible amounts in skeletal muscles, diluting out the effects of the mutant skeletal muscle alpha-actin.

  2. PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

    PubMed Central

    Sriram, Sriniwas; Tran, Jennifer A.; Guo, Xiaoqing; Hutcheon, Audrey E. K.; Lei, Hetian; Kazlauskas, Andrius; Zieske, James D.

    2017-01-01

    Purpose The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing. PMID:28245298

  3. Simvastatin inhibits transforming growth factor-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts.

    PubMed

    Mun, Je-Ho; Kim, Young-Mi; Kim, Byung-Soo; Kim, Jae-Ho; Kim, Moon-Bum; Ko, Hyun-Chang

    2014-01-01

    Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti-inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)-β1-induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α-smooth muscle actin (α-SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF-β1, and the effects of simvastatin on TGF-β1-induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α-SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Simvastatin suppressed TGF-β1-induced type I collagen, CTGF, and α-SMA production in a concentration-dependent manner. The TGF-β1-induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α-SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin-induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF-β1-induced RhoA activation. The Rho-associated coiled kinase inhibitor Y27632 abrogated TGF-β1-induced production of type I collagen

  4. Implication of fetal SMN2 expression in type I SMA pathogenesis: protection or pathological gain of function?

    PubMed

    Soler-Botija, Carolina; Cuscó, Ivón; Caselles, Lídia; López, Eva; Baiget, Montserrat; Tizzano, Eduardo F

    2005-03-01

    Spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene 1 (SMN1). The SMN2 gene, which is the highly homologous SMN1 copy that is present in all the patients, is unable to prevent the disease. Most of the SMN1 transcript is full-length, whereas a substantial proportion of the SMN2 transcript lacks exon 7 (delta7). We characterized the developmental expression of SMN2 by comparing control and SMA fetuses. The control spinal cord revealed the highest amount of FL SMN, most of which was of SMN1 origin. When analyzing the SMA spinal cord transcripts, we detected a considerable reduction in the FL/delta7 ratios due to a decrease in the FL and an increase in delta7 isoform. After immunoblot and immunohistochemistry analyses, we found that the amount of SMN2 protein in the SMA spinal cord and muscle was lower than in the controls. However, the results of the expression of SMN2 in intestine, lung, adrenal gland, kidney, and eye, which are unaffected by the disease, were the same in controls and SMA samples. In these tissues, SMN2 may compensate for the absence of SMN1, whereas in SMA motor neurons, a cell-specific dysregulation of the SMN2 expression could favor the onset of the acute form of the disease.

  5. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  6. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    SciTech Connect

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  7. Hepatoprotective effect of juglone on dimethylnitrosamine-induced liver fibrosis and its effect on hepatic antioxidant defence and the expression levels of α-SMA and collagen III.

    PubMed

    Zhou, De-Jiang; Mu, Dong; Jiang, Ming-De; Zheng, Shu-Mei; Zhang, Yong; He, Sheng; Weng, Min; Zeng, Wei-Zheng

    2015-09-01

    The present study aimed to investigate the antifibrotic effects of juglone on dimethylnitrosamine (DMN)‑induced fibrosis in rats. Juglone, which is a quinone, significantly decreased DMN‑induced rat hepatic fibrosis, which was associated with increased superoxide dismutase (SOD) activity, decreased oxidative stress and reduced levels of α‑smooth muscle actin (α‑SMA) and collagen (Col) III in the liver. Serum levels of alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, laminin, type III precollagen and type IV collagen were significantly reduced by treatment with juglone. Liver fibrosis was induced in male Sprague‑Dawley rats by subcutaneous injections of DMN solution and hepatic fibrosis was assessed using Massons trichome staining. The expression levels of α‑SMA and Col III were determined using immunohistochemical techniques. The activities of SOD and malondialdehyde in liver homogenates were also determined. The results suggested that juglone augmented the antioxidative capability of the liver, possibly by stimulating the activity of SOD, which promoted the inactivation of hepatic stellate cells (HSCs) and decreased the accumulation of extracellular matrix collagen in the liver, thereby alleviating hepatic fibrosis. Silymarin was used as a positive control for liver fibrosis protection. It was hypothesized that juglone alleviates or mitigates oxidative stress‑mediated hepatic fibrosis by upregulating the expression of peroxisome proliferator‑activated receptor γ and inhibiting the activation of HSC.

  8. αSMA Expression in Large Colonies of Colony-Forming Units-Fibroblast as an Early Predictor of Bone Marrow MSC Expandability

    PubMed Central

    Aizman, Irina; Holland, William S.; Yang, Cher; Bates, Damien

    2016-01-01

    Clinical applications of mesenchymal stromal cells (MSCs) require the manufacture of large cell lots, which involves multiple passages for cell expansion and sometimes genetic modification. MSCs from various sources, including bone marrow (BM), exhibit high donor-to-donor variability in their growth characteristics. This can lead to unpredictable manufacturing outcomes with respect to success or failure of individual lots. Early determination of lot success has the potential to reduce the cost and improve the efficiency of the MSC manufacturing process. However, methods that effectively predict lot growth potential early in the manufacturing process are currently lacking. Here we report that the growth potential of an MSC lot can be predicted a few days after BM plating based on α-smooth muscle actinSMA) protein expression in large colony-forming unit-fibroblast (CFU-f) colonies. The proposed prediction method could be a useful tool to prospectively determine MSC lot success or failure. PMID:28003933

  9. Gamma-Smooth Muscle Actin Expression Is Associated with Epithelial-Mesenchymal Transition and Stem-Like Properties in Hepatocellular Carcinoma

    PubMed Central

    Benzoubir, Nassima; Mussini, Charlotte; Lejamtel, Charlène; Dos Santos, Alexandre; Guillaume, Claire; Desterke, Christophe; Samuel, Didier; Bréchot, Christian; Bourgeade, Marie-Françoise; Guettier, Catherine

    2015-01-01

    Background and Aims The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process. Methods The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series. Results We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19. Conclusion Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression. PMID:26110787

  10. Actin organization and gene expression in Beta vulgaris seedlings under clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Y.; Shevchenko, G. V.; Artemenko, O. A.; Martyn, G. G.; Kordyum, E. L.

    2005-08-01

    Actin microfilaments (MFs) as highly dynamic structure respond by rapid reorganization to different external influences, including gravity. The object of our experiments was to examine both the actin organization and actin gene expression during growth and differentiation of root cells under clinorotation. It was shown that MFs acted as the indicator of changes caused by altered gravity in distal elongation zone (DEZ) cells, particularly actin cytoskeleton is enhanced in cortex cells. The data testify stable actin expression under altered gravity. The F-actin MFs enhancement in cortex cells of the DEZ occurred under clinorotation at the same level of the total actin content as in the stationary conditions is suggested to be caused by transformation of G-actin into F-actin.

  11. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    SciTech Connect

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  12. Expression of Muscle-Specific MiRNA 206 in the Progression of Disease in a Murine SMA Model

    PubMed Central

    Valsecchi, Valeria; Boido, Marina; De Amicis, Elena; Piras, Antonio; Vercelli, Alessandro

    2015-01-01

    Spinal muscular atrophy (SMA) is a severe neuromuscular disease, the most common in infancy, and the third one among young people under 18 years. The major pathological landmark of SMA is a selective degeneration of lower motor neurons, resulting in progressive skeletal muscle denervation, atrophy, and paralysis. Recently, it has been shown that specific or general changes in the activity of ribonucleoprotein containing micro RNAs (miRNAs) play a role in the development of SMA. Additionally miRNA-206 has been shown to be required for efficient regeneration of neuromuscular synapses after acute nerve injury in an ALS mouse model. Therefore, we correlated the morphology and the architecture of the neuromuscular junctions (NMJs) of quadriceps, a muscle affected in the early stage of the disease, with the expression levels of miRNA-206 in a mouse model of intermediate SMA (SMAII), one of the most frequently used experimental model. Our results showed a decrease in the percentage of type II fibers, an increase in atrophic muscle fibers and a remarkable accumulation of neurofilament (NF) in the pre-synaptic terminal of the NMJs in the quadriceps of SMAII mice. Furthermore, molecular investigation showed a direct link between miRNA-206-HDAC4-FGFBP1, and in particular, a strong up-regulation of this pathway in the late phase of the disease. We propose that miRNA-206 is activated as survival endogenous mechanism, although not sufficient to rescue the integrity of motor neurons. We speculate that early modulation of miRNA-206 expression might delay SMA neurodegenerative pathway and that miRNA-206 could be an innovative, still relatively unexplored, therapeutic target for SMA. PMID:26030275

  13. Expression of Muscle-Specific MiRNA 206 in the Progression of Disease in a Murine SMA Model.

    PubMed

    Valsecchi, Valeria; Boido, Marina; De Amicis, Elena; Piras, Antonio; Vercelli, Alessandro

    2015-01-01

    Spinal muscular atrophy (SMA) is a severe neuromuscular disease, the most common in infancy, and the third one among young people under 18 years. The major pathological landmark of SMA is a selective degeneration of lower motor neurons, resulting in progressive skeletal muscle denervation, atrophy, and paralysis. Recently, it has been shown that specific or general changes in the activity of ribonucleoprotein containing micro RNAs (miRNAs) play a role in the development of SMA. Additionally miRNA-206 has been shown to be required for efficient regeneration of neuromuscular synapses after acute nerve injury in an ALS mouse model. Therefore, we correlated the morphology and the architecture of the neuromuscular junctions (NMJs) of quadriceps, a muscle affected in the early stage of the disease, with the expression levels of miRNA-206 in a mouse model of intermediate SMA (SMAII), one of the most frequently used experimental model. Our results showed a decrease in the percentage of type II fibers, an increase in atrophic muscle fibers and a remarkable accumulation of neurofilament (NF) in the pre-synaptic terminal of the NMJs in the quadriceps of SMAII mice. Furthermore, molecular investigation showed a direct link between miRNA-206-HDAC4-FGFBP1, and in particular, a strong up-regulation of this pathway in the late phase of the disease. We propose that miRNA-206 is activated as survival endogenous mechanism, although not sufficient to rescue the integrity of motor neurons. We speculate that early modulation of miRNA-206 expression might delay SMA neurodegenerative pathway and that miRNA-206 could be an innovative, still relatively unexplored, therapeutic target for SMA.

  14. SMN expression is required in motor neurons to rescue electrophysiological deficits in the SMNΔ7 mouse model of SMA.

    PubMed

    McGovern, Vicki L; Iyer, Chitra C; Arnold, W David; Gombash, Sara E; Zaworski, Phillip G; Blatnik, Anton J; Foust, Kevin D; Burghes, Arthur H M

    2015-10-01

    Proximal spinal muscular atrophy (SMA) is the most frequent cause of hereditary infant mortality. SMA is an autosomal recessive neuromuscular disorder that results from the loss of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The SMN2 gene produces an insufficient amount of full-length SMN protein that results in loss of motor neurons in the spinal cord and subsequent muscle paralysis. Previously we have shown that overexpression of human SMN in neurons in the SMA mouse ameliorates the SMA phenotype while overexpression of human SMN in skeletal muscle had no effect. Using Cre recombinase, here we show that either deletion or replacement of Smn in motor neurons (ChAT-Cre) significantly alters the functional output of the motor unit as measured with compound muscle action potential and motor unit number estimation. However ChAT-Cre alone did not alter the survival of SMA mice by replacement and did not appreciably affect survival when used to deplete SMN. However replacement of Smn in both neurons and glia in addition to the motor neuron (Nestin-Cre and ChAT-Cre) resulted in the greatest improvement in survival of the mouse and in some instances complete rescue was achieved. These findings demonstrate that high expression of SMN in the motor neuron is both necessary and sufficient for proper function of the motor unit. Furthermore, in the mouse high expression of SMN in neurons and glia, in addition to motor neurons, has a major impact on survival.

  15. Cure SMA

    MedlinePlus

    ... the SMA drug pipeline. Together, we're making real progress toward a treatment and a cure. Latest ... Announce SPINRAZA (nusinersen) Meets Primary Endpoint at Interim Analysis of Phase 3 CHERISH Study Biogen and Ionis ...

  16. High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

    PubMed

    Chen, Yu-Chia; Chang, Jan-Gowth; Jong, Yuh-Jyh; Liu, Ting-Yuan; Yuo, Chung-Yee

    2015-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn-/- SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.

  17. Cardiac α-actin over-expression therapy in dominant ACTA1 disease.

    PubMed

    Ravenscroft, Gianina; McNamara, Elyshia; Griffiths, Lisa M; Papadimitriou, John M; Hardeman, Edna C; Bakker, Anthony J; Davies, Kay E; Laing, Nigel G; Nowak, Kristen J

    2013-10-01

    More than 200 mutations in the skeletal muscle α-actin gene (ACTA1) cause either dominant or recessive skeletal muscle disease. Currently, there are no specific therapies. Cardiac α-actin is 99% identical to skeletal muscle α-actin and the predominant actin isoform in fetal muscle. We previously showed cardiac α-actin can substitute for skeletal muscle α-actin, preventing the early postnatal death of Acta1 knock-out mice, which model recessive ACTA1 disease. Dominant ACTA1 disease is caused by the presence of 'poison' mutant actin protein. Experimental and anecdotal evidence nevertheless indicates that the severity of dominant ACTA1 disease is modulated by the relative amount of mutant skeletal muscle α-actin protein present. Thus, we investigated whether transgenic over-expression of cardiac α-actin in postnatal skeletal muscle could ameliorate the phenotype of mouse models of severe dominant ACTA1 disease. In one model, lethality of ACTA1(D286G). Acta1(+/-) mice was reduced from ∼59% before 30 days of age to ∼12%. In the other model, Acta1(H40Y), in which ∼80% of male mice die by 5 months of age, the cardiac α-actin transgene did not significantly improve survival. Hence cardiac α-actin over-expression is likely to be therapeutic for at least some dominant ACTA1 mutations. The reason cardiac α-actin was not effective in the Acta1(H40Y) mice is uncertain. We showed that the Acta1(H40Y) mice had endogenously elevated levels of cardiac α-actin in skeletal muscles, a finding not reported in dominant ACTA1 patients.

  18. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.

    PubMed

    Haynes, Jennifer; Srivastava, Jyoti; Madson, Nikki; Wittmann, Torsten; Barber, Diane L

    2011-12-01

    Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

  19. Increase of α-SMA(+) and CK (+) cells as an early sign of epithelial-mesenchymal transition during colorectal carcinogenesis.

    PubMed

    Valcz, Gábor; Sipos, Ferenc; Krenács, Tibor; Molnár, Jeannette; Patai, Arpád V; Leiszter, Katalin; Tóth, Kinga; Wichmann, Barna; Molnár, Béla; Tulassay, Zsolt

    2012-04-01

    Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

  20. Mucin1 expression in focal epidermal dysplasia of actinic keratosis

    PubMed Central

    Carrillo, Luz Marina; Rojas, Héctor; Ramírez, Richard; Reyes, Oscar; Suárez, Ambar; Ortega, Fabiana

    2015-01-01

    Background Actinic keratoses (AKs) are generally considered as premalignant skin lesions that can progress into squamous cell carcinoma (SCC) in situ and invasive SCC. However, its progression to SCC is still matter of debate. A transmembrane glycoprotein that contributes to the progression of certain premalignant and malignant lesions is mucin1 (MUC1). Nevertheless, their functions in the skin lesions are not yet fully clear. Therefore, the aim of this study is to ascertain whether MUC1 is present in the focal epidermal dysplasia of AK. Methods Fourteen skin biopsies from patients diagnosed with AK were selected. They were classified according to the degree of dysplasia in keratinocyte intraepidermal neoplasia (KIN) I, KIN II, and KIN III. In five biopsies the three degrees were present, in two biopsies both KIN I and KIN II, in four biopsies only KIN I, and in three biopsies only KIN III. The presence of MUC1 was assessed by immunofluorescence staining using confocal laser scanning microscopy. Results Immunostaining revealed that MUC1 was present over the entire cell surface of only a few atypical basal keratinocytes confined to the lower third of the epidermis (KIN I). While in KIN II where atypical keratinocytes occupy the lower two thirds, MUC1 was localized at the apical surface of some atypical keratinocytes and over the entire cell surface of some of them. Interestingly, in KIN III where the atypical keratinocytes extend throughout the full thickness, MUC1 was localized at the apical surface and over the entire cell surface of many of these cells. Conversely, MUC1 expression was not detected in the epidermis of normal skin. Conclusions Our findings suggest that the expression of MUC1 in AK would be induced by alteration of keratinocyte stratification and differentiation and associated to the degree of dysplasia rather than the thickness of the epidermis. PMID:26605291

  1. NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP)

    PubMed Central

    Di Zazzo, Antonio; Sgrulletta, Roberto; Cortes, Magdalena; Normando, Eduardo Maria; Lambiase, Alessandro; Bonini, Stefano

    2015-01-01

    Objective In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkANGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure. Materials and Methods Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkANGFR/p75NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkANGFR/p75NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters. Results OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75NTR and lower trkANGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75NTR. NGF exposure did not affect trkANGFR levels in early OCP-FBs while decreased both αSMA/p75NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs. Conclusions Taken together, these data are suggestive for a NGF/p75NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75NTR might be viewed as a potential therapeutic target. PMID:26569118

  2. Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, α smooth muscle actin expression and type I collagen synthesis in vitro

    PubMed Central

    Cheng, Yang; Ping, Jian; Xu, Huai-Dong; Fu, Hai-Jun; Zhou, Zhao-Hui

    2006-01-01

    AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and α smooth muscle actinSMA) expression, type I, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P = 0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P < 0.01; protein, P < 0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and typeI collagen synthesis in HSC-T6 cells than oxymatrine in vitro. PMID:16937525

  3. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    PubMed

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

  4. Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice.

    PubMed

    Wang, Yaning; Lin, Chao; Ren, Qiang; Liu, Yunqi; Yang, Xiangdong

    2015-01-01

    Numerous cytokines participate in the occurrence and development of inflammation and renal interstitial fibrosis. Previous studies confirmed that TGF-β1 overexpressed in diabetic nephropathy. As a downstream signal protein of TGF-β1 family, SMAD has an important role in the process of α-SMA mediated renal interstitial fibrosis. This study aimed to study astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissue of diabetic KKAy mice, to reveal its potential impact on renal interstitial fibrosis. 20 type II diabetic KKAy mice were randomly equally divided into model group and astragaloside group, while 10 male C57BL/6J mice were selected as the control. Astragaloside at 40 mg/(kg•d) was given when the KKAy mice fed with high-fat diet to 14 weeks old. The mice were killed at 24 weeks old and the kidney tissue samples were collected. Pathology morphological changes were observed. TGF-β1, SMAD2/3, and α-SMA expression levels were determined by immunohistochemistry. Compared with control, mice kidney in model group appeared obvious fibrosis and up-regulated blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression (P < 0.05). Mice in astragaloside group exhibited alleviated renal interstitial fibrosis compared with the model. Its blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression levels were significantly lower than the model group (P < 0.05). Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1, SMAD2/3, and α-SMA expression.

  5. SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

    PubMed Central

    Murdocca, Michela; Ciafrè, Silvia Anna; Spitalieri, Paola; Talarico, Rosa Valentina; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells. PMID:27483257

  6. SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression.

    PubMed

    Murdocca, Michela; Ciafrè, Silvia Anna; Spitalieri, Paola; Talarico, Rosa Valentina; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica

    2016-07-30

    Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.

  7. Expression of actin genes in the arrow worm Paraspadella gotoi (Chaetognatha).

    PubMed

    Yasuda, E; Goto, T; Makabe, K W; Satoh, N

    1997-12-01

    Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phylogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic cells, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm.

  8. Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein.

    PubMed

    Hofmann, Christina; Niehl, Annette; Sambade, Adrian; Steinmetz, André; Heinlein, Manfred

    2009-04-01

    The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

  9. Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P

    PubMed Central

    Bai, Fan; Caster, Hannah M.; Rubenstein, Peter A.; Dawson, John F.; Kawai, Masataka

    2014-01-01

    Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2+], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2+ sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers, and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0

  10. Immunohistochemical expression of matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9, myofibroblasts and Ki-67 in actinic cheilitis and lip squamous cell carcinoma.

    PubMed

    Bianco, Bianca C; Scotti, Fernanda M; Vieira, Daniella S C; Biz, Michelle T; Castro, Renata G; Modolo, Filipe

    2015-10-01

    Matrix metalloproteinases (MMPs), myofibroblasts (MFs) and epithelial proliferation have key roles in neoplastic progression. In this study immunoexpression of MMP-1, MMP-2 and MMP-9, presence of MFs and the epithelial proliferation index were investigated in actinic cheilitis (AC), lip squamous cell carcinoma (LSCC) and mucocele (MUC). Thirty cases of AC, thirty cases of LSCC and twenty cases of MUC were selected for immunohistochemical investigation of the proteins MMP-1, MMP-2, MMP-9, α-smooth muscle actin (α-SMA) and Ki-67. The MMP-1 expression in the epithelial component was higher in the AC than the MUC and LSCC. In the connective tissue, the expression was higher in the LSCC. MMP-2 showed lower epithelial and stromal immunostaining in the LSCC when compared to the AC and MUC. The epithelial staining for MMP-9 was higher in the AC when compared to the LSCC. However, in the connective tissue, the expression was lower in the AC compared to other lesions. The cell proliferation rate was increased in proportion to the severity of dysplasia in the AC, while in the LSCC it was higher in well-differentiated lesions compared to moderately differentiated. There were no statistically significant differences in number of MFs present in the lesions studied. The results suggest that MMPs could affect the biological behaviour of ACs and LSCCs inasmuch as they could participate in the development and progression from premalignant lesions to malignant lesions.

  11. All histological types of primary human rhabdomyosarcoma express alpha-cardiac and not alpha-skeletal actin messenger RNA.

    PubMed Central

    Schürch, W.; Bochaton-Piallat, M. L.; Geinoz, A.; d'Amore, E.; Laurini, R. N.; Cintorino, M.; Bégin, L. R.; Boivin, Y.; Gabbiani, G.

    1994-01-01

    Eleven human primary rhabdomyosarcomas (RMSs), including all histological variants, were analyzed morphologically, immunohistochemically for intermediate filament proteins and actin isoforms, and by means of Northern blots with probes specific for total actin, alpha-skeletal (SK), alpha-cardiac (CARD), and alpha-smooth muscle actin messenger (m)RNAs. All tumors disclosed ultrastructural evidence of skeletal muscle features with terminal differentiation in three cases. The RMSs contained immunohistochemically the intermediate filament proteins vimentin and desmin and reacted positively with the alpha-sarcomeric actin antibody, which recognizes alpha-SK and alpha-CARD actin isoforms. All RMSs reacted with the total actin probe, recognizing at 2.1 kb cytoplasmic actin mRNAs and at 1.7 kb alpha-actin mRNAs. With the specific probes, all RMSs expressed alpha-CARD actin mRNA, four neoplasms expressed also alpha-smooth muscle actin mRNA, whereas the probe for alpha-SK actin mRNA never produced a signal except in one case, in which the tumor masses were intermingled with non-neoplastic preexistent striated muscle fibers. Because alpha-CARD and alpha-smooth muscle actins are transiently expressed during normal skeletal muscle development, RMSs seem to follow normal skeletal myogenesis without completing the final step, consisting of alpha-SK actin mRNA expression. The use of Northern blots for alpha-CARD actin as an adjunct to conventional techniques may be helpful for the precise identification of primary RMSs compared to other soft tissue neoplasms. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8160781

  12. Alpha-SM actin expression as prognostic indicator in IgA nephropathy (Berger's disease).

    PubMed

    Pastorello, M T; Costa, R S; Ravinal, R C; Coimbra, M; dos Reis, M A; Dantas, M

    2005-01-01

    Recent studies have demonstrated that alpha-Smooth Muscle actin expression in glomerular and tubulointerstitial compartments of renal tissue could represent a prognostic marker in several renal diseases. Our objective was to identify the prognostic value of alpha-SM actin actin expression on the evolution of renal damage in Primary IgA nephropathy (Berger's disease). 43 patients followed up from 1988 to 1999 at the University Hospital, Faculty of Medicine of Ribeirão Preto, University of Sao Paulo, Brazil, was studied. Clinical-laboratory data were obtained from the medical records of the patients using a protocol containing name, race, gender, origin, profession, age at clinical presentation of the disease and personal and family history. The parameters assessed in the approach to IgA nephropathy were serum creatinine, creatinine clearance, serum albumin, total serum protein, 24 hours proteinuria, glycaemia, serum sodium, potassium, calcium and phosphorus ions, analysis of urinary sediment, serum complement profile, blood count, and renal biopsy. Morphological evaluation was performed by renal biopsy using common light and immunofluorescence microscopy. Immunohistochemical studies were performed using a murine monoclonal antibody to alpha-SM actin. Our data showed that alpha-SM actin expression in the glomerular and tubulointerstitial compartments are not correlated with unfavorable clinical course of primary IgA nephropathy.

  13. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Vyalov, S. L.; Gabbiani, G.; Kapanci, Y.

    1993-01-01

    The majority of fibroblasts in alveolar septa are characterized by the presence of cytoplasmic bundles of microfilaments that contain cytoplasmic actin isoforms; these cells have been named contractile interstitial cells or V-type myofibroblasts. In the rat, they express desmin as intermediate filament protein. In this study, we explored the possibility that modulation and replication of such septal fibroblasts result in the appearance of alpha-smooth muscle (alpha-SM) actin-positive myofibroblasts, typical of lung fibrosis. Experimental pulmonary fibrosis was produced by a unique intratracheal instillation of bleomycin to 28 rats. Eight additional rats used as controls received the equivalent volume of saline. Paraffin and frozen sections of lungs were examined at days 1, 3, 5 and 7 after treatment. Microfilaments and intermediate filaments were stained using antibodies against total actin, alpha-SM actin, desmin, vimentin, keratin, and SM myosin. Electron microscopic labeling of desmin and alpha-SM actin using immunogold technique was done on Lowicryl K4M resin-embedded specimens. alpha-SM actin appeared in desmin-positive alveolar fibroblasts as early as 24 hours after intratracheal bleomycin instillation; the modulation of alpha-SM actin in these cells was preceded by a lymphomonocytic infiltration of alveolar septa. Twenty-four hours to 3 days after bleomycin administration, a proliferation of alveolar myofibroblasts occurred. Fibrosis with laying down of collagen fibers took place after the above mentioned cellular modifications. Our results support the view that septal fibroblastic cells can modulate into typical alpha-SM actin-containing myofibroblasts during experimental bleomycin-induced pulmonary fibrosis. In such a modulation a possible role of cytokines, particularly of transforming growth factor-beta, is considered. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14

  14. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    PubMed

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  15. Succinate causes α-SMA production through GPR91 activation in hepatic stellate cells.

    PubMed

    Li, Ying Hui; Woo, Sung Hoon; Choi, Dae Hee; Cho, Eun-Hee

    2015-08-07

    Succinate acts as an extracellular signaling molecule as well as an intermediate in the citric acid cycle. It binds to and activates its specific G protein-coupled receptor 91 (GPR91). GPR91 is present in hepatic stellate cells (HSCs), but its role in hepatic fibrogenesis remains unclear. Cultured HSCs treated with succinate showed increased protein expression of GPR91 and α-smooth muscle actin (α-SMA), markers of fibrogenic response. Succinate also increased mRNA expression of α-SMA, transforming growth factor β (TGF-β), and collagen type I. Transfection of siRNA against GPR91 abrogated succinate-induced increases in α-SMA expression. Malonate, an inhibitor of succinate dehydrogenase (SDH), increased succinate levels in cultured HSCs and increased GPR91 and α-SMA expression. Feeding mice a methionine- and choline-deficient (MCD) diet is a widely used technique to create an animal model of nonalcoholic steatohepatitis (NASH). HSCs cultured in MCD media showed significantly decreased SDH activity and increased succinate concentration and GPR91 and α-SMA expression. Similarly, palmitate treatment significantly decreased SDH activity and increased GPR91 and α-SMA expression. Finally, C57BL6/J mice fed the MCD diet had elevated succinate levels in their plasma. The MCD diet also decreased SDH activity, increased succinate concentration, and increased GPR91 and α-SMA expression in isolated HSCs. Collectively, our results show that succinate plays an important role in HSC activation through GPR91 induction, and suggest that succinate and GPR91 may represent new therapeutic targets for modulating hepatic fibrosis.

  16. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA)

    PubMed Central

    Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

    2015-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA. PMID:26258776

  17. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

    PubMed

    Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

    2015-08-06

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

  18. Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord.

    PubMed

    Soler-Botija, Carolina; Cuscó, Ivón; López, Eva; Clua, Agustín; Gich, Ignasi; Baiget, Montserrat; Ferrer, Isidre; Tizzano, Eduardo F

    2005-03-01

    We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.

  19. High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development.

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

    Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

  20. Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

    PubMed

    Kitade, Yukihiro; Nakamura, Michiko; Uji, Toshiki; Fukuda, Satoru; Endo, Hirotoshi; Saga, Naotsune

    2008-10-15

    The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

  1. Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass-based surfaces.

    PubMed

    Martins, Carolina Scanavez; Ferraz, Emanuela Prado; De Castro-Raucci, Larissa Moreira Spinola; Teixeira, Lucas Novaes; Maximiano, William Marcatti Amarú; Rosa, Adalberto Luiz; De Oliveira, Paulo Tambasco

    2015-11-01

    The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals.

  2. Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis.

    PubMed

    Hoben, Gwendolyn M; Athanasiou, Kyriacos A

    2008-12-01

    Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1-100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10-100 nM) increased cartilage oligomeric matrix protein 60- to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 +/- 8.8 microg in the untreated constructs to 55.2 +/- 12.2 microg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs.

  3. Expression of two actin genes during larval development in the sea urchin Strongylocentrotus purpuratus.

    PubMed

    Cameron, R A; Britten, R J; Davidson, E H

    1989-01-01

    We report the first measurements of cell number, total RNA, and transcript accumulations for two actin genes during larval development of the sea urchin Strongylocentrotus purpuratus. At 5 weeks of feeding, when development of laboratory-raised larvae is completed, the cell number has increased about 100-fold with respect to the pluteus-stage embryo to about 150,000 +/- 50,000, and the total RNA has increased 46-fold to about 130 ng per larva. The transcripts of the Cylla cytoskeletal actin gene, which is expressed in adult tissues, continue to accumulate throughout larval development. A contrasting pattern of transcript accumulation is observed for Cyllla, a different cytoskeletal actin gene that in the embryo is expressed only in aboral ectoderm. These transcripts increase in number early in larval development, when the larval epidermis is differentiating, and then decline in quantity. It is known that at metamorphosis the larval epidermis is largely histolyzed and that the Cyllla gene is not expressed in the juvenile or adult.

  4. Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization.

    PubMed

    Miehe, Ulrich; Neumaier-Wagner, Peruka; Kadyrov, Mamed; Goyal, Pankaj; Alfer, Joachim; Rath, Werner; Huppertz, Berthold

    2005-01-01

    The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.

  5. Absence of aryl hydrocarbon receptor alters CDC42 expression and prevents actin polymerization during capacitation.

    PubMed

    Angeles-Floriano, Tania; Roa-Espitia, Ana L; Baltiérrez-Hoyos, Rafael; Cordero-Martínez, Joaquin; Elizondo, Guillermo; Hernández-González, Enrique O

    2016-11-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the toxicity of a variety of environmental chemicals. The absence of this receptor causes serious reproductive complications. Ahr-knockout (Ahr-KO) male mice, for example, are considerably less fertile: Half of the few spermatozoa they produce exhibit morphological alterations, and those with typical morphology may have pathologic modifications. We therefore investigated the consequences of AHR loss on capacitation and the acrosome reaction, and asked if these effects are a consequence of changes to actin polymerization and the expression of Cdc42, which encodes Cell division control protein 42 (CDC42), a RHO protein that controls assembly of the actin cytoskeleton in somatic cells as well as during spermatogenesis. Nearly 50% of spermatozoa produced by Ahr-KO mice had alterations in the flagellum. Ahr-KO spermatozoa were frequently capacitated, but showed reduced spontaneous and progesterone-induced acrosome reaction-which is related to low CDC42 abundance and very limited actin polymerization during capacitation. Thus, the expression of CDC42 might be regulated by AHR, and both proteins are fundamental to the development of normal spermatozoa and the acrosome reaction. Mol. Reprod. Dev. 83: 1015-1026, 2016 © 2016 Wiley Periodicals, Inc.

  6. A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

    PubMed Central

    Drummond, Meghan C.; Friderici, Karen H.

    2013-01-01

    Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as “regulated unproductive splicing and translation” (RUST). PMID:24098136

  7. CD34(low) and SMA(high) represent stromal signature in uterine cervical cancer and are markers for peritumoral stromal remodeling.

    PubMed

    Horn, Lars-Christian; Schreiter, Carolin; Canzler, Anika; Leonhardt, Karoline; Einenkel, Jens; Hentschel, Bettina

    2013-12-01

    Peritumoral desmoplastic stromal reaction (DSR) with myofibroblastic phenotype may be of prognostic impact in uterine cervical carcinoma. The present study evaluates the immunostaining (CD34 and smooth muscle actin; SMA) of 97 squamous cell cancers. Staining was scored as low/negative (<5% stroma positive), moderate (patchy/focal expression, 5%-50%), or high (diffuse expression throughout peritumoral stroma, >50%) and DSR as negative/weak and moderate/strong. The staining results were correlated to patient survival. Of the cases, 78.3% showed a decreased of CD34 (<5% stromal positivity) and 71.9% an increased SMA staining with more than 50% SMA positive stromal cells. Tumors representing moderate/strong DSR showed a significant decreased CD34 (P=.001) and an increased but not statistically significant SMA staining (P=0.345). Cases with low CD34 and high SMA staining showed reduced 5-year overall survival when compared to cases with high CD34 and low SMA positivity (59.9 vs 81.0%; P=0.025 and 64.6 vs 81.1%; P=0.243). Peritumoral stromal response in cervical carcinoma is immunohistochemically characterized by CD34(low)/SMA(high) and associated reduced overall survival.

  8. The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli.

    PubMed Central

    McDowell, J M; An, Y Q; Huang, S; McKinney, E C; Meagher, R B

    1996-01-01

    ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements. PMID:8754679

  9. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  10. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.

    PubMed Central

    Schmitt-Gräff, A.; Krüger, S.; Bochard, F.; Gabbiani, G.; Denk, H.

    1991-01-01

    It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs. Images Figure 1 Figure 2 PMID:2024709

  11. Cerebrovascular smooth muscle actin is increased in nondemented subjects with frequent senile plaques at autopsy: implications for the pathogenesis of Alzheimer disease.

    PubMed

    Hulette, Christine M; Ervin, John F; Edmonds, Yvette; Antoine, Samantha; Stewart, Nicolas; Szymanski, Mari H; Hayden, Kathleen M; Pieper, Carl F; Burke, James R; Welsh-Bohmer, Kathleen A

    2009-04-01

    We previously found that vascular smooth muscle actin (SMA) is reduced in the brains of patients with late stage Alzheimer disease (AD) compared with brains of nondemented, neuropathologically normal subjects. To assess the pathogenetic significance and disease specificity of this finding, we studied 3 additional patient groups: nondemented subjects without significant AD type pathology ("Normal"; n = 20), nondemented subjects with frequent senile plaques at autopsy ("Preclinical AD"; n = 20), and subjects with frontotemporal dementia ("FTD"; n = 10). The groups were matched for sex and age with those previously reported; SMA immunohistochemistry and image analysis were performed as previously described. Surprisingly, SMA expression in arachnoid, cerebral cortex, and white matter arterioles was greater in the Preclinical AD group than in the Normal and FTD groups. The plaques were not associated with amyloid angiopathy or other vascular disease in this group. Smooth muscle actin expression in the brains of the Normal group was intermediate between the Preclinical AD and FTD groups. All 3 groups exhibited much greater SMA expression than in our previous report. The presence of frequent plaques and increased arteriolar SMA expression in the brains of nondemented subjects suggest that increased SMA expression might represent a physiological response to neurodegeneration that could prevent or delay overt expression dementia in AD.

  12. The genomic nucleotide sequences of two differentially expressed actin-coding genes from the sea star Pisaster ochraceus.

    PubMed

    Kowbel, D J; Smith, M J

    1989-04-30

    The genomic sequences of two differentially expressed actin genes from the sea star Pisaster ochraceus are reported. The cytoplasmic actin gene (Cy) is expressed in eggs and early development. The muscle actin gene (M) is expressed in tube feet and testes. Both genes contain an 1125-nucleotide coding region interrupted by three introns at codons 41, 121 and 204. Gene M contains two additional introns at codons 150 and 267. The intron position at codon 150, although present in higher vertebrate actins, has not been reported in actin genes from invertebrates. The M gene coding region has 89.5% nucleotide homology to the Cy gene, and differs from the Cy actin gene in 13 of 375 amino acids (aa), 11 of which are found in the C-terminal half of the gene. The C-terminal half of the M gene contains a significant number of muscle isotype codons. Even though there is only 1 aa change in the first 150 codons, there have been limited substitutions at many four-fold degenerate sites which may indicate selection pressure upon the secondary structure of the mRNA and/or a biased codon usage. Variant CCAAT, TATA, and poly(A)-addition signals have been identified in the 5' and 3' flanking regions. The presence of 5' and 3' splice junction sequences in the 5' flanking region of the Cy gene suggests the potential for an intron there.

  13. Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.

    PubMed

    Li, Xue-Bao; Xu, Dan; Wang, Xiu-Lan; Huang, Geng-Qing; Luo, Juan; Li, Deng-Di; Zhang, Ze-Ting; Xu, Wen-Liang

    2010-01-01

    To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

  14. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Study of MDM2 and SUMO-1 expression in actinic cheilitis and lip cancer.

    PubMed

    Oliveira Alves, Mônica Ghislaine; da Mota Delgado, Adriana; Balducci, Ivan; Carvalho, Yasmin Rodarte; Cavalcante, Ana Sueli Rodrigues; Almeida, Janete Dias

    2014-11-01

    Actinic cheilitis exhibits a potential of malignant transformation in 10-20 % of cases. The objective of this study was to compare the expression of MDM2 and SUMO-1 proteins between actinic cheilitis (AC) and squamous cell carcinoma (SCC) of the lip. The sample consisted of lower lip mucosa specimens obtained from cases with a clinical and histopathological diagnosis of AC (n = 26) and SCC (n = 25) and specimens of labial semi-mucosa (n = 15) without clinical alterations or inflammation. The tissue samples were stained with hematoxylin-eosin and anti-MDM2 and anti-SUMO-1 antibodies. Data were analyzed by the Kruskal-Wallis and Dunn's tests (5 %). The median expression of MDM2 (kW = 36.8565; df = 3-1 = 2; p = 0.0001) and SUMO-1 (kW = 32.7080; df = 3-1 = 2; p = 0.0001) was similar in cases of AC and SCC of the lip, but differed significantly from that observed for normal labial semi-mucosa. Despite the limitations of the present study, immunohistochemistry demonstrated the overexpression of important proteins (MDM2 and SUMO-1) related to regulatory mechanisms of apoptosis in AC and SCC of the lip, but further studies are needed.

  16. A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

    PubMed Central

    Piechocki, Marie P

    2008-01-01

    Background To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA) expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1) maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro. Methods We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa. Results The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response. Conclusion This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving strategies that can dually or

  17. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

    PubMed

    Ge, Jiachun; Dong, Zhangji; Li, Jingyun; Xu, Zhiqiang; Song, Wei; Bao, Jie; Liang, Dong; Li, Junbo; Li, Kui; Jia, Wenshuang; Zhao, Muzi; Cai, Yongxiang; Yang, Jiaxin; Pan, Jianlin; Zhao, Qingshun

    2012-10-01

    Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

  18. Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns

    PubMed Central

    Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A.; Katsaros, Christos

    2016-01-01

    During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis. PMID:27656150

  19. Cytoskeleton alterations in melanoma: aberrant expression of cortactin, an actin-binding adapter protein, correlates with melanocytic tumor progression

    PubMed Central

    Xu, Xu-Zhi; Garcia, Marileila Varella; Li, Tian-yu; Khor, Li-Yan; Gajapathy, R Sujatha; Spittle, Cindy; Weed, Scott; Lessin, Stuart R; Wu, Hong

    2010-01-01

    Cortactin is a multidomain actin-binding protein important for the functions of cytoskeleton by regulating cortical actin dynamics. It is involved in a diverse array of basic cellular functions. Tumorigenesis and tumor progression involves alterations in actin cytoskeleton proteins. We sought to study the role of cortactin in melanocytic tumor progression using immunohistochemistry on human tissues. The results reveal quantitative differences between benign and malignant lesions. Significantly higher cortactin expression is found in melanomas than in nevi (P<0.0001), with levels greater in metastatic than in invasive melanomas (P<0.05). Qualitatively, tumor tissues often show aberrant cortactin localization at the cell periphery, corresponding to its colocalization with filamentous actin in cell cortex of cultured melanoma cells. This suggests an additional level of protein dysregulation. Furthermore, in patients with metastatic disease, high-level cortactin expression correlates with poor disease-specific survival. Our data, in conjunction with outcome data on several other types of human cancers and experimental data from melanoma cell lines, supports a potential role of aberrant cortactin expression in melanoma tumor progression and a rational for targeting key elements of actin-signaling pathway for developmental therapeutics in melanomas. PMID:19898426

  20. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

    PubMed Central

    1993-01-01

    Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells. PMID:8314838

  1. Spinal Muscular Atrophy (SMA)

    MedlinePlus

    ... areas of society: the arts, science, law, management, teaching — you name it. Children with SMA tend to ... or speech therapy consultations • annual flu shots • support groups for those affected, spouses, parents or other caregivers • ...

  2. Spinal Muscular Atrophy (SMA)

    MedlinePlus

    ... children with SMA develop spinal deformities, such as scoliosis (sideways curvature of the spine) and kyphosis (front- ... Magnetic Resonance Imaging (MRI) Brain and Nervous System Scoliosis Contact Us Print Resources Send to a friend ...

  3. ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

    PubMed Central

    Ahn, Young-Ho; Gibbons, Don L.; Chakravarti, Deepavali; Creighton, Chad J.; Rizvi, Zain H.; Adams, Henry P.; Pertsemlidis, Alexander; Gregory, Philip A.; Wright, Josephine A.; Goodall, Gregory J.; Flores, Elsa R.; Kurie, Jonathan M.

    2012-01-01

    Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients. PMID:22850877

  4. Expression of drebrin, an actin binding protein, in basal cell carcinoma, trichoblastoma and trichoepithelioma.

    PubMed

    Mizutani, Yoko; Iwamoto, Ikuko; Kanoh, Hiroyuki; Seishima, Mariko; Nagata, Koh-ichi

    2014-06-01

    Drebrin, an F-actin binding protein, is known to play important roles in cell migration, synaptogenesis and neural plasticity. Although drebrin was long thought to be specific for neuronal cells, its expression has recently been reported in non-neuronal cells. As for skin-derived cells, drebrin was shown to be enriched at adhering junctions (AJs) in cultured primary keratinocytes and also be highly expressed in basal cell carcinoma (BCC) cells. Since BCC and two types of benign neoplasm, trichoblastoma and trichoepithelioma, are considered to derive from the same origin, follicular germinative cells, it is sometimes difficult to morphologically distinguish BCC from trichoblastoma and trichoepithelioma. In this study, we performed immunohistochemical staining of drebrin in BCC, trichoblastoma and trichoepithelioma, to examine whether drebrin could serve as a biomarker for BCC diagnosis. In western blotting, drebrin was detected highly and moderately in the lysates from a squamous cell carcinoma cell line, DJM-1, and normal human epidermis, respectively. In immunofluorescence analyses, drebrin was colocalized with markers of AJs and tight junctions in DJM-1 cells and detected at cell-cell junction areas of human normal epidermis tissue. We then examined the distribution patterns of drebrin in BCC, trichoblastoma and trichoepithelioma. In BCC tissues, intense and homogeneous drebrin expression was observed mainly at tumor cell-cell boundaries. In contrast, drebrin was stained only weakly and non-homogeneously in trichoblastoma and trichoepthelioma tissue samples. For differential diagnosis of BCC, drebrin may be a novel and useful marker.

  5. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  6. [Expression of elongation factor-1 alpha-A and beta-actin promoters in embryos of transgenic Medaka (Oryzias latipes)].

    PubMed

    Long, Hua

    2003-06-01

    Two expression vectors with the promoter of either Medaka (Oryzias latipes) elongation factor gene or beta-actin gene were constructed based on pBluescript SK+. Both of them are linked with green-fluorescent protein (GFP) gene. And they are named as pB-EF and pB-BA, respectively. The microinjection experiments were conducted with fertilized Medaka eggs at one-cell stage. The expression of two vectors, pB-EF and pB-BA, was observed under stereo-fluorescence microscope. The detection results showed that both EF-1 alpha-A promoter and beta-actin promoter are strong. In the process of embryo development, the activity of beta-actin promoter became stronger while that of EF-1 alpha-A promoter weaker gradually. beta-actin promoter was but EF-1 alpha-A promoter distributed throughout fish body uniformly. The expression rate of two vectors, pB-EF and pB-BA, are 8.23% and 6.10%, respectively.

  7. Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae.

    PubMed

    Jin, S; Xu, R; Wei, Y; Goodwin, P H

    1999-10-01

    Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72-96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction.

  8. Spinal Muscular Atrophy (SMA)

    MedlinePlus

    ... Lessons? Visit KidsHealth in the Classroom What Other Parents Are Reading Your Child's Development (Birth to 3 Years) Feeding Your 1- to 3-Month-Old Feeding Your 4- to 7-Month-Old Feeding Your 8- to 12-Month-Old Feeding Your 1- to 2-Year-Old Spinal ... > For Parents > Spinal Muscular Atrophy (SMA) Print A A A ...

  9. Altered β-catenin expression related to cancer progression on actinic cheilitis and squamous cell carcinoma of the lip.

    PubMed

    Schussel, Juliana L; Pinto, Décio Dos Santos; Martins, Marília Trierveiler

    2011-02-01

    β-Catenin is a bifunctional protein related to cell adhesion and gene transcription when activated by Wnt pathway. Altered expression of β-catenin was related to loss of differentiation, more aggressive phenotype, increase of tumor invasion, and poor prognosis in a number of different cancers. Actinic cheilitis is caused by excessive exposure to ultraviolet radiation and has a high potential to suffer malignant transformation into squamous cell carcinoma (SCC) of the lip, the most frequent oral malignancy. Studies of oral cancer have shown the correlation of β-catenin expression and oral SCC prognosis, and loss of membrane expression may be considered as a potential marker for early tumor recurrence. Thirty-five cases of actinic cheilitis and 12 cases of SCC of the lip were select and submitted to immunohistochemical staining using β-catenin antibody. β-Catenin was positive on the membrane for all cases. Eighty-five percent of actinic cheilitis cases showed cytoplasmatic staining, and 22% nuclear staining. Eighty-three percent of SCC was positive for β-catenin, and none of them had nuclear staining. Cytoplasmatic and nuclear staining of β-catenin on studied cases point to pathway alterations. Results demonstrated that β-catenin expression is altered on epithelial dysplasia, and it is related to degree of alterations.

  10. Expression of expanded polyglutamine targets profilin for degradation and alters actin dynamics

    PubMed Central

    Burnett, Barrington G.; Andrews, Jaime; Ranganathan, Srikanth; Fischbeck, Kenneth H.; Di Prospero, Nicholas A.

    2008-01-01

    Huntington’s disease is caused by polyglutamine expansion in the huntingtin protein. Huntingtin directly interacts with profilin, a major actin monomer sequestering protein and a key integrator of signals leading to actin polymerization. We observed a progressive loss of profilin in the cerebral cortex of Huntington’s disease patients, and in cell culture and Drosophila models of polyglutamine disease. This loss of profilin is likely due to increased degradation through the ubiquitin-proteasome system. Profilin loss reduces the F/G actin ratio, indicating a shift in actin polymerization. Overexpression of profilin abolishes mutant huntingtin toxicity in cells and partially ameliorates the morphological and functional eye phenotype and extends lifespan in a transgenic polyglutamine Drosophila model. These results indicate a link between huntingtin and profilin and implicate profilin in Huntington’s disease pathogenesis. PMID:18417352

  11. Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

    PubMed

    Lu, Qing-Yi; Jin, Yu Sheng; Zhang, Zuo-Feng; Le, Anh D; Heber, David; Li, Frederick P; Dubinett, Steven M; Rao, Jian Yu

    2007-05-01

    Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.

  12. Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions

    PubMed Central

    Xiao, Chang; Ogle, Sally A.; Schumacher, Michael A.; Schilling, Neal; Tokhunts, Robert A.; Orr-Asman, Melissa A.; Miller, Marian L.; Robbins, David J.; Hollande, Frederic

    2010-01-01

    In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5Ski) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/ShhKO) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5Ski cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/ShhKO mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1. PMID:20847300

  13. Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via the reorganization of the actin cytoskeleton.

    PubMed

    Mayanagi, Taira; Morita, Tsuyoshi; Hayashi, Ken'ichiro; Fukumoto, Kentaro; Sobue, Kenji

    2008-11-07

    Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.

  14. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    PubMed

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  15. Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp.

    PubMed

    Yoshiba, Nagako; Yoshiba, Kunihiko; Ohkura, Naoto; Shigetani, Yoshimi; Takei, Erika; Hosoya, Akihiro; Nakamura, Hiroaki; Okiji, Takashi

    2012-10-01

    Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)₂ to induce a mineralized barrier at the exposed surface. After 7-42 days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42 days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.

  16. Intratumoral α-SMA enhances the prognostic potency of CD34 associated with maintenance of microvessel integrity in hepatocellular carcinoma and pancreatic cancer.

    PubMed

    Wang, Wen-Quan; Liu, Liang; Xu, Hua-Xiang; Luo, Guo-Pei; Chen, Tao; Wu, Chun-Tao; Xu, Yong-Feng; Xu, Jin; Liu, Chen; Zhang, Bo; Long, Jiang; Tang, Zhao-You; Yu, Xian-Jun

    2013-01-01

    Microvessel density (MVD) as an angiogenesis predictor is inefficient per se in cancer prognosis. We evaluated prognostic values of combining intratumoral alpha-smooth muscle actin (α-SMA)-positive stromal cell density and MVD after curative resection in hypervascular hepatocellular carcinoma (HCC) and hypovascular pancreatic cancer (PC). Tissue microarrays were constructed from tumors of 305 HCC and 57 PC patients who underwent curative resection and analyzed for α-SMA and CD34 expression by immunostaining. Prognostic values of these two proteins and other clinicopathological features were examined. Both low α-SMA density and high MVD-CD34 were associated in HCC with the presence of intrahepatic metastasis and microvascular invasion, and they were related to lymph node involvement and microvascular invasion in PC (p<0.05). Although CD34 alone, but not α-SMA, was an independent prognostic factor for overall survival and recurrence-free survival, the combination of low α-SMA and high CD34 was a predictor of worst prognosis for both types of tumors and had a better power to predict patient death and early recurrence (p<0.01). Furthermore, the results show that distribution of most of the α-SMA-positive cells and vascular endothelial cells overlap, showing major colocalization on vascular walls. Poor microvessel integrity, as indicated by high MVD, together with low perivascular α-SMA-positive cell coverage is associated with early recurrence, unfavorable metastasis, and short survival after tumor resection. This finding highlights the significance of vascular quality in tumor progression, which provides an optimized complement to vascular quantity in prognosis of postoperative patients.

  17. Arabidopsis actin capping protein (AtCP) subunits have different expression patterns, and downregulation of AtCPB confers increased thermotolerance of Arabidopsis after heat shock stress.

    PubMed

    Wang, Jue; Qian, Dong; Fan, Tingting; Jia, Honglei; An, Lizhe; Xiang, Yun

    2012-09-01

    As a heterodimer actin-binding protein, capping protein is composed of α and β subunits, and can stabilize the actin filament cytoskeleton by binding to F-actin ends to inhibit G-actin addition or loss from that end. Until now, studies on plant capping protein have focused on biochemical functions in vitro, and so the expression patterns and physiological functions of actin capping protein in Arabidopsis (AtCP) are poorly understood. In the present study, real-time quantitative PCR and Western blot analysis showed that although AtCP α and β subunits (i.e. AtCPA and AtCPB) were expressed in various tissues, their expression patterns were significantly different. GUS staining further indicated they were present in different parts of the same organs. We also demonstrated that the expression levels of both subunits were induced by heat shock stress. However, only the atcpβ-mutant showed enhanced thermotolerance, and confocal microscopy showed that the actin filaments of the atcpβ-mutant were much more complete than that in the wild-type and the atcpα-mutant after heat treatment at 45 °C for 40 and 45 min. In conclusion, these results demonstrated that AtCPA and AtCPB showed distinct expression patterns in vivo, and that downregulation of AtCPB conferred increased plant thermotolerance after heat shock stress.

  18. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  19. ROCK/actin/MRTF signaling promotes the fibrogenic phenotype of fibroblast-like synoviocytes derived from the temporomandibular joint

    PubMed Central

    Yokota, Seiji; Chosa, Naoyuki; Kyakumoto, Seiko; Kimura, Hitomichi; Ibi, Miho; Kamo, Masaharu; Satoh, Kazuro; Ishisaki, Akira

    2017-01-01

    Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast-like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho-associated coiled-coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α-smooth muscle actin (α-SMA) and type I collagen; and ii) an inhibitor against the actin-polymerizing agent ROCK, Y-27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor-regulated transcription, CCG-100602, clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor-1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ-OA and may aid in identifying drug targets for treating this condition at the molecular level. PMID:28259960

  20. Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells.

    PubMed

    Li, Ying Hui; Choi, Dae Hee; Lee, Eun Hye; Seo, Su Ryeon; Lee, Seungkoo; Cho, Eun-Hee

    2016-05-06

    Sirtuin 3 (SIRT3) is an NAD(+)-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment.

  1. Yap/Taz transcriptional activity is essential for vascular regression via Ctgf expression and actin polymerization

    PubMed Central

    Nagasawa-Masuda, Ayumi; Terai, Kenta

    2017-01-01

    Vascular regression is essential to remove redundant vessels during the formation of an efficient vascular network that can transport oxygen and nutrient to every corner of the body. However, no mechanism is known to explain how major blood vessels regress during development. Here we use the dorsal part of the caudal vein plexus (dCVP) in Zebrafish to investigate the mechanism of regression and discover a new role of Yap/Taz in vascular regression. During regression, Yap/Taz is activated by blood circulation in the endothelial cells. This leads to induction of Ctgf and actin polymerization. Interference with Yap/Taz activation decreased Ctgf production, which decreased actin polymerization and vascular regression. These results implicate a novel role of Yap/Taz in vascular regression. PMID:28369143

  2. Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells.

    PubMed

    Wang, Ping; Li, Ji-Cheng

    2007-09-15

    Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of gamma-actin mRNA preceded that of beta-actin; alpha- and beta-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.

  3. Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

    PubMed

    Goncharenko, A V; Malyuchenko, N V; Moisenovich, A M; Kotlyarova, M S; Arkhipova, A Yu; Kon'kov, A S; Agapov, I I; Molochkov, A V; Moisenovich, M M; Kirpichnikov, M P

    2016-09-01

    3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

  4. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  5. EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

    PubMed

    Gu, Yanping; Wang, Congying; Li, Guangwen; Huang, Li-Yen Mae

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

  6. Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

    PubMed

    Peixoto da-Silva, Janaína; Lourenço, Silvia; Nico, Marcello; Silva, Filomena H; Martins, Marília Trierveiler; Costa-Neves, Adriana

    2012-10-15

    The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, β1 subunits of α3β1 heterodimer and β4 subunit of integrin α6β4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of β1, β4 and α3 integrins, and SISCCs lacked β1, β4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin β1, β4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype.

  7. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão, Joana N.; Mota, Luís Jaime; Franco, Irina S.

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  8. The β-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

    PubMed

    Barman, Hirak Kumar; Mohanta, Ramya; Patra, Swagat Kumar; Chakrapani, Vemulawada; Panda, Rudra Prasanna; Nayak, Swapnarani; Jena, Sasmita; Jayasankar, Pallipuram; Nandanpawar, Priyanka

    2015-06-01

    We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

  9. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

    PubMed Central

    Duggirala, Aparna; Kimura, Tomomi E.; Sala-Newby, Graciela B.; Johnson, Jason L.; Wu, Yih-Jer; Newby, Andrew C.; Bond, Mark

    2015-01-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  10. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.

    PubMed

    Duggirala, Aparna; Kimura, Tomomi E; Sala-Newby, Graciela B; Johnson, Jason L; Wu, Yih-Jer; Newby, Andrew C; Bond, Mark

    2015-02-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  11. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

  12. Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

    PubMed

    Muscat, G E; Kedes, L

    1987-11-01

    Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and

  13. Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

    PubMed

    Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2012-04-01

    This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

  14. Hypothermia improves disease manifestations in SMA mice via SMN augmentation.

    PubMed

    Tsai, Li-Kai; Chen, Chien-Lin; Tsai, Yi-Chieh; Ting, Chen-Hung; Chien, Yin-Hsio; Lee, Ni-Chong; Hwu, Wuh-Liang

    2016-02-15

    Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by a deficiency of survival motor neuron (SMN) protein. In this study, we evaluated the efficacy of intermittent transient hypothermia in a mouse model of SMA. SMA mice were exposed to ice for 50 s to achieve transient hypothermia (below 25°C) daily beginning on postnatal day 1. Neonatal SMA mice (Smn(-/-)SMN2(+/-)) who received daily transient hypothermia exhibited reduced motor neuron degeneration and muscle atrophy and preserved the architecture of neuromuscular junction when compared with untreated controls at day 8 post-treatment. Daily hypothermia also prolonged the lifespan, increased body weight and improved motor coordination in SMA mice. Quantitative polymerase chain reaction and western blot analyses showed that transient hypothermia led to an increase in SMN transcript and protein levels in the spinal cord and brain. In in vitro studies using an SMN knockdown motor neuron-like cell-line, transient hypothermia increased intracellular SMN protein expression and length of neurites, confirming the direct effect of hypothermia on motor neurons. These data indicate that the efficacy of intermittent transient hypothermia in improving outcome in an SMA mouse model may be mediated, in part, via an upregulation of SMN levels in the motor neurons.

  15. SMA DOE Student Fellowship Initiative

    SciTech Connect

    Steel Manufacturers Association

    2004-12-24

    Steel companies in many areas of the country have found it increasingly difficult to attract talented recent graduates of college and university engineering and applied science programs to the Electric Arc Furnace iron & steel industry. College student involvement in co-operative programs at steel companies can attract needed talent to the industry. Additionally, certain R & D needs identified in the Steel Industry Technology Roadmap are addressed as co-operative program activities. The Steel Manufacturers Association (''SMA'') therefore established a co-operative education program for selected college students who have completed the first or second year of a four or five-year college program, to be recognized as SMA Co-Operative Fellows, in regard to their summer and fall semester projects with SMA's member companies.

  16. Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs

    PubMed Central

    Karl, Alexandra; Berner, Arne; Schmitz, Paul; Koch, Matthias; Nerlich, Michael; Mueller, Michael B.

    2016-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism. PMID:27843458

  17. [Actin in the wound healing process].

    PubMed

    Nowak, Dorota; Popow-Woźniak, Agnieszka; Raźnikiewicz, Linda; Malicka-Błaszkiewicz, Maria

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field.

  18. Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton.

    PubMed

    Eyster, Craig A; Duggins, Quwanza S; Olson, Ann Louise

    2005-05-06

    The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.

  19. Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

    PubMed

    Torres-Cruz, Francisco M; Rodríguez-Cruz, Fanny; Escobar-Herrera, Jaime; Barragán-Andrade, Norma; Basurto-Islas, Gustavo; Ripova, Daniela; Ávila, Jesús; Garcia-Sierra, Francisco

    2016-03-21

    Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

  20. NADPH OXIDASE 4 MEDIATES TGF-β-INDUCED SMOOTH MUSCLE α-ACTIN VIA p38MAPK AND SRF

    PubMed Central

    Martin-Garrido, Abel; Brown, David I.; Lyle, Alicia N.; Dikalova, Anna; Seidel-Rogol, Bonnie; Lassègue, Bernard; Martín, Alejandra San; Griendling, Kathy K.

    2010-01-01

    In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β. PMID:21074607

  1. An actin-binding protein, CAP, is expressed in a subset of rat taste bud cells.

    PubMed

    Ishimaru, Y; Yasuoka, A; Asano-Miyoshi, M; Abe, K; Emori, Y

    2001-02-12

    Single cell cDNA libraries were constructed from taste bud cells of rat circumvallate papillae. Using three steps of screening, including differential hybridization, sequence analyses and in situ hybridization, a clone encoding a rat homolog of yeast adenylyl cyclase-associated protein (CAP) was identified to be highly expressed in a subset of taste bud cells.

  2. No effect of 60 Hz electromagnetic fields on MYC or {beta}-actin expression in human leukemic cells

    SciTech Connect

    Lacy-Hulbert, A.; Wilkins, R.C.; Hesketh, T.R.

    1995-10-01

    Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and {beta}actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 {mu}T for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods. 70 refs., 2 figs., 1 tab.

  3. Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo.

    PubMed

    Feng, Jun; Li, Guang; Liu, Xin; Wang, Jing; Wang, Yi-Quan

    2014-10-01

    Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.

  4. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  5. Twist1 in tumor cells and α-smooth muscle actin in stromal cells are possible biomarkers for metastatic giant basal cell carcinoma.

    PubMed

    Motegi, Sei-ichiro; Yamada, Kazuya; Ishikawa, Osamu

    2013-08-01

    We previously reported a case of giant basal cell carcinoma (BCC) in a 75-year-old Japanese man, who subsequently developed a pulmonary metastasis. With regard to the pathogenesis of metastasis of BCC, recently, it has been reported that high levels of expression of Twist1 and N-cadherin in primary and metastatic tumor cells, suggesting that Twist1 expression and an epithelial-mesenchymal transition (EMT) of tumor cells are important for the promotion of tumor invasion and subsequent metastasis. In this report, we identified the expressions of Twist1 in tumor cells and α-smooth muscle actin (α-SMA) in stromal cells in the primary and metastatic sites of giant BCC. These results suggest that Twist1-induced EMT of tumor cells might have been associated with distant organ metastasis in our case, and the presence of α-SMA-positive myofibroblasts surrounding a BCC nest can be one of hallmarks of the aggressiveness of BCC.

  6. Types of SMA (Spinal Muscular Atrophy)

    MedlinePlus

    ... Trials Research Publications Support & Care For Newly Diagnosed Care Packages Information Packets Equipment Pool Living With SMA Medical Issues Palliative Breathing Orthopedics Nutrition Equipment Daily Life At School At Home Adults With SMA Play Travel Grief & Loss Community & ...

  7. Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

    PubMed Central

    Huang, Fu; Paulson, Ariel; Dutta, Arnob; Venkatesh, Swaminathan; Smolle, Michaela

    2014-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. PMID:25512562

  8. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  9. R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells.

    PubMed

    Cerda, S R; Wilkinson, J; Thorgeirsdottir, S; Broitman, S A

    1999-01-01

    Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

  10. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

    Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

    2016-01-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  11. SMA actuators for morphing wings

    NASA Astrophysics Data System (ADS)

    Brailovski, V.; Terriault, P.; Georges, T.; Coutu, D.

    An experimental morphing laminar wing was developed to prove the feasibility of aircraft fuel consumption reduction through enhancement of the laminar flow regime over the wing extrados. The morphing wing prototype designed for subsonic cruise flight conditions (Mach 0.2 … 0.3; angle of attack - 1 … +2∘), combines three principal subsystems: (1) flexible extrados, (2) rigid intrados and (3) an actuator group located inside the wing box. The morphing capability of the wing relies on controlled deformation of the wing extrados under the action of shape memory alloys (SMA) actuators. A coupled fluid-structure model of the morphing wing was used to evaluate its mechanical and aerodynamic performances in different flight conditions. A 0.5 m chord and 1 m span prototype of the morphing wing was tested in a subsonic wind tunnel. In this work, SMA actuators for morphing wings were modeled using a coupled thermo-mechanical finite element model and they were windtunnel validated. If the thermo-mechanical model of SMA actuators presented in this work is coupled with the previously developed structureaerodynamic model of the morphing wing, it could serve for the optimization of the entire morphing wing system.

  12. CAP2 is a regulator of the actin cytoskeleton and its absence changes infiltration of inflammatory cells and contraction of wounds.

    PubMed

    Kosmas, Kosmas; Eskandarnaz, Ali; Khorsandi, Arya B; Kumar, Atul; Ranjan, Rajeev; Eming, Sabine A; Noegel, Angelika A; Peche, Vivek S

    2015-01-01

    Cyclase associated protein (CAP) is a highly conserved protein with roles in actin dynamics and many cellular processes. Two isoforms exist in higher eukaryotes, CAP1 and CAP2. CAP1 is ubiquitously expressed whereas CAP2 shows restricted tissue distribution. In mice, ablation of CAP2 leads to development of cardiomyopathy. CAP2 is expressed in skin. In human skin its expression is increased in wounds. To elucidate the role of CAP2 in skin upon injury, we studied the wound healing in CAP2 deficient mice and found altered wound healing response presumably resulting from reduced levels of α-SMA, decreased macrophage infiltration and slower neovascularization. In vitro cultured Cap2 deficient keratinocytes showed reduced velocity and a delay in scratch closure. The analysis of primary mutant fibroblasts also showed reduced velocity and less contractibility. They had extended protrusions and more focal adhesions. In addition the F-actin content was increased keeping the total actin content unaltered. Mutant fibroblasts furthermore exhibited an altered response during recovery from drug-induced disruption of the actin cytoskeleton. Interestingly, CAP1 was upregulated in knockout unwounded skin and in wounds which might partially compensate for the loss of CAP2. Taken together, our studies reveal a role for CAP2 in wound healing which may be based on its function as a regulator of the actin cytoskeleton.

  13. Protein expression of MMP-2 and MT1-MMP in actinic keratosis, squamous cell carcinoma of the skin, and basal cell carcinoma.

    PubMed

    de Oliveira Poswar, Fabiano; de Carvalho Fraga, Carlos Alberto; Gomes, Emisael Stênio Batista; Farias, Lucyana Conceição; Souza, Linton Wallis Figueiredo; Santos, Sérgio Henrique Souza; Gomez, Ricardo Santiago; de-Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena

    2015-02-01

    Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are 2 skin neoplasms with distinct potentials to invasion and metastasis. Actinic keratosis (AK) is a precursor lesion of SCC. Immunohistochemistry was performed to evaluate the expression of MMP-2 and MT1-MMP in samples of BCC (n = 29), SCC (n = 12), and AK (n = 13). The ratio of positive cells to total cells was used to quantify the staining. Statistical significance was considered under the level P < .05. We found a higher expression of MMP-2 in tumor stroma and parenchyma of SCC as compared to BCC. The expression of this protein was also similar between SCC and its precursor actinic keratosis, and it was higher in the stroma of high-risk BCC when compared to low-risk BCC. MT1-MMP, which is an activator of MMP-2, was similarly expressed in all groups. Our results suggest that MMP-2 expression may contribute to the distinct invasive patterns seen in SCC and BCC.

  14. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

    PubMed

    Jiwani, Shahanawaz; Alvarado, Stephenie; Ohr, Ryan J; Romero, Adriana; Nguyen, Brenda; Jewett, Travis J

    2013-02-01

    All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

  15. A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire

    PubMed Central

    2011-01-01

    Background The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. Results We use expression microarrays to identify the lola-dependent transcriptome in the Drosophila embryo. We find that lola regulates the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, we find lola to be a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. We show that spire is expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. We further show that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. Conclusions These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis. PMID:22129300

  16. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  17. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    PubMed Central

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2015-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

  18. Study on loss mechanism of SMA tracheal stent subjected to cough excitation.

    PubMed

    Zhu, Zhiwen; Li, Xinmiao; Xu, Jia

    2015-01-01

    A kind of Ti-Ni shape memory alloy (SMA) hysteretic nonlinear model is developed, and the loss mechanism of a SMA tracheal stent subjected to cough excitation is studied in this paper. Nonlinear differential items are introduced to express the hysteretic phenomena of Ti-Ni SMA, and the fitting effect of the SMA constitutive model on the experimental data is proved by the partial least-square regression method. The nonlinear dynamic model of a Ti-Ni SMA tracheal stent subjected to cough excitation is developed, and the system's dynamic response is obtained. The numerical results show that the system's vibration is little in weak excitation, becomes large with the increase of the stochastic excitation, and finally becomes little again with the further increase of the stochastic excitation; the stochastic resonance phenomenon occurs in the process, which may cause stent fracture or loss.

  19. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization*

    PubMed Central

    Hien, Tran Thi; Turczyńska, Karolina M.; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F.; Albinsson, Sebastian

    2016-01-01

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. PMID:26683376

  20. Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

    The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration

  1. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects.

  2. Two SMA-Actuated Miniature Mechanisms

    NASA Technical Reports Server (NTRS)

    Willey, Cliff E.

    2005-01-01

    The figures depict two miniature mechanisms actuated by strips made of shape-memory alloy (SMA). A typical SMA is a nickel-titanium alloy known by the trade name "Flexinol" or "Nitinol." In preparation for a typical application, a suitably sized and shaped piece of an SMA is deformed by a predetermined amount at the lower of two operating temperatures, then mounted in a mechanism. When stroking of the mechanism in one direction is desired, the piece of SMA is heated above a transition temperature to make it return to the "remembered" undeformed state. When stroking of the mechanism in the opposite direction is desired, the SMA is cooled below the transition temperature to make it return to the deformed state. Also, the SMA alloy chosen for a specific application is one that has a transition temperature somewhat above the ambient temperature, so that stroking in one direction or the opposite direction can be achieved by heating the SMA, or refraining from heating the SMA, respectively, above the transition temperature. In the present mechanisms as in typical other SMA mechanisms, the heating is effected by electric currents applied via electrical contacts at the ends of the SMA strips. The purpose served by the mechanism of Figure 1 is to lock or release a flexible latch attachment. In preparation for use in this mechanism, two initially straight SMA strips are deformed into curved springs that, when mounted in the mechanism at ambient temperature, clamp the knob at the lower end of the flexible latch attachment. When heated above their transition temperature by an electric current, the SMA strips return to their original straight configuration, thereby releasing the knob. This mechanism is redundant in the sense that as long as at least one of the two SMA strips straightens when commanded to do so, the knob is released. The mechanism of Figure 2 is suited to any of a variety of applications in which there are requirements for a small mechanism that affords

  3. SMA Syndrome Treated by Single Incision Laparoscopic Duodenojejunostomy.

    PubMed

    Kim, Sungsoo; Kim, Yoo Seok; Min, Young-Don

    2014-01-01

    Superior mesenteric artery (SMA) syndrome is a mechanical duodenal obstruction by the SMA. The traditional approach to SMA syndrome was open bypass surgery. Nowadays, a conventional approach has been replaced by laparoscopic surgery. But single incision laparoscopic approach for SMA syndrome is rare. Herein, we report the first case of SMA syndrome patient who was treated by single incision laparoscopic duodenojejunostomy.

  4. A novel function for the MAP kinase SMA-5 in intestinal tube stability.

    PubMed

    Geisler, Florian; Gerhardus, Harald; Carberry, Katrin; Davis, Wayne; Jorgensen, Erik; Richardson, Christine; Bossinger, Olaf; Leube, Rudolf E

    2016-12-01

    Intermediate filaments are major cytoskeletal components whose assembly into complex networks and isotype-specific functions are still largely unknown. Caenorhabditis elegans provides an excellent model system to study intermediate filament organization and function in vivo. Its intestinal intermediate filaments localize exclusively to the endotube, a circumferential sheet just below the actin-based terminal web. A genetic screen for defects in the organization of intermediate filaments identified a mutation in the catalytic domain of the MAP kinase 7 orthologue sma-5(kc1) In sma-5(kc1) mutants, pockets of lumen penetrate the cytoplasm of the intestinal cells. These membrane hernias increase over time without affecting epithelial integrity and polarity. A more pronounced phenotype was observed in the deletion allele sma-5(n678) and in intestine-specific sma-5(RNAi) Besides reduced body length, an increased time of development, reduced brood size, and reduced life span were observed in the mutants, indicating compromised food uptake. Ultrastructural analyses revealed that the luminal pockets include the subapical cytoskeleton and coincide with local thinning and gaps in the endotube that are often enlarged in other regions. Increased intermediate filament phosphorylation was detected by two-dimensional immunoblotting, suggesting that loss of SMA-5 function leads to reduced intestinal tube stability due to altered intermediate filament network phosphorylation.

  5. A novel function for the MAP kinase SMA-5 in intestinal tube stability

    PubMed Central

    Geisler, Florian; Gerhardus, Harald; Carberry, Katrin; Davis, Wayne; Jorgensen, Erik; Richardson, Christine; Bossinger, Olaf; Leube, Rudolf E.

    2016-01-01

    Intermediate filaments are major cytoskeletal components whose assembly into complex networks and isotype-specific functions are still largely unknown. Caenorhabditis elegans provides an excellent model system to study intermediate filament organization and function in vivo. Its intestinal intermediate filaments localize exclusively to the endotube, a circumferential sheet just below the actin-based terminal web. A genetic screen for defects in the organization of intermediate filaments identified a mutation in the catalytic domain of the MAP kinase 7 orthologue sma-5(kc1). In sma-5(kc1) mutants, pockets of lumen penetrate the cytoplasm of the intestinal cells. These membrane hernias increase over time without affecting epithelial integrity and polarity. A more pronounced phenotype was observed in the deletion allele sma-5(n678) and in intestine-specific sma-5(RNAi). Besides reduced body length, an increased time of development, reduced brood size, and reduced life span were observed in the mutants, indicating compromised food uptake. Ultrastructural analyses revealed that the luminal pockets include the subapical cytoskeleton and coincide with local thinning and gaps in the endotube that are often enlarged in other regions. Increased intermediate filament phosphorylation was detected by two-dimensional immunoblotting, suggesting that loss of SMA-5 function leads to reduced intestinal tube stability due to altered intermediate filament network phosphorylation. PMID:27733627

  6. SMA microactuators for microvalve applications

    NASA Astrophysics Data System (ADS)

    Kohl, M.; Liu, Y.; Krevet, B.; Dürr, S.; Ohtsuka, M.

    2004-06-01

    This paper presents a study of the mechanical and thermal properties of SMA microvalves, which are driven by a microactuator of a NiTi foil or a Ni2MnGa thin film. By coupled finite element simulations, von Mises stress profiles and resulting actuation forces are determined, which are compared with experimental force-displacement characteristics. The relatively high phase transformation temperatures of the used Ni2MnGa specimens Mf / Af of 92 / 125 circC, give rise to cooling times of about 25 ms compared to 90 ms for NiTi. Thus, Ni2MnGa microvalves show a superior switching performance. However, due to brittleness and buckling instability, Ni2MnGa microvalves only allow a maximum controllable pressure difference of 70 kPa, which is more than a factor of 7 smaller compared to NiTi microvalves.

  7. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-XL expression

    PubMed Central

    2011-01-01

    Background Thymoquinone (TQ), the major active component of the medicinal herb Nigella sativa Linn., has been described as a chemopreventive and chemotherapeutic compound. Methods In this study, we investigated the effect of TQ on survival, actin cytoskeletal reorganization, proliferation and signal transduction in multiple myeloma (MM) cells. Results We found that TQ induces growth arrest in both MDN and XG2 cells in a dose- and time-dependent manner. TQ also inhibited CXC ligand-12 (CXCL-12)-mediated actin polymerization and cellular proliferation, as shown by flow cytometry. The signal transducer and activator of transcription (STAT) and B-cell lymphoma-2 (Bcl-2) signaling pathways may play important roles in the malignant transformation of a number of human malignancies. The constitutive activation of the STAT3 and Bcl-2 pathways is frequently observed in several cancer cell lines, including MM cells. Using flow cytometry, we found that TQ markedly decreased STAT3 phosphorylation and Bcl-2 and Bcl-XL expression without modulating STAT5 phosphorylation in MM cells. Using western blotting, we confirmed the inhibitory effect of TQ on STAT3 phosphorylation and Bcl-2 and Bcl-XL expression. Conclusions Taken together, our data suggests that TQ could potentially be applied toward the treatment of MM and other malignancies. PMID:22177381

  8. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

    Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

    2012-01-15

    2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.

  9. Functional dissociation of pre-SMA and SMA-proper in temporal processing.

    PubMed

    Schwartze, Michael; Rothermich, Kathrin; Kotz, Sonja A

    2012-03-01

    The ability to assess temporal structure is crucial in order to adapt to an ever-changing environment. Increasing evidence suggests that the supplementary motor area (SMA) is involved in both sensory and sensorimotor processing of temporal structure. However, it is not entirely clear whether the structural differentiation of the SMA translates into functional specialization, and how the SMA relates to other systems that engage in temporal processing, namely the cerebellum and cortico-striatal circuits. Anatomically, the SMA comprises at least two subareas, the rostral pre-SMA and the caudal SMA-proper. Each displays a characteristic pattern of connections to motor and non-motor structures. Crucially, these connections establish a potential hub among cerebellar and cortico-striatal systems, possibly forming a dedicated subcortico-cortical temporal processing network. To further explore the functional role of each SMA subarea, we performed a meta-analysis of functional neuroimaging studies by contrasting activations according to whether they linked with either sensory, sensorimotor, sequential, non-sequential, explicit, non-explicit, subsecond, or suprasecond temporal processing. This procedure yielded a set of functional differences, which mirror the rostro-caudal anatomical dimension. Activations associated with sensory, non-sequential, and suprasecond temporal processing tend to locate to the rostral SMA, while the opposite is true for the caudal SMA. These findings confirm a functional dissociation of pre-SMA and SMA-proper in temporal processing.

  10. Mouse models of SMA: tools for disease characterization and therapeutic development.

    PubMed

    Bebee, Thomas W; Dominguez, Catherine E; Chandler, Dawn S

    2012-08-01

    Mouse models of human disease are an important tool for studying disease mechanism and manifestation in a way that is physiologically relevant. Spinal muscular atrophy (SMA) is a neurodegenerative disease that is caused by deletion or mutation of the survival motor neuron gene (SMN1). The SMA disease is present in a spectrum of disease severities ranging from infant mortality, in the most severe cases, to minor motor impairment, in the mildest cases. The variability of disease severity inversely correlates with the copy number, and thus expression of a second, partially functional survival motor neuron gene, SMN2. Correspondingly, a plethora of mouse models has been developed to mimic these different types of SMA. These models express a range of SMN protein levels and extensively cover the severe and mild types of SMA, with neurological and physiological manifestation of disease supporting the relevance of these models. The SMA models provide a strong background for studying SMA and have already shown to be useful in pre-clinical therapeutic studies. The purpose of this review is to succinctly summarize the genetic and disease characteristic of the SMA mouse models and to highlight their use for therapeutic testing.

  11. RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria

    PubMed Central

    2011-01-01

    Background The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. Conclusion This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals. PMID:21679422

  12. The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

    PubMed Central

    Belmont, Lisa D.; Drubin, David G.

    1998-01-01

    Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables. PMID:9732289

  13. Actin of Beta vulgaris seedlings under the clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Ye.

    We study the influence of altered gravity on actin expression in roots of Beta vulguris seedlings grown on the horizontal clinostat (2 rpm) from seed germination for three days. It is shown that the total actin quantity was not influenced. Three actin isoforms are revealed; a relative protein quantity of these isoforms was similar both in clinorotated seedlings and in ones grown in norm. This point to stable expression of actin under the altered gravity conditions.

  14. Lubricin and smooth muscle α-actin-containing myofibroblasts in the pseudomembranes around loose hip and knee prostheses.

    PubMed

    Cheriyan, Thomas; Ready, John E; Brick, Gregory W; Martin, Scott D; Martin, Tamara L; Schmid, Thomas M; Padera, Robert F; Spector, Myron

    2013-03-01

    The objective was to evaluate the presence and distribution of the lubricating and anti-adhesion glycoprotein lubricin and cells containing the contractile isoform smooth muscle α-actin (SMA) in pseudomembranes around loose hip prostheses. Periprosthetic tissue was obtained at revision arthroplasty of eight aseptic, loose hip implants, and for comparison three loose knee prostheses. Immunohistochemical analysis was performed in 3 zones: zone 1, within 300μm of the edge of the implant-tissue interface; zone 2, between zones 1 and 3; zone 3, within 300μm of the resected/trimmed edge. The presence of lubricin was extensive in all samples: (1) as a discrete layer at the implant-tissue interface; (2) within the extracellular matrix (ECM); (3) intracellularly. There was significantly more high grade (>50%) lubricin surface staining at the implant-tissue interface compared with the resected edge. While there was also a significant effect of location of high grade ECM lubricin staining, there was no significant effect of implant type (i.e. hip versus knee). All but two hip pseudomembrane samples showed the presence of many SMA-containing cells. There was a significant effect of location on the number of SMA-expressing cells, but not of implant type. These findings might explain why the management of loose prosthesis is so challenging.

  15. VPA response in SMA is suppressed by the fatty acid translocase CD36.

    PubMed

    Garbes, Lutz; Heesen, Ludwig; Hölker, Irmgard; Bauer, Tim; Schreml, Julia; Zimmermann, Katharina; Thoenes, Michaela; Walter, Michael; Dimos, John; Peitz, Michael; Brüstle, Oliver; Heller, Raoul; Wirth, Brunhilde

    2013-01-15

    Functional loss of SMN1 causes proximal spinal muscular atrophy (SMA), the most common genetic condition accounting for infant lethality. Hence, the hypomorphic copy gene SMN2 is the only resource of functional SMN protein in SMA patients and influences SMA severity in a dose-dependent manner. Consequently, current therapeutic approaches focus on SMN2. Histone deacetylase inhibitors (HDACi), such as the short chain fatty acid VPA (valproic acid), ameliorate the SMA phenotype by activating the SMN2 expression. By analyzing blood SMN2 expression in 16 VPA-treated SMA patients, about one-third of individuals were identified as positive responders presenting increased SMN2 transcript levels. In 66% of enrolled patients, a concordant response was detected in the respective fibroblasts. Most importantly, by taking the detour of reprograming SMA patients' fibroblasts, we showed that the VPA response was maintained even in GABAergic neurons derived from induced pluripotent stem cells (iPS) cells. Differential expression microarray analysis revealed a complete lack of response to VPA in non-responders, which was associated with an increased expression of the fatty acid translocase CD36. The pivotal role of CD36 as the cause of non-responsiveness was proven in various in vitro approaches. Most importantly, knockdown of CD36 in SMA fibroblasts converted non- into pos-responders. In summary, the concordant response from blood to the central nervous system (CNS) to VPA may allow selection of pos-responders prior to therapy. Increased CD36 expression accounts for VPA non-responsiveness. These findings may be essential not only for SMA but also for other diseases such as epilepsy or migraine frequently treated with VPA.

  16. The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

    PubMed

    Hosseinibarkooie, Seyyedmohsen; Peters, Miriam; Torres-Benito, Laura; Rastetter, Raphael H; Hupperich, Kristina; Hoffmann, Andrea; Mendoza-Ferreira, Natalia; Kaczmarek, Anna; Janzen, Eva; Milbradt, Janine; Lamkemeyer, Tobias; Rigo, Frank; Bennett, C Frank; Guschlbauer, Christoph; Büschges, Ansgar; Hammerschmidt, Matthias; Riessland, Markus; Kye, Min Jeong; Clemen, Christoph S; Wirth, Brunhilde

    2016-09-01

    Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

  17. Recombinant alpha-actin for specific fluorescent labeling.

    PubMed

    Iwane, Atsuko H; Morimatsu, Masatoshi; Yanagida, Toshio

    2009-01-01

    Until recently, actin was thought to act merely as a passive track for its motility partner, myosin, during actomyosin interactions. Yet a recent report having observed dynamical conformational changes in labeled skeletal muscle alpha-actin suggests that actin has a more active role. Because the labeling technique was still immature, however, conclusions regarding the significance of the different conformations are difficult to make. Here, we describe the preparation of fully active alpha-actin obtained from a baculovirus expression system. We developed alpha-actin recombinants, of which subdomains 1 and 2 have specific sites for fluorescent probes. This specific labeling technique offers to significantly expand the information acquired from actin studies.

  18. Actin-related protein Arp6 influences H2A.Z-dependent and -independent gene expression and links ribosomal protein genes to nuclear pores.

    PubMed

    Yoshida, Takahito; Shimada, Kenji; Oma, Yukako; Kalck, Véronique; Akimura, Kazumi; Taddei, Angela; Iwahashi, Hitoshi; Kugou, Kazuto; Ohta, Kunihiro; Gasser, Susan M; Harata, Masahiko

    2010-04-15

    Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Delta) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Delta strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression.

  19. S&MA Requirements Tool (SMART)

    NASA Technical Reports Server (NTRS)

    Kulpa, Vyga

    2004-01-01

    In FY03 QS10 began building an S&MA web based data management tool,"Safety & Mission Assurance Requirements Tool" (SMART) that identifies S&MA requirements, tailors requirements to IAW project/program categories, tracks implementation, and provides a template for developing requirements and tracking waivers. This report provides a SMART process flow, typical application, typical data requirement deliverables, progress in 03, and FY04 activities.

  20. Increased IGF-1 in muscle modulates the phenotype of severe SMA mice.

    PubMed

    Bosch-Marcé, Marta; Wee, Claribel D; Martinez, Tara L; Lipkes, Celeste E; Choe, Dong W; Kong, Lingling; Van Meerbeke, James P; Musarò, Antonio; Sumner, Charlotte J

    2011-05-01

    Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

  1. Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

    PubMed Central

    Bosch-Marcé, Marta; Wee, Claribel D.; Martinez, Tara L.; Lipkes, Celeste E.; Choe, Dong W.; Kong, Lingling; Van Meerbeke, James P.; Musarò, Antonio; Sumner, Charlotte J.

    2011-01-01

    Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1NEG and mIGF-1POS SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients. PMID:21325354

  2. An assessment of the malignant potential of actinic keratoses and Bowen's disease: p53 and PCNA expression pattern correlate with the number of desmosomes.

    PubMed

    Ramzi, Saeef Taher; Maruno, Motoyoshi; Khaskhely, Noor Mohammad; Khan, Mohammed Abul Kasem; Takamiyagi, Atsushi; Uezato, Hiroshi; Nonaka, Shigeo

    2002-09-01

    Actinic keratoses (AK) and Bowen's disease (BD), both intraepidermal skin tumors, have a potential progression to squamous cell carcinoma (SCC). To evaluate the malignant potential of AK and BD, the expression pattern of p53 protein and proliferating cell nuclear antigen (PCNA) were examined in five types of AK and BD by immunohistochemistry. The ultrastructural difference of epidermal cells between AK and BD lesions was investigated. In the study of p53 and PCNA expression, the atrophic and acantholytic types of AK showed lower positive rates compared to others. These two types did not demonstrate all layers expression pattern. The number of desmosomes of the epidermal cells was significantly reduced in BD, and in the bowenoid and hypertrophic types of AK compared with other types of AK The number of hemi-desmosomes showed greatest reduction in BD and the bowenoid type of AK On the basis of our findings, it is hypothesized that atrophic and acantholytic types of AK may have the lowest, and the bowenoid type of AK and BD may have the highest, malignant potential.

  3. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  4. The nuclear actin-related protein ARP6 is a pleiotropic developmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering in Arabidopsis.

    PubMed

    Deal, Roger B; Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Meagher, Richard B

    2005-10-01

    Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.

  5. Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

    PubMed

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.

  6. Mediatory role of interleukin-6 in α smooth muscle actin induction and myofibroblast formation around silicone tissue expander.

    PubMed

    Joseph, Josna; Variathu, Kumary Thrikkovil; Mohanty, Mira

    2013-10-01

    Materials used for medical devices are usually tested for their biocompatibility, before use. However, it is known that long-term implantation in the body may lead to degradation of the material leading to an adverse tissue response. The failure of silicone breast implants due to excessive fibrosis and contracture has led to studies to delineate the cause of fibrosis around this material. To detect the biological moieties involved, conditioned media from RAW 264.7 macrophages seeded over commercially available silicone tissue expander material was added to L929 fibroblasts. Ultrahigh-molecular-weight polyethylene and tissue culture grade polystyrene served as the control materials. The gene expression of fibrogenic cytokines, interleukin-6 (IL-6), and transforming growth factor beta (TGFβ) in the RAW macrophages and myofibroblast marker alpha smooth muscle actinSMA) in L929 cells were quantitated by real time polymerase chain reaction. Protein expression analysis of αSMA was carried out by immunocytochemical staining and confocal microscopy. An in vitro degradation study of silicone expander material in pseudoextracellular fluid (PECF) and the αSMA expression in fibroblasts incubated with the silicone extract containing PECF has revealed the role of silicone leachants in induction of myofibroblasts. This in vitro expression study revealed the additional profibrotic role of IL-6 in fibroblast to myofibroblast transition and the synergy between material aspects and biomolecules in regulating fibrosis around Silicone implants. These findings may help in targeting newer biological moieties in the profibrotic pathway and in devising better manufacturing processes aiding the life of millions of patients.

  7. F-actin aggregates in transformed cells

    PubMed Central

    1981-01-01

    Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait. PMID:6270163

  8. Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro.

    PubMed Central

    Bains, W; Ponte, P; Blau, H; Kedes, L

    1984-01-01

    We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated. Images PMID:6493226

  9. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  10. Disrupting pre-SMA activity impairs facial happiness recognition: an event-related TMS study.

    PubMed

    Rochas, Vincent; Gelmini, Lauriane; Krolak-Salmon, Pierre; Poulet, Emmanuel; Saoud, Mohamed; Brunelin, Jerome; Bediou, Benoit

    2013-07-01

    It has been suggested that the left pre-supplementary motor area (pre-SMA) could be implicated in facial emotion expression and recognition, especially for laughter/happiness. To test this hypothesis, in a single-blind, randomized crossover study, we investigated the impact of transcranial magnetic stimulation (TMS) on performances of 18 healthy participants during a facial emotion recognition task. Using a neuronavigation system based on T1-weighted magnetic resonance imaging of each participant, TMS (5 pulses, 10 Hz) was delivered over the pre-SMA or the vertex (control condition) in an event-related fashion after the presentation of happy, fear, and angry faces. Compared with performances during vertex stimulation, we observed that TMS applied over the left pre-SMA specifically disrupted facial happiness recognition (FHR). No difference was observed between the 2 conditions neither for fear and anger recognition nor for reaction times (RT). Thus, interfering with pre-SMA activity with event-related TMS after stimulus presentation produced a selective impairment in the recognition of happy faces. These findings provide new insights into the functional implication of the pre-SMA in FHR, which may rely on the mirror properties of pre-SMA neurons.

  11. Pathological impact of SMN2 mis-splicing in adult SMA mice.

    PubMed

    Sahashi, Kentaro; Ling, Karen K Y; Hua, Yimin; Wilkinson, John Erby; Nomakuchi, Tomoki; Rigo, Frank; Hung, Gene; Xu, David; Jiang, Ya-Ping; Lin, Richard Z; Ko, Chien-Ping; Bennett, C Frank; Krainer, Adrian R

    2013-10-01

    Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose-response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.

  12. JNK1 and JNK2 regulate α-SMA in hepatic stellate cells during CCl4 -induced fibrosis in the rat liver.

    PubMed

    Hong, Il-Hwa; Park, Sang-Joon; Goo, Moon-Jung; Lee, Hye-Rim; Park, Jin-Kyu; Ki, Mi-Ran; Kim, Sang-Hyeob; Lee, Eun-Mi; Kim, Ah-Young; Jeong, Kyu-Shik

    2013-10-01

    Following liver injuries, hepatic stellate cells (HSCs) express α-SMA. Mitogen activated protein kinase (MAPK) signaling pathways mediate α-SMA expression in distinct cell types. However, the regulation of α-SMA expression by MAPKs in HSCs has been rarely studied. We aimed to study the role of MAPKs in the activation of HSCs during liver fibrosis. Liver fibrosis of rats was induced by carbon tetrachloride. HSC-T6 cells, murine embryonic fibroblasts, JNK1(-/-) and JNK2(-/-) cells were used for in vitro studies. Immunohistochemistry and immunoblot analysis were used. We have found that the expression of JNK and α-SMA co-localized in HSCs during liver fibrosis, but ERK and p38 expressed in macrophages. The expression of α-SMA was up-regulated by JNK1 and JNK2 in non-stress condition. Under TGF-β stimulation, however, the level α-SMA expression was increased by only JNK1, but not significantly changed by JNK2. We suggest that JNKs are responsible for α-SMA regulation, and especially JNK1 has a major role in up-regulation of α-SMA expression in HSCs under stress condition induced by TGF-β during liver fibrosis.

  13. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  14. TMS of supplementary motor area (SMA) facilitates mental rotation performance: Evidence for sequence processing in SMA.

    PubMed

    Cona, G; Marino, G; Semenza, C

    2017-02-01

    In the present study we applied online transcranial magnetic stimulation (TMS) bursts at 10Hz to the supplementary motor area (SMA) and primary motor cortex to test whether these regions are causally involved in mental rotation. Furthermore, in order to investigate what is the specific role played by SMA and primary motor cortex, two mental rotation tasks were used, which included pictures of hands and abstract objects, respectively. While primary motor cortex stimulation did not affect mental rotation performance, SMA stimulation improved the performance in the task with object stimuli, and only for the pairs of stimuli that had higher angular disparity between each other (i.e., 100° and 150°). The finding that the effect of SMA stimulation was modulated by the amount of spatial orientation information indicates that SMA is causally involved in the very act of mental rotation. More specifically, we propose that SMA mediates domain-general sequence processes, likely required to accumulate and integrate information that are, in this context, spatial. The possible physiological mechanisms underlying the facilitation of performance due to SMA stimulation are discussed.

  15. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  16. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

  17. Short Term Exposure of Beta Cells to Low Concentrations of Interleukin-1β Improves Insulin Secretion through Focal Adhesion and Actin Remodeling and Regulation of Gene Expression*

    PubMed Central

    Arous, Caroline; Ferreira, Pedro G.; Dermitzakis, Emmanouil T.; Halban, Philippe A.

    2015-01-01

    Type 2 diabetes involves defective insulin secretion with islet inflammation governed in part by IL-1β. Prolonged exposure of islets to high concentrations of IL-1β (>24 h, 20 ng/ml) impairs beta cell function and survival. Conversely, exposure to lower concentrations of IL-1β for >24 h improves these same parameters. The impact on insulin secretion of shorter exposure times to IL-1β and the underlying molecular mechanisms are poorly understood and were the focus of this study. Treatment of rat primary beta cells, as well as rat or human whole islets, with 0.1 ng/ml IL-1β for 2 h increased glucose-stimulated (but not basal) insulin secretion, whereas 20 ng/ml was without effect. Similar differential effects of IL-1β depending on concentration were observed after 15 min of KCl stimulation but were prevented by diazoxide. Studies on sorted rat beta cells indicated that the enhancement of stimulated secretion by 0.1 ng/ml IL-1β was mediated by the NF-κB pathway and c-JUN/JNK pathway acting in parallel to elicit focal adhesion remodeling and the phosphorylation of paxillin independently of upstream regulation by focal adhesion kinase. Because the beneficial effect of IL-1β was dependent in part upon transcription, gene expression was analyzed by RNAseq. There were 18 genes regulated uniquely by 0.1 but not 20 ng/ml IL-1β, which are mostly involved in transcription and apoptosis. These results indicate that 2 h of exposure of beta cells to a low but not a high concentration of IL-1β enhances glucose-stimulated insulin secretion through focal adhesion and actin remodeling, as well as modulation of gene expression. PMID:25586177

  18. The testis-specific VAD1.3/AEP1 interacts with {beta}-actin and syntaxin 1 and directs peri-nuclear/Golgi expression with bipartite nucleus localization (BNL) sequence

    SciTech Connect

    Zuo, Yan; Gao, Jing; Yeung, William S.B.; Lee, Kai-Fai

    2010-10-15

    Research highlights: {yields} VAD1.3 interacts {beta}-actin and syntaxin 1. {yields} VAD1.3 colocalizes {beta}-actin in spermatids. {yields} The bipartite nucleus localization (BNL) signal is important for peri-nuclear/Golgi expression in transfected cells. {yields} The C-terminal region of VAD1.3 direct nuclei localization. -- Abstract: VAD1.3 (AEP1), a novel testis-specific gene, was first isolated from the testis of a retinol-treated vitamin-A-deficient (VAD) rat model. It is expressed at the acrosomal region of spermatids from postnatal day 25. VAD1.3 immunoreactivity is present in rat, human, monkey and porcine spermatids and spermatozoa, suggesting that VAD1.3 may play a role in acrosome formation. However, direct evidence on the detailed sub-cellular localization of the VAD1.3 protein in the acrosome and how VAD1.3 is involved in acrosome formation remains largely unknown. Here, we isolated and identified VAD1.3 interacting proteins by immunoprecipitation followed by mass spectrometry, and determined the functional motifs of VAD1.3 that were important for its specific sub-cellular location in vitro. We found that VAD1.3 bound to syntaxin 1 and {beta}-actin proteins in vitro. Immunogold electron microscopic study localized VAD1.3 immunoreactivity to the acrosome membranes and matrix, and colocalized it with the {beta}-actin protein. The full-length GFP-VAD (1-3601) and GFP-VAD (1-730) fusion proteins that contain the bipartite nucleus localization (BNL) signal were located in the peri-nucleus/Golgi of the transfected cells. In addition, the GFP signal colocalized with the endoplasmic reticulum marker and the syntaxin 1 protein in the transfected HeLa and GC-2spd cells. The C-terminal GFP-VAD (1770-3601) was expressed in the nucleus. Taken together, VAD1.3 interacts with {beta}-actin and syntaxin 1 in vitro. The BNL signal may mediate the peri-nuclei localization of the protein that may interact with syntaxin 1 and {beta}-actin for acrosome formation in

  19. Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β

    PubMed Central

    Papke, Christina L.; Cao, Jiumei; Kwartler, Callie S.; Villamizar, Carlos; Byanova, Katerina L.; Lim, Soon-Mi; Sreenivasappa, Harini; Fischer, Grant; Pham, John; Rees, Meredith; Wang, Miranda; Chaponnier, Christine; Gabbiani, Giulio; Khakoo, Aarif Y.; Chandra, Joya; Trache, Andreea; Zimmer, Warren; Milewicz, Dianna M.

    2013-01-01

    Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2−/− mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2−/− SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations. PMID:23591991

  20. Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β.

    PubMed

    Papke, Christina L; Cao, Jiumei; Kwartler, Callie S; Villamizar, Carlos; Byanova, Katerina L; Lim, Soon-Mi; Sreenivasappa, Harini; Fischer, Grant; Pham, John; Rees, Meredith; Wang, Miranda; Chaponnier, Christine; Gabbiani, Giulio; Khakoo, Aarif Y; Chandra, Joya; Trache, Andreea; Zimmer, Warren; Milewicz, Dianna M

    2013-08-01

    Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2(-/-) mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2(-/-) SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.

  1. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  2. Expression patterns of sarcomeric α-actin, α-actinin and UCP2 in the myocardium of Kunming mice after exposure to c-terminal polypeptide of cardiotrophin-1.

    PubMed

    Chen, Shu-fen; Rao, Li-ya; Wei, Tao-zhi; Xu, Min-guang; Dong, Zhan-ling

    2014-12-01

    Cardiotrophin-1 (CT-1) activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expression pattern of sarcomeric contractile protein α-actin, specialized cytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 (UCP2) in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide (CP) of CT-1 (CT-1-CP, 500 μg·kg(-1)· day(-1)) for 1, 2, 3 and 4 week (s), respectively (4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group). Those injected with physiological saline for 4 weeks served as controls (n=10, with 5 males and 5 females). The heart tissues of mice were harvested at 1, 2, 3 or 4 week (s). Immunohistochemistry (IHC) and Western blotting (WB) were used to detect the distribution and expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyocytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The expression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group (IHC: χ(2)=6.125; WB: F=0.249, P>0.05) and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups (χ (2)=7.386, P>0.05). But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group (F=2.912; q=4.203, P<0.05). Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group (ICH: χ (2)=21.977; WB: F=50.388; P<0.01). The expression of UCP2

  3. Desmoplastic stromal response as defined by positive α-smooth muscle actin staining is predictive of invasion in adenocarcinoma of the uterine cervix.

    PubMed

    Jordan, Sara M; Watanabe, Tawny; Osann, Kathryn; Monk, Bradley J; Lin, Fritz; Rutgers, Joanne K L

    2012-07-01

    The objective of this research was to examine the immunohistochemical profiles of adenocarcinoma in situ (AIS) and early invasive adenocarcinoma (AC) to identify biomarkers that enhance the accurate diagnosis of early invasive glandular lesions of the cervix. The University of California, Irvine, and Long Beach Memorial tumor registries were used to identify 20 women with AIS or early AC treated between 1990 and 2008. An immunohistochemical study was performed, and the primary endpoint measured was the correlation between biomarker expression and invasive disease as diagnosed on hematoxylin and eosin examination. The biomarkers studied included α-smooth muscle actin (α-SMA), estrogen receptor, carcinoembryonic antigen, Ki67, p16, cyclooxygenase-2, and cluster of differentiation 1a. Stains were described on the basis of (1) positive or negative staining; (2) intensity; (3) percentage of positive cells; and (4) pattern of staining. Statistical analysis was performed using SYSTAT v. 11.0. Fisher exact test, Mann-Whitney nonparametric test, κ statistic, and intraclass correlation coefficient were used to evaluate results and interpreter agreement. A statistically significant increase in the staining of the periglandular stroma for α-SMA was seen in AC as compared with AIS. The intensity was 2.2 versus 1.2 (P=0.04) and the percent of positive-staining cells was 44% versus 18% (P=0.05) in AC and AIS, respectively. The presence of a desmoplastic stromal response as identified by the increased periglandular staining for α-SMA is useful in identifying invasive glandular lesions of the endocervix. Further studies are necessary to establish biologically relevant cut-off values for α-SMA staining.

  4. Dynamic response of a frame with SMA bracing

    NASA Astrophysics Data System (ADS)

    Sun, Shuangshuang; Rajapakse, R. K. N. D.

    2003-08-01

    This paper investigates the dynamic response of a frame structure with pre-strained SMA bracing elements. The constitutive model proposed by Brinson is used to simulate the axial response of SMA bracing elements. A non-linear transient finite element model incorporated with Newmark's time integration scheme is used to analyze the dynamic response of a structure. The time histories of displacements and hysteresis loops of SMA tendons are computed under harmonic loading. The effect of forcing amplitude and initial pre-strains of SMA tendons on transient dynamic response of a structure is discussed. The suitability of pseudoelastic SMA for energy dissipation is briefly discussed.

  5. Anthropomorphic finger antagonistically actuated by SMA plates.

    PubMed

    Engeberg, Erik D; Dilibal, Savas; Vatani, Morteza; Choi, Jae-Won; Lavery, John

    2015-08-20

    Most robotic applications that contain shape memory alloy (SMA) actuators use the SMA in a linear or spring shape. In contrast, a novel robotic finger was designed in this paper using SMA plates that were thermomechanically trained to take the shape of a flexed human finger when Joule heated. This flexor actuator was placed in parallel with an extensor actuator that was designed to straighten when Joule heated. Thus, alternately heating and cooling the flexor and extensor actuators caused the finger to flex and extend. Three different NiTi based SMA plates were evaluated for their ability to apply forces to a rigid and compliant object. The best of these three SMAs was able to apply a maximum fingertip force of 9.01N on average. A 3D CAD model of a human finger was used to create a solid model for the mold of the finger covering skin. Using a 3D printer, inner and outer molds were fabricated to house the actuators and a position sensor, which were assembled using a multi-stage casting process. Next, a nonlinear antagonistic controller was developed using an outer position control loop with two inner MOSFET current control loops. Sine and square wave tracking experiments demonstrated minimal errors within the operational bounds of the finger. The ability of the finger to recover from unexpected disturbances was also shown along with the frequency response up to 7 rad s(-1). The closed loop bandwidth of the system was 6.4 rad s(-1) when operated intermittently and 1.8 rad s(-1) when operated continuously.

  6. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent.

    PubMed

    Kim, Hak Rim; Gallant, Cynthia; Leavis, Paul C; Gunst, Susan J; Morgan, Kathleen G

    2008-09-01

    Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

  7. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  8. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  9. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  10. Combination of keratins and alpha-smooth muscle actin distinguishes secretory coils from ducts of eccrine sweat glands.

    PubMed

    Li, Haihong; Zhang, Xiang; Zeng, Shaopeng; Li, Xuexue; Zhang, Bingna; Chen, Lu; Lin, Changmin; Zhang, Mingjun; Tang, Shijie; Fu, Xiaobing

    2015-04-01

    Eccrine sweat glands are comprised of secretory coils and ducts, which are distinct in morphology and function. To better understand the roles of the two parts in development, homeostasis, wound repair and regeneration of eccrine sweat glands, we must distinguish between them. In this study, the localization of keratins and alpha-SMA in human eccrine sweat glands was examined by immunofluorescence staining. Based on the differential localization of keratins and alpha-SMA in different cell types, four pairs of antibodies (K5/K7, K5/alpha-SMA, K14/K7 and K14/alpha-SMA) were used to differentiate secretory coils from ducts by double-immunofluorescence staining. Immunofluorescence staining showed that myoepithelial cells of secretory coils expressed K5, K14 and alpha-SMA, whereas secretory cells of secretory coils expressed K7, K8, K15, K18 and K19. Ductal cells expressed K5, K8, K14 and K19. Double-staining showed that the secretory coils were K5(+)/K7(+), K5(+)/alpha-SMA(+), K14(+)/K7(+) and K14(+)/alpha-SMA(+), whereas ducts were K5(+)/K7(-), K5(+)/alpha-SMA(-), K14(+)/K7(-) and K14(+)/alpha-SMA(-). In conclusion, by combining use of keratins and alpha-SMA antibodies, secretory coils can be easily differentiated from ducts in morphology.

  11. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  12. Indoxyl sulfate-induced activation of (pro)renin receptor is involved in expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells.

    PubMed

    Saito, Shinichi; Shimizu, Hidehisa; Yisireyili, Maimaiti; Nishijima, Fuyuhiko; Enomoto, Atsushi; Niwa, Toshimitsu

    2014-05-01

    Activation of (pro)renin receptor (PRR) is involved in the progression of chronic kidney disease. However, the role of indoxyl sulfate, a uremic toxin, in the activation of PRR is not clear. The present study aimed to clarify the role of indoxyl sulfate in activation of PRR, in relation to renal expression of fibrotic genes. Renal expression of PRR and renin/prorenin was up-regulated in chronic kidney disease rats compared with normal rats, whereas AST-120 suppressed these expression by reducing serum levels of indoxyl sulfate. Furthermore, administration of indoxyl sulfate to normotensive and hypertensive rats increased renal expression of PRR and renin/prorenin. Indoxyl sulfate induced expression of PRR and prorenin in cultured human proximal tubular cells (HK-2 cells). Indoxyl sulfate-induced PRR expression was inhibited by small interfering RNAs of signal transducer and activator of transcription 3 (Stat3) and nuclear factor-κB p65 in proximal tubular cells. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed indoxyl sulfate-induced PRR expression in proximal tubular cells. N-acetylcysteine prevented indoxyl sulfate-induced phosphorylation of Stat3 in proximal tubular cells. PRR small interfering RNA inhibited indoxyl sulfate-induced expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Taken together, indoxyl sulfate-induced up-regulation of prorenin expression and activation of PRR through production of reactive oxygen species and activation of Stat3 and nuclear factor-κB play an important role in the expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.

  13. Integrin α3 blockade enhances microtopographical down-regulation of α-smooth muscle actin: role of microtopography in ECM regulation.

    PubMed

    Ayala, Perla; Desai, Tejal A

    2011-07-01

    Development of functional engineered matrices for regenerative therapies can benefit from an understanding of how physical cues at the microscale affect cell behavior. In this work, we use microfabricated systems to study how stiffness and microscale topographical cues in the form of "micropegs" affect extracellular matrix synthesis. Previous work from our lab has shown that microtopographical cues in 2D and 3D systems decrease cellular proliferation and regulate matrix synthesis. In this work, the combined role of stiffness and topography on ECM synthesis is investigated in a 2D micropeg system. These studies show that fibroblasts cultured on polydimethylsiloxane (PDMS) substrates with micropegs have reduced expression of collagen type I (Col I) and collagen type VI (Col VI) compared to fibroblasts cultured on flat substrates. In addition, cells on micropegged substrates exhibit down-regulation of other important regulators of ECM synthesis such as α-smooth muscle actin (α-SMA), and integrin α3 (Int α3). Interestingly, this effect is dependent on the contractility and adhesion of the cells. When cultured in the presence of RhoA kinase (ROCK) and myosin light chain kinase (MLCK) inhibitors, no significant differences in the expression of collagen, α-SMA, Int α3, and TGFB1 are observed. Additionally, disruptions in cell adhesion prevent microtopographical regulation of ECM synthesis. When using an antibody to block the extracellular domain of Int α3, no differences in the expression of collagen are observed and blocking Int α3 results in enhanced down-regulation of α-SMA on the stiffer micropegged substrates. These findings demonstrate that regulation of extracellular matrix production by cells on a synthetic substrate can be guided via physical cues at the microscale, and add to the body of knowledge on the role of integrin-mediated mechanotransduction.

  14. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  15. Actin and myosin isoforms in aneural and malformed chick hearts.

    PubMed

    Kirby, M L; Shimizu, N; Gagnon, J; Toyofuku, T; Kennedy, J; Conrad, D C; Zak, R

    1990-09-01

    Although it is generally accepted that actin and myosin isoforms adapt to their functional requirements, the sequence of expression of these proteins in hearts developing abnormally is unknown. In the chick embryo it is possible to change various aspects of heart development without direct manipulation of the cardiovascular system, by removing various regions of the neural crest from early embryos. The neural crest provides both neural (sympathetic and parasympathetic) and ectomesenchymal components to the heart, and selective removal of various areas results in embryos with sympathetically aneural hearts, or persistent truncus arteriosus with or without parasympathetic denervation. Myosin isoform expression was studied in each of these types of hearts using an array of myosin antibodies specific for atrium, ventricle or the conduction system. Myosin expression in experimental hearts was found to follow the normal pattern of development using these antibodies. Actin expression was studied using cDNA probes for the 3' untranslated region of actin mRNA of the alpha-skeletal, alpha-cardiac and beta-actin isoforms. Using slot-blot hybridization analysis, the pattern of actin expression in atrium and ventricle was followed throughout the period of incubation in normal hearts. The pattern of actin expression was found to be abnormal in hearts which were sympathetically aneural and those which had persistent truncus arteriosus combined with parasympathetic denervation. ATPase activity was increased only in atria of hearts with persistent truncus arteriosus. It appears from these experiments that actin isoform expression is influenced in the chick heart by autonomic innervation.

  16. Correlating interfacial properties with stress transfer in SMA composites

    SciTech Connect

    Kline, G.E.; Jonnalagadda, K.; Sottos, N.R.

    1995-12-31

    Shape memory alloy (SMA) wires have been proposed as large strain actuators for use in smart structures. SMA wires can be embedded in a host material to alter the stiffness or modal response and provide vibration control. The interaction between the embedded SMA and the host material is critical to applications requiring transfer of loads or strain from the wire to the host. Paine, Jones and Rogers have asserted the importance of interfacial adhesion between embedded SMA wires and the host material. When the SMA wires are actuated, large shear strains are generated at the SMA/host interface. The stronger the interface, the greater the transfer of strain from the actuator to the host material. Although there has been a significant amount of research dedicated to characterizing and modeling the response of SMA alone, little work has been done to understand the behavior of embedded SMA wires. Maximum displacement, load transfer and repeatability of actuation of the embedded wire are particularly critical in assessing the effects of the host material. This work continues to investigate the interaction between SMA wires and a host polymer matrix. High resolution photoelasticity was utilized to study the internal stresses induced during actuation of an embedded shape memory alloy wire in a polymer matrix. The influence of several wire surface treatments on the resulting stresses and load transfer was investigated. Four different surface treatments were considered: untreated, acid etched, hand sanded and sandblasted. Pull-out data indicated that sandblasting of wires increased the SMA/polymer interfacial bond strength while hand sanding and acid cleaning actually decreased the bond strength. Wires with greater adhesion (sandblasted) resulted in higher stresses induced in the polymer while those with lower adhesion transferred less load. Overall, properties of the SMA/polymer interface were shown to significantly affect the performance of the embedded SMA actuator.

  17. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  18. Fabrication and Characterization of SMA Hybrid Composites

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.; Lach, Cynthia L.; Cano, Robert J.

    2001-01-01

    Results from an effort to fabrication shape memory alloy hybrid composite (SMAHC) test specimens and characterize the material system are presented in this study. The SMAHC specimens are conventional composite structures with an embedded SMA constituent. The fabrication and characterization work was undertaken to better understand the mechanics of the material system, address fabrication issues cited in the literature, and provide specimens for experimental validation of a recently developed thermomechanical model for SMAHC structures. Processes and hardware developed for fabrication of the SMAHC specimens are described. Fabrication of a SMA14C laminate with quasi-isotropic lamination and ribbon-type Nitinol actuators embedded in the 0' layers is presented. Beam specimens are machined from the laminate and are the focus of recent work, but the processes and hardware are readily extensible to more practical structures. Results of thermomechanical property testing on the composite matrix and Nitinol ribbon are presented. Test results from the Nitinol include stress-strain behavior, modulus versus temperature. and constrained recovery stress versus temperature and thermal cycle. Complex thermomechanical behaviors of the Nitinol and composite matrix are demonstrated, which have significant implications for modeling of SMAHC structures.

  19. Targeting RNA-splicing for SMA treatment.

    PubMed

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

    The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease.

  20. 3-D diffusion tensor axonal tracking shows distinct SMA and pre-SMA projections to the human striatum.

    PubMed

    Lehéricy, Stéphane; Ducros, Mathieu; Krainik, Alexandre; Francois, Chantal; Van de Moortele, Pierre-François; Ugurbil, Kamil; Kim, Dae-Shik

    2004-12-01

    Studies in non-human primates have shown that medial premotor projections to the striatum are characterized as a set of distinct circuits conveying different type of information. This study assesses the anatomical projections from the supplementary motor area (SMA), pre-SMA and motor cortex (MC) to the human striatum using diffusion tensor imaging (DTI) axonal tracking. Eight right-handed volunteers were studied at 1.5 T using DTI axonal tracking. A connectivity matrix was computed, which tested for connections between cortical areas (MC, SMA and pre-SMA) and subcortical areas (posterior, middle and anterior putamen and the head of the caudate nucleus) in each hemisphere. Pre-SMA projections to the striatum were located rostral to SMA projections to the striatum. The SMA and the MC were similarly connected to the posterior and middle putamen and not to the anterior striatum. These data show that the MC and SMA have connections with similar parts of the sensorimotor compartment of the human striatum, whereas the pre-SMA sends connections to more rostral parts of the striatum, including the associative compartment.

  1. Atrophy of myoepithelial cells in parotid glands of diabetic mice; detection using skeletal muscle actin, a novel marker☆

    PubMed Central

    Nashida, Tomoko; Yoshie, Sumio; Haga-Tsujimura, Maiko; Imai, Akane; Shimomura, Hiromi

    2013-01-01

    In mouse parotid glands, we found expression of skeletal muscle actin (actin-α1) protein and mRNA. We isolated myoepithelial cells from the mouse parotid glands and investigated their actin-α1 expression because smooth muscle actin (actin-α2) has been used as a marker for myoepithelial cells. We used actin-α1 expression to identify pathological changes in diabetic non-obese diabetic (NOD; NOD/ShiJcl) mice—a mouse model for Sjögren's syndrome—and found myoepithelial cells to be decreased or atrophied in the diabetic state. PMID:23772384

  2. PKCδ as a regulator for TGFβ1-induced α-SMA production in a murine nonalcoholic steatohepatitis model.

    PubMed

    Lee, Su Jin; Kang, Jeong Han; Choi, Soo Young; Suk, Ki Tae; Kim, Dong Joon; Kwon, Oh-Shin

    2013-01-01

    The precise mechanism of TGFβ1 signaling in the progression of non-alcoholic steatohepatitis (NASH) has remained unclear. In particular, a potential regulatory mechanism by which PKCδ affects profibrogenic gene expression had never been explored. In this study, therefore, the role of PKCδ in TGFβ1 mediated α-SMA expression was investigated using NASH model mice. In preparation of the NASH model, male C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 3 weeks, after which time they were intraperitoneally injected with lipopolysaccharide (LPS). In addition, Tlr4(Lps-d) (CH3/HeJ) mice were used to demonstrate the TGFβ1 signaling's dependency on TLR4 induction. Liver histology and hepatic hepatitis markers were investigated, and hepatic gene expression levels were determined by real-time PCR. Acute liver injury by LPS injection specifically elevated not only α-SMA expression but also phospho-PKCδ in this model. In contrast, Tlr4(Lps-d) (CH3/HeJ) and blockade of TGFβ1 receptor by SB431542 resulted in a significant reduction of PKCδ activation and α-SMA expression. Moreover, the TGFβ1-induced α-SMA production was significantly reduced by a specific PKCδ inhibitor. These findings suggested that PKCδ plays a critical role in TGFβ1-induced α-SMA production in a NASH model. Thus, this was the first demonstration of the involvement of PKCδ in the regulation of α-SMA expression in NASH liver tissues, and the impaired induction of PKCδ phosphorylation by LPS in a steatohepatitis condition. Interestingly, treatment by PKCδ inhibitor caused dramatic reduction of myofibroblast activation, indicating that PKCδ represents a promising target for treating NASH.

  3. Switchable Shape Memory Alloys (SMA) Thermal Materials Project

    NASA Technical Reports Server (NTRS)

    Falker, John; Zeitlin, Nancy; Williams, Martha; Fesmire, James

    2014-01-01

    Develop 2-way switchable thermal systems for use in systems that function in cold to hot temperature ranges using different alloy designs for SMA system concepts. In this project, KSC will specifically address designs of two proof of concept SMA systems with transition temperatures in the 65-95 C range and investigate cycle fatigue and "memory loss" due to thermal cycling.

  4. SMA actuators: a viable practical technology (Presentation Video)

    NASA Astrophysics Data System (ADS)

    Browne, Alan L.; Brown, Jeffrey; Hodgson, Darel E.

    2015-04-01

    Diverse products either based solely on or incorporating Shape Memory Alloys (SMA) have and are being made in a wide range of industries, and IP is being captured. Why then compared to SE (superelastic) Nitinol, and especially conventional technology, do so few ideas reach production? This presentation delves deeply into this topic in reaching the final assessment that SMA actuators are indeed now a viable practical technology. The presentation begins with an introduction to and description of the fundamental basis of SMA actuator technology. Examples of multiple commercially available geometric forms of SMA actuators are given and the functionalities that they provide are described. This is followed by examples of multiple commercial products incorporating such SMA actuators. Given that there are literally millions of commercial products incorporating conventional actuator technologies, indications are given as to why there are their less than 1000 that utilize SMA. Experience based challenges to the commercial use of SMA actuators are described. Besides having to compete with existing non-SMA technology which is quite mature additional challenges that are unique to SM actuators are indicated these including a wider than expected set of technical engineering problems and challenges and that a broader scope of dynamics is required.

  5. Investigation on low velocity impact resistance of SMA composite material

    NASA Astrophysics Data System (ADS)

    Hu, Dianyin; Zhang, Long; Wang, Rongqiao; Zhang, Xiaoyong

    2016-04-01

    A method to improve low velocity impact resistance of aeroengine composite casing using shape memory alloy's properties of shape memory(SM) and super-elasticity(SE) is proposed in this study. Firstly, a numerical modeling of SMA reinforced composite laminate under low velocity impact load with impact velocity of 10 m/s is established based on its constitutive model implemented by the VUMAT subroutine of commercial software ABAQUS. Secondly, the responses of SMA composite laminate including stress and deflection distributions were achieved through transient analysis under low velocity impact load. Numerical results show that both peak stress and deflection values of SMA composite laminate are less than that without SMA, which proves that embedding SMA into the composite structure can effectively improve the low velocity impact performance of composite structure. Finally, the influence of SM and SE on low velocity impact resistance is quantitatively investigated. The values of peak stress and deflection of SMA composite based on SM property decrease by 18.28% and 9.43% respectively, compared with those without SMA, instead of 12.87% and 5.19% based on SE. In conclusion, this proposed model described the impact damage of SMA composite structure and turned to be a more beneficial method to enhance the impact resistance by utilizing SM effect.

  6. Directed actin assembly and motility.

    PubMed

    Boujemaa-Paterski, Rajaa; Galland, Rémi; Suarez, Cristian; Guérin, Christophe; Théry, Manuel; Blanchoin, Laurent

    2014-01-01

    The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

  7. Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized acti...

  8. SMN deficiency does not induce oxidative stress in SMA iPSC-derived astrocytes or motor neurons.

    PubMed

    Patitucci, Teresa N; Ebert, Allison D

    2016-02-01

    Spinal muscular atrophy (SMA) is a genetic disorder characterized by loss of motor neurons in the spinal cord leading to muscle atrophy and death. Although motor neurons (MNs) are the most obviously affected cells in SMA, recent evidence suggest dysfunction in multiple cell types. Astrocytes are a crucial component of the motor circuit and are intimately involved with MN health and maintenance. We have previously shown that SMA astrocytes are altered both morphologically and functionally early in disease progression, though it is unclear what causes astrocytes to become reactive. Oxidative stress is a common feature among neurodegenerative diseases. Oxidative stress can both induce apoptosis in neurons and can cause astrocytes to become reactive, which are features observed in the SMA induced pluripotent stem cell (iPSC) cultures. Therefore, we asked if oxidative stress contributes to SMA astrocyte pathology. We examined mitochondrial bioenergetics, transcript and protein levels of oxidative and anti-oxidant factors, and reactive oxygen species (ROS) production and found little evidence of oxidative stress. We did observe a significant increase in endogenous catalase expression in SMA iPSCs. While catalase knockdown in SMA iPSCs increased ROS production above basal levels, levels of ROS remained lower than in controls, further arguing against robust oxidative stress in this system. Viral delivery of survival motor neuron (SMN) reversed astrocyte activation and restored catalase levels to normal, without changing mitochondrial respiration or expression of oxidative stress markers. Taken together, these data indicate that SMN deficiency induces astrocyte reactivity, but does not do so through an oxidative stress-mediated process.

  9. Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

    PubMed

    Merritt, Raymond C; Manor, Uri; Salles, Felipe T; Grati, M'hamed; Dose, Andrea C; Unrath, William C; Quintero, Omar A; Yengo, Christopher M; Kachar, Bechara

    2012-02-21

    Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

  10. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  11. Preliminary characterization and modeling of SMA-based textile composites

    NASA Astrophysics Data System (ADS)

    Masuda, Arata; Ni, Qing-Qing; Sone, Akira; Zhang, Run-Xin; Yamamura, Takahiko

    2004-07-01

    In this paper, we conduct a feasibility study to investigate the future potential of textile composites with shape memory alloys. Two different types of SMA-based textile composites are presented. First, a composite plate with embedded woven SMA layer is fabricated, and the stiffness tuning capability is evaluated by impact vibration tests. The results are not favorable, but may be improved by increasing the volume fraction of SMA, and by controlling the prestrain more accurately during the lamination process. The modeling and analysis methodology for woven SMA-based composites are briefly discussed. Then, the possibility of textile composites with SMA stitching is discussed, that is expected to give the composites multi-functions such as tunable stiffness, shape control and sensing capability, selectively distributed on demand.

  12. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    PubMed Central

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. PMID:28129393

  13. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  14. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  15. Actinic keratosis. Current treatment options.

    PubMed

    Jeffes, E W; Tang, E H

    2000-01-01

    Actinic keratoses are hyperkeratotic skin lesions that represent focal abnormal proliferation of epidermal keratinocytes. Some actinic keratoses evolve into squamous cell carcinoma of the skin, while others resolve spontaneously. The conversion rate of actinic keratosis to squamous cell carcinoma is not accurately known, but appears to be in the range of 0.25 to 1% per year. Although there is a low rate of conversion of actinic keratoses to squamous cell carcinoma, 60% of squamous cell carcinomas of the skin probably arise from actinic keratoses. The main cause of actinic keratoses in otherwise healthy Caucasians appears to be the sun. Therapy for actinic keratoses begins with prevention which starts with sun avoidance and physical protection. Sunprotection with sunscreens actually slows the return of actinic keratoses in patients already getting actinic keratoses. Interestingly, a few studies are available that demonstrate that a high fat diet is associated with the production of more actinic keratoses than is a low fat diet. One of the mainstays of therapy has been local destruction of the actinic keratoses with cryotherapy, and curettage and electrodesiccation. A new addition to this group of therapies to treat individual actinic keratoses is photodynamic therapy with topical aminolevulinic acid and light. In patients who have numerous actinic keratoses in an area of severely sun damaged skin, therapies which are applied to the whole actinic keratosis area are used. The goal of treating such an area of skin is to treat all of the early as well as the numerous clinically evident actinic keratoses at the same time. The classical approaches for treating areas of photodamaged skin without treating actinic keratoses individually include: the use of topically applied fluorouracil cream, dermabrasion, and cutaneous peels with various agents like trichloroacetic acid. Both topically as well as orally administered retinoids have been used to treat actinic keratoses but

  16. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  17. Dynamic regulation of platelet-derived growth factor receptor α expression in alveolar fibroblasts during realveolarization.

    PubMed

    Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn; Perl, Anne-Karina T

    2012-10-01

    Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial

  18. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  19. Relationship between input power and power density of SMA spring

    NASA Astrophysics Data System (ADS)

    Park, Cheol Hoon; Ham, Sang Yong; Son, Young Su

    2016-04-01

    The important required characteristics of an artificial muscle for a human arm-like manipulator are high strain and high power density. From this viewpoint, an SMA (shape memory alloy) spring is a good candidate for the actuator of a robotic manipulator that utilizes an artificial muscle. In this study, the maximum power density of an SMA spring was evaluated with respect to the input power. The spring samples were fabricated from SMA wires of different diameters ranging between 0.1 and 0.3 mm. For each diameter, two types of wires with different transition temperatures were used. The relationship between the transition temperature and maximum power density was also evaluated. Each SMA spring was stretched downward by an attached weight and the temperature was increased through the application of an electric current. The displacement, velocity, and temperature of the SMA spring were measured by laser displacement sensors and a thermocouple. Based on the experimental data, it was determined that the maximum power densities of the different SMA springs ranged between 1,300 and 5,500 W/kg. This confirmed the applicability of an SMA spring to human arm-like robotic manipulators. The results of this study can be used as reference for design.

  20. Periostin expression induced by oxidative stress contributes to myocardial fibrosis in a rat model of high salt-induced hypertension

    PubMed Central

    WU, HAN; CHEN, LIANG; XIE, JUN; LI, RAN; LI, GUAN-NAN; CHEN, QIN-HUA; ZHANG, XIN-LIN; KANG, LI-NA; XU, BIAO

    2016-01-01

    Periostin is an extracellular matrix protein involved in fibrosis. The present study investigated the importance of periostin in hypertension-induced myocardial fibrosis. Rats were randomly divided into either the normal group (0.4% NaCl diet; n=8) or hypertension group (8% NaCl diet; n=8). For 36 weeks, the blood pressure and heart rate of the rats were monitored. At week 36, the hearts were extracted for further analysis. Masson's staining and western blotting were performed to determine the levels of periostin protein expression, oxidative stress and fibrosis. In addition, fibroblasts were isolated from adult rats and cultured in vitro, and following treatment with angiotensin II (Ang II) and N-acetyl-L-cysteine (NAC), western blotting, immunofluorescence and 2′,7′ dichlorodihydrofluorescin staining were performed to examine reactive oxygen species production, and periostin and α-smooth muscle actin (α-SMA) expression levels. The results demonstrated that periostin expression and oxidative stress were increased in hypertensive hearts compared with normal hearts. The in vitro experiments demonstrated that Ang II upregulated the expression levels of periostin and α-SMA compared with the control, whereas, pretreatment with NAC inhibited oxidative stress, periostin and α-SMA expression in fibroblasts. In conclusion, the results of the current study suggested that oxidative stress-induced periostin is involved in myocardial fibrosis and hypertension. The present study demonstrated that periostin inhibition may be a promising approach for the inhibition of hypertension-induced cardiac remodeling. PMID:27220372

  1. Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection*

    PubMed Central

    Gordón-Alonso, Mónica; Rocha-Perugini, Vera; Álvarez, Susana; Ursa, Ángeles; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Muñoz-Fernández, María A.; Sánchez-Madrid, Francisco

    2013-01-01

    HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1. PMID:23926103

  2. β- and γ-Actins in the nucleus of human melanoma A375 cells.

    PubMed

    Migocka-Patrzałek, Marta; Makowiecka, Aleksandra; Nowak, Dorota; Mazur, Antonina J; Hofmann, Wilma A; Malicka-Błaszkiewicz, Maria

    2015-11-01

    Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle β- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that β- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both β- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that β-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.

  3. Interaction of actin and the chloroplast protein import apparatus.

    PubMed

    Jouhet, Juliette; Gray, John C

    2009-07-10

    Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

  4. Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium.

    PubMed

    Furukawa, Ruth; Maselli, Andrew; Thomson, Susanne A M; Lim, Rita W L; Stokes, John V; Fechheimer, Marcus

    2003-01-01

    The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

  5. Miniature High-Force, Long-Stroke SMA Linear Actuators

    NASA Technical Reports Server (NTRS)

    Cummin, Mark A.; Donakowski, William; Cohen, Howard

    2008-01-01

    Improved long-stroke shape-memory-alloy (SMA) linear actuators are being developed to exert significantly higher forces and operate at higher activation temperatures than do prior SMA actuators. In these actuators, long linear strokes are achieved through the principle of displacement multiplication, according to which there are multiple stages, each intermediate stage being connected by straight SMA wire segments to the next stage so that relative motions of stages are additive toward the final stage, which is the output stage. Prior SMA actuators typically include polymer housings or shells, steel or aluminum stages, and polymer pads between successive stages of displacement-multiplication assemblies. Typical output forces of prior SMA actuators range from 10 to 20 N, and typical strokes range from 0.5 to 1.5 cm. An important disadvantage of prior SMA wire actuators is relatively low cycle speed, which is related to actuation temperature as follows: The SMA wires in prior SMA actuators are typically made of a durable nickel/titanium alloy that has a shape-memory activation temperature of 80 C. An SMA wire can be heated quickly from below to above its activation temperature to obtain a stroke in one direction, but must then be allowed to cool to somewhat below its activation temperature (typically, less than or equal to 60 C in the case of an activation temperature of 80 C) to obtain a stroke in the opposite direction (return stroke). At typical ambient temperatures, cooling times are of the order of several seconds. Cooling times thus limit cycle speeds. Wires made of SMA alloys having significantly higher activation temperatures [denoted ultra-high-temperature (UHT) SMA alloys] cool to the required lower return-stroke temperatures more rapidly, making it possible to increase cycle speeds. The present development is motivated by a need, in some applications (especially aeronautical and space-flight applications) for SMA actuators that exert higher forces, operate

  6. Highly-Compact SMA Actuators A Feasibiltiy Study of Fuel-Powered and Thermoelectric SMA Actuators

    DTIC Science & Technology

    2003-12-01

    TEM top surface TEM DC Power source Finned Heat Sink (FHS) DAQ Thermocouple(s) TEM bottom surface Config 1: single TEM ( Melcor ) Config 2: stacked TEM...strained to 3% before testing. A PWM based actuation control scheme was used to resistively heat the SMA actuator. TEMs were acquired from Melcor and...Output 1.70W 2.06W Avg. Energy Output 0.17J 0.103J Stacked (2) TEMs Energy Input 72J 36J Power Input 1.6W Power Input 501.6W 501.6W Weight 2.2gr

  7. Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila.

    PubMed

    Fernández, Beatriz García; Gaspar, Pedro; Brás-Pereira, Catarina; Jezowska, Barbara; Rebelo, Sofia Raquel; Janody, Florence

    2011-06-01

    The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. Here, we report that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control.

  8. Seismic Protection of an Ancient Aqueduct Using SMA Devices

    SciTech Connect

    Chrysostomou, Christis Z.; Demetriou, Themos; Stassis, Andreas; Hamdaoui, Karim

    2008-07-08

    The effectiveness of the use of Cu-based shape memory alloy (SMA) prestressing devices on an ancient aqueduct is examined in this paper. The dynamic characteristics of the aqueduct were measured within the span of three years and computational models were developed that matched very closely its dynamic behaviour. Using this as a bench mark, SMA prestressing devices were applied on the structure and the effects on its dynamic characteristics were assessed. It was noted that the SMA prestressing devices have a significant effect on the dynamic response of the structure. This is attributed to the stiffening of the structure due to the increase in contact between the masonry units and hence the increase of its stiffness through the increase of the modulus of elasticity of the masonry matrix. It can be concluded that the SMA prestressing devices can provide an inconspicuous means of stiffening masonry structures and increase their resistance to earthquake loads.

  9. Seismic Protection of an Ancient Aqueduct Using SMA Devices

    NASA Astrophysics Data System (ADS)

    Chrysostomou, Christis Z.; Demetriou, Themos; Stassis, Andreas; Hamdaoui, Karim

    2008-07-01

    The effectiveness of the use of Cu-based shape memory alloy (SMA) prestressing devices on an ancient aqueduct is examined in this paper. The dynamic characteristics of the aqueduct were measured within the span of three years and computational models were developed that matched very closely its dynamic behaviour. Using this as a bench mark, SMA prestressing devices were applied on the structure and the effects on its dynamic characteristics were assessed. It was noted that the SMA prestressing devices have a significant effect on the dynamic response of the structure. This is attributed to the stiffening of the structure due to the increase in contact between the masonry units and hence the increase of its stiffness through the increase of the modulus of elasticity of the masonry matrix. It can be concluded that the SMA prestressing devices can provide an inconspicuous means of stiffening masonry structures and increase their resistance to earthquake loads.

  10. Shape correction of composite beams with SMA wires

    NASA Astrophysics Data System (ADS)

    Maji, Arup K.; Rochin, Refugio

    2001-07-01

    Shape Memory Alloys (SMA) are increasingly used as smart devices ina erospace applicatons.Their priry advnatage over other smart materials z9piezo-ceramics adnpiezo-polymers) is in their ability to undergo large strains and dispalcements that enables the devlopment of smart mechanissm. The specific objective of this research was toinvestigate the use of embedded SMA for active shape correctionof flexibl emirror substrates.Active shape correction is a possiblemeans of mitigating thrmally induced distortions in space based optical iaging systems inorder to understand and quantify the designvalraibles that lead to that goal, embedded SMA wires we used toa cctuate a series of composite bmeasm.Themoemvent induced byactutionw as monitored with theMoire inteferietery method,and the rsutls wer compared witha n anlaytical model.The repeatabiltya nd rlaibltiyof a possible acutation systmeand the propertis of the SMa wires was also studied by testing the stress-strian anthe stress reo ery behavior under controlled conditons.110

  11. Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis▿

    PubMed Central

    Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen

    2009-01-01

    As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role. PMID:19710142

  12. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells.

    PubMed

    Sheahan, Michael B; Staiger, Chris J; Rose, Ray J; McCurdy, David W

    2004-12-01

    The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

  13. Damper-controlled switch for SMA motion smoothing

    NASA Astrophysics Data System (ADS)

    Aguayo, Clover; Utter, Brent; Luntz, Jonathan; Gonzalez, Richard; Brei, Diann; Johnson, Nancy L.; Alexander, Paul W.

    2014-04-01

    While the use of SMA-actuated devices continues to grow in many industries, current device limitations pose a challenge to successful adoption for certain classes of applications. SMA-actuated devices typically demonstrate motion with non-constant velocity due to the non-linear thermo-mechanically coupled behavior of SMA material transformation, and motion sensitivity to external factors such as voltage and load. This variation in motion can lead to the perception of poor device quality, limiting SMA-actuated devices to applications hidden from the sight of the product user, or requiring them to be augmented with higher cost controls to improve the motion quality. Therefore, a need exists for simple, passive, low-cost device technologies that enable the designer to prescribe desired motion characteristics with relative insensitivity to fluctuation in operating conditions. This paper presents a Damper Controlled Switch (DCS) mechanism that delivers constant velocity and relative insensitivity to operating conditions when combined with a standard SMA wire actuator. The DCS includes a damper that acts against a spring to open a switch when the velocity exceeds a tunable threshold. To validate the ability of the DCS to provide the desired motion quality, experiments were conducted comparing the normal motion of the SMA actuator to the motion produced when the same actuator was fitted with a DCS prototype. The addition of the DCS produced nearly constant actuator velocity, performing significantly better than the SMA actuator alone. The tunability of the DCS was demonstrated producing a wide range of attainable constant velocities. Finally, a set of experiments explored the DCS's sensitivity to voltage and load, indicating a low sensitivity to a wide range of operating parameters for which the operating limits were identified. The DCS represents a simple, compact technology based on passive, low-cost components, providing a very practical solution that will enable

  14. Evaluation of eligibility criteria in living donor liver transplantation for hepatocellular carcinoma by α-SMA-positive cancer-associated fibroblasts.

    PubMed

    Takamura, Hiroyuki; Nakanuma, Shinichi; Hayashi, Hironori; Tajima, Hidehiro; Kakinoki, Kaheita; Sakai, Seisyo; Makino, Isamu; Nakagawara, Hisatoshi; Miyashita, Tomoharu; Okamoto, Koichi; Nakamura, Keishi; Oyama, Katunobu; Inokuchi, Masashi; Ninomiya, Itasu; Kitagawa, Hirohisa; Fushida, Sachio; Fujimura, Takashi; Ohnishi, Ichiro; Kayahara, Masato; Tani, Takashi; Arai, Kuniaki; Yamashita, Taro; Yamashita, Tatsuya; Kitamura, Hoshiko; Ikeda, Hiroko; Kaneko, Shuichi; Nakanuma, Yasuni; Matsui, Osamu; Ohta, Tetsuo

    2013-10-01

    The eligibility criteria of liver transplantation (LT) for hepatocellular carcinoma (HCC) must clearly confirm the prognosis not only from pathological diagnosis but also from pre-operative imaging diagnosis. In the present study, we evaluated published eligibility criteria for LT based on both pre-operative imaging diagnosis and pathological diagnosis using living donor liver transplantation (LDLT) recipients at our hospital by α-smooth muscle actin (SMA)-positive cancer-associated fibroblasts (CAFs) in HCC. The Up-to-seven (Up-to-7), Asan and Tokyo criteria were evaluated, in both overall survival and HCC disease-free survival, to be statistically significantly beneficial criteria to define post-LDLT prognosis. Recipients only within Up-to-7 criteria based on both pre-operative imaging diagnosis and pathological diagnosis survived without HCC recurrence. Recipients with proliferation of α-SMA-positive CAFs in HCC had significantly poorer prognosis. All survival recipients without HCC recurrence, who were above the Up-to-7 criteria in pathological diagnosis, had no proliferation of α-SMA-positive CAFs. As a result of multivariate analysis, the significant independent factors defining prognosis of recipients after LDLT for HCC were Up-to-7 criteria and proliferation of α-SMA-positive CAFs. The ideal eligibility criteria for LDLT with HCC is Up-to-7 criteria and α-SMA-positive CAFs was considered to be an important factor in HCC recurrence. LDLT should be limited to recipients within Up-to-7 criteria or without proliferation of α-SMA-positive CAFs.

  15. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  16. Villin severing activity enhances actin-based motility in vivo.

    PubMed

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel; Robine, Sylvie

    2007-03-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

  17. Villin Severing Activity Enhances Actin-based Motility In Vivo

    PubMed Central

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel

    2007-01-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition. PMID:17182858

  18. The Drosophila javelin Gene Encodes a Novel Actin-Associated Protein Required for Actin Assembly in the Bristle ▿

    PubMed Central

    Shapira, Shira; Bakhrat, Anna; Bitan, Amir; Abdu, Uri

    2011-01-01

    The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development. PMID:21930794

  19. Organization and regulation of the actin cytoskeleton in the pollen tube

    PubMed Central

    Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Liu, Xiaonan; Zhang, Ruihui; Huang, Shanjin

    2015-01-01

    Proper organization of the actin cytoskeleton is crucial for pollen tube growth. However, the precise mechanisms by which the actin cytoskeleton regulates pollen tube growth remain to be further elucidated. The functions of the actin cytoskeleton are dictated by its spatial organization and dynamics. However, early observations of the distribution of actin filaments at the pollen tube apex were quite perplexing, resulting in decades of controversial debate. Fortunately, due to improvements in fixation regimens for staining actin filaments in fixed pollen tubes, as well as the adoption of appropriate markers for visualizing actin filaments in living pollen tubes, this issue has been resolved and has given rise to the consensus view of the spatial distribution of actin filaments throughout the entire pollen tube. Importantly, recent descriptions of the dynamics of individual actin filaments in the apical region have expanded our understanding of the function of actin in regulation of pollen tube growth. Furthermore, careful documentation of the function and mode of action of several actin-binding proteins expressed in pollen have provided novel insights into the regulation of actin spatial distribution and dynamics. In the current review, we summarize our understanding of the organization, dynamics, and regulation of the actin cytoskeleton in the pollen tube. PMID:25620974

  20. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

    PubMed

    Skillman, Kristen M; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L David

    2011-10-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  1. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

    Skillman, Kristen M.; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  2. Evidence for changes in beta- and gamma-actin proportions during inner ear hair cell life.

    PubMed

    Andrade, Leonardo R

    2015-06-01

    Cytoplasmic actin isoforms beta (β-) and gamma (γ-) perform crucial physiological roles in inner ear hair cells (HC). The stereocilium, which is structured by parallel actin filaments composed of both isoforms, is the responsive organelle to mechanical stimuli such as sound, gravity and head movements. Modifications in isoform proportions affect the function of the stereocilia as previously shown in genetic studies of mutant mice. Here, immunogold labeling TEM studies in mice showed that both β- and γ-actin isoforms colocalize throughout stereocilia actin filaments, adherens junctions and cuticular plates as early as embryonic stage 16.5. Gold-particle quantification indicated that there was 40% more γ- actin than β-actin at E16.5. In contrast, β- and γ-actin were equally concentrated in adult stereocilia of cochlear and vestibular HC. Interestingly, all actin-based structures presented almost five-fold more β-actin than γ-actin in 22 month- old mice, suggesting that γ-actin is probably under-expressed during the aging process. These data provide evidence of dynamic modifications of the actin isoforms in stereocilia, cuticular plates and cell junctions during the whole HC life.

  3. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  4. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

    PubMed Central

    1995-01-01

    The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere. PMID:7822423

  5. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

    PubMed

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-08-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

  6. Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury.

    PubMed Central

    Ogawa, K.; Suzuki, J.; Mukai, H.; Mori, M.

    1986-01-01

    Immunohistochemical investigations were carried out on the properties of the cells and extracellular matrix (ECM) in focal hepatic injuries. A liquid nitrogen-cooled syringe needle was thrust into the rat liver. Necrotic areas became permeated with plasma within 24-hour period. Areas became strongly positive for fibronectin and were infiltrated with inflammatory cells positive for lysozyme. By the third day, Ito cells were proliferated in the peripheral portions of the damaged areas. These Ito cells showed enhanced immunostaining for desmin and actin but were negative for lysozyme. Interstitial fibers which were immunochemically positive for Types I and IV collagens, laminin, and fibronectin, began to increase from Day 3. They appeared on the rim of the hepatocytes adjacent to the damaged areas and extended into the injured regions with the Ito cells. An increase in basal laminas associated with capillaries and bile ducts also increased with a 1-day delay. The damaged areas were replaced by granulation tissue by Day 5. A rapid diminution then occurred in the granulation tissue, and normal hepatic tissue was restored in 7-10 days. These observations demonstrate that ECM changed in a sequential manner and then finally disappeared from the damaged site within 10 days. Although various cells, including parenchymal cells, macrophages, endothelial cells, and cholangiolar cells contributed to the healing of the damaged area, Ito cells, which exhibit unique phenotypic changes, presumably had a major role in the process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3799820

  7. Simulation-Based Design of a Rotatory SMA Drive

    NASA Astrophysics Data System (ADS)

    Dilthey, Sascha; Meier, Horst

    2009-08-01

    The design and optimization of a rotatory drive powered by shape memory alloy (SMA) actuators is described in this paper. SMA actuators used in technical applications are parameterized by the use of trial-and-error methods, because there is a lack of computer-aided design tools for this active material. A numerical modeling approach was developed to design and optimize the geometry and the load and heating conditions of SMA actuators in a technical system to achieve a good dynamic and a high reliability. The shape memory effect used in most technical systems is the extrinsic two way effect (2WE). This effect can be simulated with the numerical model which was implemented in MATLAB/SIMULINK. The focus of the model is on the activation behavior of the SMA actuator, which defines its rate of heating and cooling. Different load conditions and various actuator geometries and shapes, e.g. wire or spring actuator, are simulated by the calculation of the energetic balance of the whole system. The numerical model can be used to simulate time variant heating currents in order to obtain an optimal system performance. The model was used to design a rotatory SMA-drive system, which is based on the moving concept of a wave drive gear set. In contrast to the conventional system, which is driven by an electric motor, the SMA drive consists of a strain wave gear and SMA wire actuators that are applied circularly to generate a rotatory movement. Special characteristics of this drive system are a high torque density and a high positioning accuracy.

  8. The ADF/cofilin family: actin-remodeling proteins.

    PubMed

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

  9. Actin dynamics at sites of extracellular matrix degradation.

    PubMed

    Baldassarre, Massimiliano; Ayala, Inmaculada; Beznoussenko, Galina; Giacchetti, Giada; Machesky, Laura M; Luini, Alberto; Buccione, Roberto

    2006-12-01

    The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.

  10. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

    PubMed Central

    Savage, C; Das, P; Finelli, A L; Townsend, S R; Sun, C Y; Baird, S E; Padgett, R W

    1996-01-01

    Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers. Images Fig. 1 PMID:8570636

  11. Evidence for {gamma}-actin as a Z disc component in skeletal myofibers

    SciTech Connect

    Papponen, Hinni; Kaisto, Tuula; Leinonen, Sanna; Kaakinen, Mika; Metsikkoe, Kalervo

    2009-01-15

    We investigated the targeting of the {gamma}-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged {gamma}-actin in rat flexor digitorum brevis myofibers. We found that the {gamma}-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific {alpha}-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the {gamma}-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of {gamma}-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells.

  12. Age-Dependent Expression of Collagen Receptors and Deformation of Type I Collagen Substrates by Rat Cardiac Fibroblasts

    PubMed Central

    Wilson, Christopher G.; Stone, John W.; Fowlkes, Vennece; Morales, Mary O.; Murphy, Catherine J.; Baxter, Sarah C.; Goldsmith, Edie C.

    2014-01-01

    Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR) 1, DDR2, and β1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of β1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age. PMID:21740617

  13. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    PubMed Central

    Schreiber, Joerg; Végh, Marlene J.; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T.; De Zeeuw, Chris I.; Kneussel, Matthias; Meredith, Rhiannon M.; Smit, August B.

    2015-01-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3−/− mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

  14. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    PubMed

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction.

  15. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

    PubMed

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-09-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

  16. Ring closure in actin polymers

    NASA Astrophysics Data System (ADS)

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-03-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  17. Metallothionein immunolocalization in actinic skin nonmelanoma carcinomas.

    PubMed

    Borges Júnior, Paulo C; Ribeiro, Rosy I M A; Cardoso, Sérgio V; Berbet, Alceu L C; Rocha, Ademir; Espindola, Foued S; Loyola, Adriano M

    2007-06-01

    Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent skin cancer. Its pathogeny is linked to genotoxic effects of actinic radiation exposure, especially to ultraviolet wavelength. Metallothionein (MT) is a low-molecular weight protein with high affinity for heavy metal. Its intracellular function has been related to heavy metals and free-radical detoxification, although many studies linked MT to protective action against actinic mutagenesis. In other way, overexpression in malignant tumors has been related to worse prognosis. We aimed to evaluate MT immunohistochemical expression in skin cancer associated to actinic radiation. Twenty-six BCC cases, 20 SCC, and 6 normal skin fragments were investigated. Immunohistochemical assay were performed by streptavidin-biotin-peroxidase technique with standard monoclonal antibody (E9). In normal skin, immunostaining was observed in basal layer of the epithelium. In the epithelium adjacent to tumors, suprabasal layer was also intensely labeled. Mean MT immunostaining indices were 18.5+21.2% for BCC and 69.1+14.4% for SCC. This difference was statistically significant. Higher MT expression in SCC as compared with BCC suggests association with tumoral aggressiveness.

  18. Prosthetic leg powered by MR brake and SMA wires

    NASA Astrophysics Data System (ADS)

    Nguyen, The; Munguia, Vicente; Calderon, Jose

    2014-04-01

    Current knee designs for prosthetic legs rely on electric motors for both moving and stationary states. The electric motors draw an especially high level of current to sustain a fixed position. The advantage of using magnetorheological (MR) fluid is that it requires less current and can have a variable braking torque. Besides, the proposed prosthetic leg is actuated by NiTinol wire, a popular shape memory alloy (SMA). The incorporation of NiTinol gives the leg more realistic weight distribution with appropriate arrangement of the batteries and wires. The prosthesis in this research was designed with MR brake as stopping component and SMA wire network as actuating component at the knee. The MR brake was designed with novel non-circular shape for the rotor that improved the braking torque while minimizing the power consumption. The design also helped simplify the control of braking process. The SMA wire network was design so that the knee motion was actively rotated in both directions. The SMA wires were arranged and played very similar role as the leg's muscles. The study started with the overall solid design of the knee including both MR and SMA parts. Theoretical models were derived and programmed in Simulink for both components. The simulation was capable of predicting the power required for moving the leg or hold it in a fixed position for a certain amount of time. Subsequently, the design was prototyped and tested to validate the theoretical prediction. The theoretical models were updated accordingly to correlate with the experimental data.

  19. Seismic response control of frame structure braced with SMA tendons

    NASA Astrophysics Data System (ADS)

    Yan, Shi; Song, Gangbing; Huo, Linsheng; Gu, Haichang

    2007-04-01

    This paper presents studies of seismic response control of a frame structure braced with SMA (Shape Memory Alloy) tendons through both numerical and experimental approaches. Based on the Brinson one-dimensional constitutive law for SMAs, a two-story frame structure braced diagonally with SMA tendons is used as an example to simulate numerically the vibration control process. By considering the temperature, different initial states and thermal properties of the SMA tendon, and the variable intensity and frequency of earthquake input, the parameters of the system were analyzed during the numerically simulation. The time histories of the displacement and hysteretic loops of the SMA tendons were simulated under earthquake ground motion by using finite element method (FEM). To validate the efficiency of the simulation, a shaking table test for the frame structure was conducted. Both numerical simulation and experimental results show that the actively controlled martensite SMA tendons can effectively suppress the vibration of the multi-story frame structure during an earthquake.

  20. Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP).

    PubMed

    Kato-Minoura, Takako

    2011-09-09

    Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

  1. SWAP-70 identifies a transitional subset of actin filaments in motile cells.

    PubMed

    Hilpelä, Pirta; Oberbanscheidt, Pia; Hahne, Penelope; Hund, Martin; Kalhammer, Georg; Small, J Victor; Bähler, Martin

    2003-08-01

    Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

  2. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  3. Actin isoform specificity is required for the maintenance of lactation

    PubMed Central

    Weymouth, Nate; Shi, Zengdun; Rockey, Don C.

    2014-01-01

    Smooth muscle α-actin (Acta2) is one of six highly conserved mammalian actin isoforms that appear to exhibit functional redundancy. Nonetheless, we have postulated a specific functional role for the smooth muscle specific isoform. Here, we show that Acta2 deficient mice have a remarkable mammary phenotype such that dams lacking Acta2 are unable to nurse their offspring effectively. The phenotype was rescued in cross fostering experiments with wild type mice, excluding a developmental defect in Acta2 null pups. The mechanism for the underlying phenotype is due to myoepithelial dysfunction postpartum resulting in precocious involution. Further, we demonstrate a specific defect in myoepithelial cell contractility in Acta2 null mammary glands, despite normal expression of cytoplasmic actins. We conclude that Acta2 specifically mediates myoepithelial cell contraction during lactation and that this actin isoform therefore exhibits functional specificity. PMID:22123032

  4. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  5. CRMP-5 interacts with actin to regulate neurite outgrowth

    PubMed Central

    GONG, XIAOBING; TAN, MINGHUI; GAO, YUAN; CHEN, KEEN; GUO, GUOQING

    2016-01-01

    CRMP family proteins (CRMPs) are abundantly expressed in the developing nervous system mediating growth cone guidance, neuronal polarity and axon elongation. CRMP-5 has been indicated to serve a critical role in neurite outgrowth. However, the detailed mechanisms of how CRMP-5 regulates neurite outgrowth remain unclear. In the current study, co-immunoprecipitation was used to identify the fact that CRMP-5 interacted with the actin and tubulin cytoskeleton networks in the growth cones of developing hippocampal neurons. CRMP-5 exhibited increased affinity towards actin when compared with microtubules. Immunocytochemistry was used to identify the fact that CRMP-5 colocalized with actin predominantly in the C-domain and T-zone in growth cones. In addition, genetic inhibition of CRMP-5 by siRNA suppressed the expression of actin, growth cone development and neurite outgrowth. Overexpression of CRMP-5 promoted the interaction with actin, growth cone development and hippocampal neurite outgrowth. Taken together, these data suggest that CRMP-5 is able to interact with the actin cytoskeleton network in the growth cone and affect growth cone development and neurite outgrowth via this interaction in developing hippocampal neurons. PMID:26677106

  6. Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins.

    PubMed

    Dovas, Athanassios; Gligorijevic, Bojana; Chen, Xiaoming; Entenberg, David; Condeelis, John; Cox, Dianne

    2011-02-14

    Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

  7. Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens.

    PubMed Central

    Kim, Mijung; Robich, Rebecca M.; Rinehart, Joseph P.; Denlinger, David L.

    2007-01-01

    Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25°C and in diapausing mosquitoes reared at 18°C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7–10 days post-eclosion were exposed to low temperature (-5°C for 12h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1month post-eclosion) to low temperature (−5°C for 12h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton. PMID:17078965

  8. Finite element simulation of adaptive aerospace structures with SMA actuators

    NASA Astrophysics Data System (ADS)

    Frautschi, Jason; Seelecke, Stefan

    2003-07-01

    The particular demands of aerospace engineering have spawned many of the developments in the field of adaptive structures. Shape memory alloys are particularly attractive as actuators in these types of structures due to their large strains, high specific work output and potential for structural integration. However, the requisite extensive physical testing has slowed development of potential applications and highlighted the need for a simulation tool for feasibility studies. In this paper we present an implementation of an extended version of the M'ller-Achenbach SMA model into a commercial finite element code suitable for such studies. Interaction between the SMA model and the solution algorithm for the global FE equations is thoroughly investigated with respect to the effect of tolerances and time step size on convergence, computational cost and accuracy. Finally, a simulation of a SMA-actuated flexible trailing edge of an aircraft wing modeled with beam elements is presented.

  9. FE Simulation of SMA Seal for Mars Sample Return

    NASA Technical Reports Server (NTRS)

    Bao, Xiaoqi; Younse, Paulo; Bhandari, Pradeep

    2013-01-01

    Several NASA rovers and landers have been on Mars and performed successful in-situ exploration. Returning Martian samples to Earth for extensive analysis is of great interest to the planetary science community. Current Mars sample return architecture would require leaving the acquired samples on Mars for years before being retrieved by subsequent mission. Each sample would be sealed securely to keep its integrity. A reliable seal technique that does not affect the integrity of the samples and uses a simple low-mass tool is required. The shape memory alloy (SMA) seal technique is a promising candidate. A study of the thermal performances of several primary designs of a SMA seal for sample tubes by finite element (FE) simulation are presented in this paper. The results show sealing the sample tube by SMA plugs and controlling the sample temperature below the allowed temperature level are feasible.

  10. Graphical design for thin-film SMA microactuators

    NASA Astrophysics Data System (ADS)

    Ishida, A.; Sato, M.; Yoshikawa, W.; Tabata, O.

    2007-10-01

    Simple graphical designs for bridge- and diaphragm-type actuators using shape-memory alloy (SMA) thin films are proposed. The performance (force and displacement) of these actuators is predicted on the basis of the stress-strain curves of an SMA film by introducing the material parameter K that represents the mechanical properties of both the austenite and martensite films. The methods of designing actuators are presented along with numerical examples and their validity is discussed, referring to some actual devices such as a microvalve and a micropump. A comparison of theory and actual device performance shows that the proposed design gives a useful approximation for practical applications. The advantage of the proposed design method is that, once the value of K is obtained for a given SMA film, one can design various actuators with various dimensions without detailed information about its mechanical properties.

  11. Relaxin Modulates the Expression of MMPs and TIMPs in Fibroblasts of Patients with Carpal Tunnel Syndrome

    PubMed Central

    Kang, Young-Mi; Lee, Hwan-Mo; Moon, Seong-Hwan; Kang, Ho

    2017-01-01

    Purpose The aim of this study was to investigate the anti-fibrotic effect of relaxin in subsynovial fibroblasts activated by transforming growth factor beta (TGF-β). Materials and Methods To test the anti-fibrotic effect of an adenovirus-relaxin construct (Ad-RLN) on subsynovial fibroblasts in vitro, cells from subsynovial connective tissue of patients with carpal tunnel syndrome were activated with TGF-β1 and exposed to Ad-RLN (as a therapeutic gene) or adenovirus-lacZ construct (as a marker gene) for four hours. Subsynovial fibroblast cultures without adenoviral exposure served as controls. Results We observed induction of gene expressions of collagen I, III and IV, as well as the abatement of alpha-smooth muscle actin (a-SMA) synthesis, Smad2 phosphorylation, and fibronectin at the protein level, in comparison to controls. In addition, protein expressions of matrix metalloproteinase (MMP) I was significantly induced, whereas the protein expressions of tissue inhibitor of metalloproteinases (TIMP) I and IV were reduced due to relaxin expression. Conclusion RLN prevents excessive synthesis of extracellular matrix by reducing the expressions of its components, such as fibronectin, a-SMA, and phosphorylated Smad2, by increasing the expression of MMPs; and by decreasing the expression of TIMPs. PMID:28120574

  12. The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation.

    PubMed

    Shi, Jun-Wen; Liu, Wei; Zhang, Ting-Ting; Wang, Sheng-Chun; Lin, Xiao-Lin; Li, Jing; Jia, Jun-Shuang; Sheng, Hong-Fen; Yao, Zhi-Fang; Zhao, Wen-Tao; Zhao, Zun-Lan; Xie, Rao-Ying; Yang, Sheng; Gao, Fei; Fan, Quan-Rong; Zhang, Meng-Ya; Yue, Min; Yuan, Jin; Gu, Wei-Wang; Yao, Kai-Tai; Xiao, Dong

    2013-04-01

    In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.

  13. Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells

    PubMed Central

    Simiczyjew, Aleksandra; Mazur, Antonina Joanna; Dratkiewicz, Ewelina; Nowak, Dorota

    2017-01-01

    Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by non-muscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers–ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes. PMID:28333953

  14. Adaptive and energy efficient SMA-based handling systems

    NASA Astrophysics Data System (ADS)

    Motzki, P.; Kunze, J.; Holz, B.; York, A.; Seelecke, S.

    2015-04-01

    Shape Memory Alloys (SMA's) are known as actuators with very high energy density. This fact allows for the construction of very light weight and energy-efficient systems. In the field of material handling and automated assembly process, the avoidance of big moments of inertia in robots and kinematic units is essential. High inertial forces require bigger and stronger robot actuators and thus higher energy consumption and costs. For material handling in assembly processes, many different individual grippers for various work piece geometries are used. If one robot has to handle different work pieces, the gripper has to be exchanged and the assembly process is interrupted, which results in higher costs. In this paper, the advantages of using high energy density Shape Memory Alloy actuators in applications of material-handling and gripping-technology are explored. In particular, light-weight SMA actuated prototypes of an adaptive end-effector and a vacuum-gripper are constructed via rapid-prototyping and evaluated. The adaptive end-effector can change its configuration according to the work piece geometry and allows the handling of multiple different shaped objects without exchanging gripper tooling. SMA wires are used to move four independent arms, each arm adds one degree of freedom to the kinematic unit. At the tips of these end-effector arms, SMA-activated suction cups can be installed. The suction cup prototypes are developed separately. The flexible membranes of these suction cups are pulled up by SMA wires and thus a vacuum is created between the membrane and the work piece surface. The self-sensing ability of the SMA wires are used in both prototypes for monitoring their actuation.

  15. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

    Saito, S; Watabe, S; Ozaki, H; Kigoshi, H; Yamada, K; Fusetani, N; Karaki, H

    1996-09-01

    Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

  16. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

    Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

  17. Micro-Ball-Lens Optical Switch Driven by SMA Actuator

    NASA Technical Reports Server (NTRS)

    Yang, Eui-Hyeok

    2003-01-01

    The figure is a simplified cross section of a microscopic optical switch that was partially developed at the time of reporting the information for this article. In a fully developed version, light would be coupled from an input optical fiber to one of two side-by-side output optical fibers. The optical connection between the input and the selected output fiber would be made via a microscopic ball lens. Switching of the optical connection from one output fiber to another would be effected by using a pair of thin-film shape-memory-alloy (SMA) actuators to toggle the lens between two resting switch positions. There are many optical switches some made of macroscopic parts by conventional fabrication techniques and some that are microfabricated and, hence, belong to the class of microelectromechanical systems (MEMS). Conventionally fabricated optical switches tend to be expensive. MEMS switches can be mass-produced at relatively low cost, but their attractiveness has been diminished by the fact that, heretofore, MEMS switches have usually been found to exhibit high insertion losses. The present switch is intended to serve as a prototype of low-loss MEMS switches. In addition, this is the first reported SMA-based optical switch. The optical fibers would be held in V grooves in a silicon frame. The lens would have a diameter of 1 m; it would be held by, and positioned between, the SMA actuators, which would be made of thin films of TiNi alloy. Although the SMA actuators are depicted here as having simple shapes for the sake of clarity of illustration, the real actuators would have complex, partly net-like shapes. With the exception of the lens and the optical fibers, the SMA actuators and other components of the switch would be made by microfabrication techniques. The components would be assembled into a sandwich structure to complete the fabrication of the switch. To effect switching, an electric current would be passed through one of the SMA actuators to heat it above

  18. Actin cytoskeleton: putting a CAP on actin polymerization.

    PubMed

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  19. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  20. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  1. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  2. The in vivo expression of actin/salt-resistant hyperactive DNase I inhibits the development of anti-ssDNA and anti-histone autoantibodies in a murine model of systemic lupus erythematosus

    PubMed Central

    Manderson, Anthony P; Carlucci, Francesco; Lachmann, Peter J; Lazarus, Robert A; Festenstein, Richard J; Cook, H Terence; Walport, Mark J; Botto, Marina

    2006-01-01

    Systemic lupus erythematosus (SLE) is characterised by the production of autoantibodies against ubiquitous antigens, especially nuclear components. Evidence makes it clear that the development of these autoantibodies is an antigen-driven process and that immune complexes involving DNA-containing antigens play a key role in the disease process. In rodents, DNase I is the major endonuclease present in saliva, urine and plasma, where it catalyses the hydrolysis of DNA, and impaired DNase function has been implicated in the pathogenesis of SLE. In this study we have evaluated the effects of transgenic over-expression of murine DNase I endonucleases in vivo in a mouse model of lupus. We generated transgenic mice having T-cells that express either wild-type DNase I (wt.DNase I) or a mutant DNase I (ash.DNase I), engineered for three new properties – resistance to inhibition by G-actin, resistance to inhibition by physiological saline and hyperactivity compared to wild type. By crossing these transgenic mice with a murine strain that develops SLE we found that, compared to control non-transgenic littermates or wt.DNase I transgenic mice, the ash.DNase I mutant provided significant protection from the development of anti-single-stranded DNA and anti-histone antibodies, but not of renal disease. In summary, this is the first study in vivo to directly test the effects of long-term increased expression of DNase I on the development of SLE. Our results are in line with previous reports on the possible clinical benefits of recombinant DNase I treatment in SLE, and extend them further to the use of engineered DNase I variants with increased activity and resistance to physiological inhibitors. PMID:16606442

  3. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  4. Participation of Actin on Giardia lamblia Growth and Encystation

    PubMed Central

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Perez Rangel, Armando; Cortes Zarate, Rafael; Garcia Tovar, Carlos; Hernandez, Jose Manuel

    2009-01-01

    Background Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. Methodology and Principal Findings By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of flourescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). Conclusions and Significance All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression. PMID:19774081

  5. [Photodynamic therapy for actinic cheilitis].

    PubMed

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  6. In Vivo Imaging and Characterization of Actin Microridges

    PubMed Central

    Lam, Pui-ying; Mangos, Steve; Green, Julie M.; Reiser, Jochen; Huttenlocher, Anna

    2015-01-01

    Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo. PMID:25629723

  7. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  8. Modeling of piezo-SMA composites for thermal energy harvester

    NASA Astrophysics Data System (ADS)

    Namli, Onur Cem; Lee, Jae-Kon; Taya, Minoru

    2007-04-01

    A hydrid composite comprised of shape memory alloy (SMA) fibers with piezoelectric ceramic is designed to transform thermomechanical energy into electrical energy that can be stored or used to power other devices. SMA fiber, after its shape is memorized and prestrained at martensitic phase, extends to its original length upon heating to austenitic finish temperature. The compressive residual stress of the composite is induced at austenitic phase, and then by cooling to martensitic finish temperature, SMA will shrink and the residual stress will reduce. By direct effect of the piezoelectric matrix material the mechanical energy which was induced by temperature change can be converted to electrical energy. 1-D and 3-D models for the energy harvesting mechanism of the composite have been proposed. Eshelby formulation with Mori-Tanaka mean field theory modification is used to determine the effective thermo-electro-mechanical properties of the composite. Attention is focused on the constrained recovery behavior of SMA phase in this study. Electrical model is examined and the electrical energy stored in the piezoelectric matrix as a result of stress fluctuation is estimated. Numerical example is given that illustrate the ability of the composite to convert the thermomechanical energy into electrical energy.

  9. Fabricating Composite-Material Structures Containing SMA Ribbons

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.; Cano, Roberto J.; Lach, Cynthia L.

    2003-01-01

    An improved method of designing and fabricating laminated composite-material (matrix/fiber) structures containing embedded shape-memory-alloy (SMA) actuators has been devised. Structures made by this method have repeatable, predictable properties, and fabrication processes can readily be automated. Such structures, denoted as shape-memory-alloy hybrid composite (SMAHC) structures, have been investigated for their potential to satisfy requirements to control the shapes or thermoelastic responses of themselves or of other structures into which they might be incorporated, or to control noise and vibrations. Much of the prior work on SMAHC structures has involved the use SMA wires embedded within matrices or within sleeves through parent structures. The disadvantages of using SMA wires as the embedded actuators include (1) complexity of fabrication procedures because of the relatively large numbers of actuators usually needed; (2) sensitivity to actuator/ matrix interface flaws because voids can be of significant size, relative to wires; (3) relatively high rates of breakage of actuators during curing of matrix materials because of sensitivity to stress concentrations at mechanical restraints; and (4) difficulty of achieving desirable overall volume fractions of SMA wires when trying to optimize the integration of the wires by placing them in selected layers only.

  10. Rho GTPases, phosphoinositides, and actin

    PubMed Central

    Croisé, Pauline; Estay-Ahumada, Catherine; Gasman, Stéphane; Ory, Stéphane

    2014-01-01

    Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments. PMID:24914539

  11. Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects.

    PubMed

    Costa, Céline F; Rommelaere, Heidi; Waterschoot, Davy; Sethi, Kamaljit K; Nowak, Kristen J; Laing, Nigel G; Ampe, Christophe; Machesky, Laura M

    2004-07-01

    Mutations in the gene encoding alpha-skeletal-muscle actin, ACTA1, cause congenital myopathies of various phenotypes that have been studied since their discovery in 1999. Although much is now known about the clinical aspects of myopathies resulting from over 60 different ACTA1 mutations, we have very little evidence for how mutations alter the behavior of the actin protein and thus lead to disease. We used a combination of biochemical and cell biological analysis to classify 19 myopathy mutants and found a range of defects in the actin. Using in vitro expression systems, we probed actin folding and actin's capacity to interact with actin-binding proteins and polymerization. Only two mutants failed to fold; these represent recessive alleles, causing severe myopathy, indicating that patients produce nonfunctional actin. Four other mutants bound tightly to cyclase-associated protein, indicating a possible instability in the nucleotide-binding pocket, and formed rods and aggregates in cells. Eleven mutants showed defects in the ability to co-polymerize with wild-type actin. Some of these could incorporate into normal actin structures in NIH 3T3 fibroblasts, but two of the three tested also formed aggregates. Four mutants showed no defect in vitro but two of these formed aggregates in cells, indicating functional defects that we have not yet tested for. Overall, we found a range of defects and behaviors of the mutants in vitro and in cultured cells, paralleling the complexity of actin-based muscle myopathy phenotypes.

  12. Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

    PubMed

    Wang, Xiu-Ling; Gao, Xin-Qi; Wang, Xue-Chen

    2011-08-01

    Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

  13. Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

    PubMed Central

    Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

    2016-01-01

    Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution.

  14. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  15. Seasonal immunohistochemical reactivity of S-100 and α-smooth muscle actin proteins in the epididymis of dromedary camel, Camelus dromedarius.

    PubMed

    Ibrahim, Z H; Joshi, D; Singh, S K

    2016-08-10

    The S-100 and alpha smooth muscle actin (α-SMA) proteins have been localised in epididymal tissue of several mammalian species, but there have been no data for a seasonal work in camel. The aim of this study was to investigate the immunoreactivities of S-100 and α-SMA proteins in the epididymis of dromedary camel during breeding and nonbreeding seasons. The immunopositive signals for both proteins were observed in different regions of camel epididymis. S-100-immunopositive signals were noted in both the epididymal epithelium and the intertubular connective tissue, while α-SMA signals were confined to the intertubular connective tissue, especially in the peritubular smooth muscle coat and the blood vessels. This study showed an increase in the intensity of S-100 and α-SMA immunoreactions during the breeding season in different regions of camel epididymis than that seen in the nonbreeding season. In conclusion, epididymis might be considered as a source of S-100 and α-SMA proteins in the camel and the secretion of these proteins showed distinct seasonal variations. Further, S-100 and α-SMA may affect the structural and physiological states of the epididymal duct.

  16. Actin complexes in the cell nucleus: new stones in an old field.

    PubMed

    Castano, E; Philimonenko, V V; Kahle, M; Fukalová, J; Kalendová, A; Yildirim, S; Dzijak, R; Dingová-Krásna, H; Hozák, P

    2010-06-01

    Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.

  17. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  18. Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

    PubMed Central

    1994-01-01

    Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin. PMID:8163553

  19. Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

    PubMed Central

    Hilbert, Lennart; Bates, Genevieve; Roman, Horia N.; Blumenthal, Jenna L.; Zitouni, Nedjma B.; Sobieszek, Apolinary; Mackey, Michael C.; Lauzon, Anne-Marie

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics. PMID:24204225

  20. Oestrogen receptor-alpha and -beta expression in breast implant capsules: experimental findings and clinical correlates.

    PubMed

    Persichetti, Paolo; Segreto, Francesco; Carotti, Simone; Marangi, Giovanni Francesco; Tosi, Daniele; Morini, Sergio

    2014-03-01

    Myofibroblasts provide a force to decrease the surface area of breast implant capsules as the collagen matrix matures. 17-β-Oestradiol promotes myofibroblast differentiation and contraction. The aim of the study was to investigate the expression of oestrogen receptors α and β in capsular tissue. The study enrolled 70 women (80 capsules) who underwent expander or implant removal, following breast reconstruction. Specimens were stained with haematoxylin/eosin, Masson trichrome and immunohistochemistry and immunofluorescence stainings for alpha-smooth muscle actin (α-SMA), oestrogen receptor-alpha (ER-α) and oestrogen receptor-beta (ER-β). The relationship between anti-oestrogenic therapy and capsular severity was evaluated. A retrospective analysis of 233 cases of breast reconstruction was conducted. Myofibroblasts expressed ER-α, ER-β or both. In the whole sample, α-SMA score positively correlated with ER-α (p = 0.022) and ER-β expression (p < 0.004). ER-β expression negatively correlated with capsular thickness (p < 0.019). In capsules surrounding expanders α-SMA and ER-α, expressions negatively correlated with time from implantation (p = 0.002 and p = 0.016, respectively). The incidence of grade III-IV contracture was higher in patients who did not have anti-oestrogenic therapy (p < 0.036); retrospective analysis of 233 cases confirmed this finding (p < 0.0001). This study demonstrates the expression of oestrogen receptors in myofibroblasts of capsular tissue. A lower contracture severity was found in patients who underwent anti-oestrogenic therapy.

  1. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  2. The Role of the Supplementary Motor Region in Overt Reading: Evidence for Differential Processing in SMA-Proper and Pre-SMA as a Function of Task Demands.

    PubMed

    Cummine, Jacqueline; Hanif, Wahab; Dymouriak-Tymashov, Inna; Anchuri, Kavya; Chiu, Stephanie; Boliek, Carol A

    2017-03-04

    A differentiation in function between the pre-SMA (i.e., cognitive load) and the SMA-proper (i.e., motor execution) has been described (Zhang et al., Cereb Cortex 22:99-111, 2012). These differential SMA functions may be influential in overt reading tasks. The present study examined the relationships between various segments of the SMA and overt reading through the modulation of task demands in an effort to explore the complexity of the print-to-speech network. Skilled reading adults (N = 15) took part in five overt reading tasks: pure regular word reading, pure exception word reading, mixed regular word and exception word reading, go/no-go reading with nonword foils and go/no-go reading with pseudohomophone foils. Five regions of interest that spanned the pre-SMA to the SMA-proper were isolated. Behaviour-function relationships were tested to examine the associations between performance (response time) and brain activity (percent signal change). Further, the coherence between feedforward (SMA) and feedback (supramarginal gyrus) regions were explored to further refine the print-to-speech network. We found that the pre-SMA was related to cognitively demanding tasks (go/no-go with pseudohomophones), whereas the SMA-proper was related to an automatized task (pure regular words). Notably, only those tasks that required information from the feedback system (i.e., mixed word lists, go/no-go tasks) showed connections between SMA regions and the supramarginal gyrus, which is in line with the role of feedback and feedforward systems in the print-to-speech network. Together, these results support the notion that the pre-SMA and SMA-proper are sensitive to reading tasks that differentially invoke higher cognitive resources (mixed word lists, go/no-go) versus automatized articulation (pure lists), respectively. We discuss our findings in the context of print-to-speech neural networks.

  3. Columbia SMA Project: A Randomized, Control Trial of the Effects of Exercise on Motor Function and Strength in Patients With Spinal Muscular Atrophy (SMA)

    DTIC Science & Technology

    2011-06-01

    www.CQillmbiasma.org No. 1994 ?. 2 Musc::ular Dystrophy Assoc1at1on (hUAJ Clinic Spinal Muscular Atrophy Clinical (SMA) Research Center Nan<:y E. Strauss, M.D...3 ~ COLUMBIA UNIVERSIT~ 0 ~ Muscutar Dystrophy Association (MDA) Clinic \\WI M C Spinal Muscular Atrophy Clinical {SMA) - EDICAL ENTER Research...9lu mbi~_rlli:’U~t9 0000000000 p.1 Muscular Dystrophy Association (MDA) Clinic Spinal Muscular Atrophy Clinical (SMA) Research Center Nancy E

  4. Nucleus-associated actin in Amoeba proteus.

    PubMed

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  5. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  6. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.

    PubMed

    Higashida, Chiharu; Suetsugu, Shiro; Tsuji, Takahiro; Monypenny, James; Narumiya, Shuh; Watanabe, Naoki

    2008-10-15

    mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

  7. Reconstitution and dissection of the 600-kDa Srv2/CAP complex: roles for oligomerization and cofilin-actin binding in driving actin turnover.

    PubMed

    Quintero-Monzon, Omar; Jonasson, Erin M; Bertling, Enni; Talarico, Lou; Chaudhry, Faisal; Sihvo, Maarit; Lappalainen, Pekka; Goode, Bruce L

    2009-04-17

    Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.

  8. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  9. SMA Hybrid Composites for Dynamic Response Abatement Applications

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.

    2000-01-01

    A recently developed constitutive model and a finite element formulation for predicting the thermomechanical response of Shape Memory Alloy (SMA) hybrid composite (SMAHC) structures is briefly described. Attention is focused on constrained recovery behavior in this study, but the constitutive formulation is also capable of modeling restrained or free recovery. Numerical results are shown for glass/epoxy panel specimens with embedded Nitinol actuators subjected to thermal and acoustic loads. Control of thermal buckling, random response, sonic fatigue, and transmission loss are demonstrated and compared to conventional approaches including addition of conventional composite layers and a constrained layer damping treatment. Embedded SMA actuators are shown to be significantly more effective in dynamic response abatement applications than the conventional approaches and are attractive for combination with other passive and/or active approaches.

  10. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  11. Optimization of SMA layers in composite structures to enhance damping

    NASA Astrophysics Data System (ADS)

    Haghdoust, P.; Cinquemani, S.; Lecis, N.; Bassani, P.

    2016-04-01

    The performance of lightweight structures can be severely affected by vibration. New design concepts leading to lightweight, slender structural components can increase the vulnerability of the components to failure due to excessive vibration. The intelligent approach to address the problem would be the use of materials which are more capable in dissipating the energy due to their high value of loss factor. Among the different materials available to achieve damping, much attention has been attached to the use of shape memory alloys (SMAs) because of their unique microstructure, leading to good damping capacity. This work describes the design and optimization of a hybrid layered composite structure for the passive suppression of flexural vibrations in slender and light structures. Embedding the SMA layers in composite structure allows to combine different properties: the lightness of the base composite (e.g. fiber glass), the mechanical strength of the insert of metallic material and the relevant damping properties of SMA, in the martensitic phase. In particular, we put our attention on embedding the CuZnAl in the form of thin sheet in a layered composite made by glass fiber reinforced epoxy. By appropriately positioning of the SMA sheets so that they are subjected to the maximum curvature, the damping of the hybrid system can be considerably enhanced. Accordingly analytical method for evaluating the energy dissipation of the thin sheets with different shapes and patterns is developed and is followed by a shape optimization based on genetic algorithm. Eventually different configurations of the hybrid beam structure with different patterns of SMA layer are proposed and compared in the term of damping capacity.

  12. Development of a Simple Structured Artificial Muscle Using SMA Wire

    SciTech Connect

    Ibuki, Ryuta; Maruyama, Shigenao; Komiya, Atsuki

    2006-05-05

    Artificial heart muscle using SMA wire is developed to assist weaken heartbeat. Simple structure design was adopted for large output force, large displacement and rapid cyclic motion of the actuator. The actuator was designed and fabricated from the viewpoint of heat transfer. Moving performance of the actuator was experimentally measured under 10N of loading condition. Under the maximum efficiency condition, the actuator shows cyclic motion with 1mm of displacement and time period of about 2 seconds in one cycle.

  13. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  14. Analysis of SMA Hybrid Composite Structures using Commercial Codes

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.; Patel, Hemant D.

    2004-01-01

    A thermomechanical model for shape memory alloy (SMA) actuators and SMA hybrid composite (SMAHC) structures has been recently implemented in the commercial finite element codes MSC.Nastran and ABAQUS. The model may be easily implemented in any code that has the capability for analysis of laminated composite structures with temperature dependent material properties. The model is also relatively easy to use and requires input of only fundamental engineering properties. A brief description of the model is presented, followed by discussion of implementation and usage in the commercial codes. Results are presented from static and dynamic analysis of SMAHC beams of two types; a beam clamped at each end and a cantilevered beam. Nonlinear static (post-buckling) and random response analyses are demonstrated for the first specimen. Static deflection (shape) control is demonstrated for the cantilevered beam. Approaches for modeling SMAHC material systems with embedded SMA in ribbon and small round wire product forms are demonstrated and compared. The results from the commercial codes are compared to those from a research code as validation of the commercial implementations; excellent correlation is achieved in all cases.

  15. STUDY OF THE RHIC BPM SMA CONNECTOR FAILURE PROBLEM

    SciTech Connect

    LIAW,C.; SIKORA, R.; SCHROEDER, R.

    2007-06-25

    About 730 BPMs are mounted on the RHIC CQS and Triplet super-conducting magnets. Semi-rigid coaxial cables are used to bring the electrical signal from the BPM feedthroughs to the outside flanges. at the ambient temperature. Every year around 10 cables will lose their signals during the operation. The connection usually failed at the warm end of the cable. The problems were either the solder joint failed or the center conductor retracted out of the SMA connector. Finite element analyses were performed to understand the failure mechanism of the solder joint. The results showed that (1) The SMA center conductor can separate from the mating connector due to the thermal retraction. (2) The maximum thermal stress at the warm end solder joint can exceed the material strength of the Pb37/Sn63 solder material and (3) The magnet ramping frequency (-10 Hz), during the machine startup, can possibly resonant the coaxial cable and damage the solder joints, especially when a fracture is initiated. Test results confirmed that by using the silver bearing solder material (a higher strength material) and by crimping the cable at the locations close to the SMA connector (to prevent the center conductor from retracting) can effectively resolve the connector failure problem.

  16. Passive base isolation with superelastic nitinol SMA helical springs

    NASA Astrophysics Data System (ADS)

    Huang, Bin; Zhang, Haiyang; Wang, Han; Song, Gangbing

    2014-06-01

    Seismic isolation of structures such as multi-story buildings, nuclear reactors, bridges, and liquid storage tanks should be designed to preserve structural integrity. By implementing seismic isolation technology, the deformation of superstructures can be dramatically reduced, consequently helping to protect their safety as well. In this paper, an innovative type of passive base isolation system, which is mainly composed of superelastic nitinol SMA helical springs, is developed. In order to verify the effectiveness of the proposed system, a two-story experimental steel frame model is constructed, and two superelastic SMA helical springs are thermo-mechanically built in the laboratory. To describe the nonlinear mechanical properties of the superelastic SMA helical springs under reciprocating load, a phenomenological model is presented in terms of a series of tensile tests. Afterwards, a numerical model of the two-story frame with the suggested isolation system is set up to simulate the response of the isolated frame subjected to an earthquake. Both the experimental and the numerical simulation results indicate that the proposed base isolation system can remarkably suppress structural vibrations and has improved isolation effects when compared with a steel spring isolation system. Due to the capabilities of energy dissipation as well as fully re-centering, it is very applicable to utilize the suggested isolation system in base isolated structures to resist earthquakes.

  17. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  18. Optimal treatment of actinic keratoses

    PubMed Central

    Uhlenhake, Elizabeth E

    2013-01-01

    The most compelling reason and primary goal of treating actinic keratoses is to prevent malignant transformation into invasive squamous cell carcinoma, and although there are well established guidelines outlining treatment modalities and regimens for squamous cell carcinoma, the more commonly encountered precancerous actinic lesions have no such standard. Many options are available with variable success and patient compliance rates. Prevention of these lesions is key, with sun protection being a must in treating aging patients with sun damage as it is never too late to begin protecting the skin. PMID:23345970

  19. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    PubMed

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  20. The Differential Formation of the LINC-Mediated Perinuclear Actin Cap in Pluripotent and Somatic Cells

    PubMed Central

    Khatau, Shyam B.; Kusuma, Sravanti; Hanjaya-Putra, Donny; Mali, Prashant; Cheng, Linzhao; Lee, Jerry S. H.; Gerecht, Sharon; Wirtz, Denis

    2012-01-01

    The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape. PMID:22574215

  1. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  2. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  3. Active Chemical Thermodynamics promoted by activity of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Rao, Madan

    2011-03-01

    The spatial distribution and dynamics of formation and breakup of the nanoclusters of cell surface proteins is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we have proposed a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. We study the consequences of such active actin-based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that the active remodeling of cortical actin, can give rise to a dramatic increase in efficiency and extent of conformational spread, even at low levels of expression at the cell surface. We define a activity temperature (τa) arising due to actin activities which can be used to describe chemical thermodynamics of the system. We plot TTT (time-temparature-transformation) curves and compute the Arrhenius factors which depend on τa . With this, the active asters can be treated as enzymes whose enzymatic reaction rate can be related to the activity.

  4. Antibodies to Actin in Autoimmune Neutropenia

    DTIC Science & Technology

    1990-02-01

    protein as actin. Purified Acanthamoeba actin by anti-neutrophil antibodies in autoimmune neutropenia, comigrated with the protein and was specifically...anti-rabbit IgG were obtained from ICN Immunobiolog- formed using purified Acanthamoeba actin (gift of Dr Blair Bowers. icals, Naperville, IL. Cells...preparations𔃼 1 - was the protein recognized by these anti-neutrophil antibody 6 .2- positive sera, lgG, and F(ab’) 2. Purified Acanthamoeba actin

  5. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  6. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  7. Actinic cheilitis in dental practice.

    PubMed

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  8. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin

    PubMed Central

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-01-01

    ABSTRACT Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation. PMID:25315833

  9. α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

    PubMed Central

    Sousa, Vítor L.; Bellani, Serena; Giannandrea, Maila; Yousuf, Malikmohamed; Valtorta, Flavia; Meldolesi, Jacopo

    2009-01-01

    The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration. PMID:19553474

  10. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  11. CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

    PubMed

    Lin, Chien-Huang; Shih, Chung-Huang; Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.

  12. CXCL12 Induces Connective Tissue Growth Factor Expression in Human Lung Fibroblasts through the Rac1/ERK, JNK, and AP-1 Pathways

    PubMed Central

    Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression. PMID:25121739

  13. Actin crosslinkers: repairing the sense of touch.

    PubMed

    Sun, Sean X; Walcott, Sam

    2010-10-26

    Cells use actin bundles infused with myosin to exert contractile forces on the extracellular environment. This active tension is essential for cellular mechanosensation. Now, the role of actin crosslinkers in stabilizing and repairing the actin bundles is coming into clearer view.

  14. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  15. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    PubMed

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice.

  16. Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Wong, Chris K. C.; Han, Daishu; Lee, Will M.

    2015-01-01

    During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics. PMID:25901598

  17. Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle.

    PubMed

    Sevdali, Maria; Kumar, Vikash; Peckham, Michelle; Sparrow, John

    2013-03-01

    Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

  18. Fatigue of NiTi SMA-pulley system using Taguchi and ANOVA

    NASA Astrophysics Data System (ADS)

    Mohd Jani, Jaronie; Leary, Martin; Subic, Aleksandar

    2016-05-01

    Shape memory alloy (SMA) actuators can be integrated with a pulley system to provide mechanical advantage and to reduce packaging space; however, there appears to be no formal investigation of the effect of a pulley system on SMA structural or functional fatigue. In this work, cyclic testing was conducted on nickel-titanium (NiTi) SMA actuators on a pulley system and a control experiment (without pulley). Both structural and functional fatigues were monitored until fracture, or a maximum of 1E5 cycles were achieved for each experimental condition. The Taguchi method and analysis of the variance (ANOVA) were used to optimise the SMA-pulley system configurations. In general, one-way ANOVA at the 95% confidence level showed no significant difference between the structural or functional fatigue of SMA-pulley actuators and SMA actuators without pulley. Within the sample of SMA-pulley actuators, the effect of activation duration had the greatest significance for both structural and functional fatigue, and the pulley configuration (angle of wrap and sheave diameter) had a greater statistical significance than load magnitude for functional fatigue. This work identified that structural and functional fatigue performance of SMA-pulley systems is optimised by maximising sheave diameter and using an intermediate wrap-angle, with minimal load and activation duration. However, these parameters may not be compatible with commercial imperatives. A test was completed for a commercially optimal SMA-pulley configuration. This novel observation will be applicable to many areas of SMA-pulley system applications development.

  19. Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.

    PubMed

    Gumienny, Tina L; Macneil, Lesley; Zimmerman, Cole M; Wang, Huang; Chin, Lena; Wrana, Jeffrey L; Padgett, Richard W

    2010-05-20

    Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

  20. Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

    PubMed

    Morrison, Kerrie A; Akram, Aneel; Mathews, Ashlyn; Khan, Zoeya A; Patel, Jaimin H; Zhou, Chumin; Hardy, David J; Moore-Kelly, Charles; Patel, Roshani; Odiba, Victor; Knowles, Tim J; Javed, Masood-Ul-Hassan; Chmel, Nikola P; Dafforn, Timothy R; Rothnie, Alice J

    2016-12-01

    The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

  1. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  2. STK16 regulates actin dynamics to control Golgi organization and cell cycle

    PubMed Central

    Liu, Juanjuan; Yang, Xingxing; Li, Binhua; Wang, Junjun; Wang, Wenchao; Liu, Jing; Liu, Qingsong; Zhang, Xin

    2017-01-01

    STK16 is a ubiquitously expressed, myristoylated, and palmitoylated serine/threonine protein kinase with underexplored functions. Recently, it was shown to be involved in cell division but the mechanism remains unclear. Here we found that human STK16 localizes to the Golgi complex throughout the cell cycle and plays important roles in Golgi structure regulation. STK16 knockdown or kinase inhibition disrupts actin polymers and causes fragmented Golgi in cells. In vitro assays show that STK16 directly binds to actin and regulates actin dynamics in a concentration- and kinase activity-dependent way. In addition, STK16 knockdown or kinase inhibition not only delays mitotic entry and prolongs mitosis, but also causes prometaphase and cytokinesis arrest. Therefore, we revealed STK16 as a novel actin binding protein that resides in the Golgi, which regulates actin dynamics to control Golgi structure and participate in cell cycle progression. PMID:28294156

  3. Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

    PubMed

    Weins, Astrid; Schlondorff, Johannes S; Nakamura, Fumihiko; Denker, Bradley M; Hartwig, John H; Stossel, Thomas P; Pollak, Martin R

    2007-10-09

    Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.

  4. The large GTPase dynamin regulates actin comet formation and movement in living cells

    PubMed Central

    Orth, James D.; Krueger, E. W.; Cao, H.; McNiven, Mark A.

    2002-01-01

    The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2ΔPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility. PMID:11782546

  5. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  6. NASA Keynote at the 2015 Trilateral SMA Conference, Frascati, Italy

    NASA Technical Reports Server (NTRS)

    Groen, Frank

    2015-01-01

    The purpose of this presentation is to illustrate some new directions within NASA's safety and mission function in response to changes in missions, technology, and practices. The presentation lists last year's highlights from NASA's human and robotic spaceflight missions, and discusses anticipated highlights for the coming year taken from existing Agency presentations. It will highlight changes to NASA's mission and the way NASA does business, as described in the 2014 strategic plan. It will then discuss how these changes pose challenges to trusted SMA practices, and provide some examples of initiatives NASA is taking action to address these challenges.

  7. Actin Dynamics: From Nanoscale to Microscale

    PubMed Central

    Carlsson, Anders E.

    2010-01-01

    The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research. PMID:20462375

  8. Computational Tension Mapping of Adherent Cells Based on Actin Imaging.

    PubMed

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.

  9. Role of actin filaments in fusopod formation and osteoclastogenesis.

    PubMed

    Wang, Yongqiang; Brooks, Patricia Joyce; Jang, Janet Jinyoung; Silver, Alexandra Shade; Arora, Pamma D; McCulloch, Christopher A; Glogauer, Michael

    2015-07-01

    Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.

  10. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  11. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  12. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  13. Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling.

    PubMed

    Pakhomov, Andrei G; Xiao, Shu; Pakhomova, Olga N; Semenov, Iurii; Kuipers, Marjorie A; Ibey, Bennett L

    2014-12-01

    Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

  14. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    PubMed

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  15. Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans.

    PubMed

    Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine; Meijer, Harold J G

    2015-04-01

    Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.

  16. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    PubMed

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  17. Keeping it all together: auxin-actin crosstalk in plant development.

    PubMed

    Zhu, Jinsheng; Geisler, Markus

    2015-08-01

    Polar auxin transport and the action of the actin cytoskeleton are tightly interconnected, which is documented by the finding that auxin transporters reach their final destination by active movement of secretory vesicles along F-actin tracks. Moreover, auxin transporter polarity and flexibility is thought to depend on transporter cycling that requires endocytosis and exocytosis of vesicles. In this context, we have reviewed the current literature on an involvement of the actin cytoskeleton in polar auxin transport and identify known similarities and differences in its structure, function and dynamics in comparison to non-plant organisms. By describing how auxin modulates actin expression and actin organization and how actin and its stability affects auxin-transporter endocytosis and recycling, we discuss the current knowledge on regulatory auxin-actin feedback loops. We focus on known effects of auxin and of auxin transport inhibitors on the stability and organization of actin and examine the functionality of auxin and/or auxin transport inhibitor-binding proteins with respect to their suitability to integrate auxin/auxin transport inhibitor action. Finally, we indicate current difficulties in the interpretation of organ, time and concentration-dependent auxin/auxin transport inhibitor treatments and formulate simple future experimental guidelines.

  18. An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.

    PubMed

    Gao, Ningning; Wadhwani, Parvesh; Mühlhäuser, Philipp; Liu, Qiong; Riemann, Michael; Ulrich, Anne S; Nick, Peter

    2016-07-01

    Ginkbilobin is a short antifungal protein that had been purified and cloned from the seeds of the living fossil Ginkgo biloba. Homologues of this protein can be detected in all seed plants and the heterosporic fern Selaginella and are conserved with respect to domain structures, peptide motifs, and specific cysteine signatures. To get insight into the cellular functions of these conserved motifs, we expressed green fluorescent protein fusions of full-length and truncated ginkbilobin in tobacco BY-2 cells. We show that the signal peptide confers efficient secretion of ginkbilobin. When this signal peptide is either cleaved or masked, ginkbilobin binds and visualizes the actin cytoskeleton. This actin-binding activity of ginkbilobin is mediated by a specific subdomain just downstream of the signal peptide, and this subdomain can also coassemble with actin in vitro. Upon stable overexpression of this domain, we observe a specific delay in premitotic nuclear positioning indicative of a reduced dynamicity of actin. To elucidate the cellular response to the binding of this subdomain to actin, we use chemical engineering based on synthetic peptides comprising different parts of the actin-binding subdomain conjugated with the cell-penetrating peptide BP100 and with rhodamine B as a fluorescent reporter. Binding of this synthetic construct to actin efficiently induces programmed cell death. We discuss these findings in terms of a working model, where ginkbilobin can activate actin-dependent cell death.

  19. Fibrillar Type I Collagen Enhances the Differentiation and Proliferation of Myofibroblasts by Lowering α2β1 Integrin Expression in Cardiac Fibrosis

    PubMed Central

    Hong, Jian; Chu, Ming; Qian, Lijun; Wang, Junhong; Guo, Yan

    2017-01-01

    Many studies have shown that α2β1 integrin plays an important role in the development of cardiac fibrosis. However, the mechanism of how α2β1 integrin regulates the differentiation and proliferation of myofibroblasts in cardiac fibrosis through fibrillar collagen (FC) remains uncertain. We established that FC mimicked the 3-dimensional extracellular matrix (ECM) of fibroblasts from post-myocardial infarction (MI) patients in vivo. This allowed us to explore the differentiation and proliferation of cardiac fibroblasts on FC. Here, we report that low expression of α2β1 integrin increased protein kinase B (AKT) activation and α-smooth muscle actin (α-SMA) expression. This occurred due to the instability of phosphatase and tensin homolog (PTEN) in myofibroblasts on FC. We also demonstrated that FC reduced protein phosphatase type 2A (PP2A) activity of myofibroblasts, which was coincident with low α2β1 integrin expression and activation of AKT, but not mitogen-activated protein kinase (ERK). In addition, knock-down of both β1 integrin and PP2A in fibroblasts promoted differentiation and proliferation via AKT activation and increased α-SMA expression. In summary, our study demonstrated that low α2β1 integrin expression regulated its downstream targets PTEN and AKT via crosstalk with PP2A, a critical cell signaling pathway that permits aberrant differentiation and proliferation of myofibroblasts on FC. PMID:28251149

  20. Superior mesenteric artery (SMA) syndrome: an unusual cause of intestinal obstruction in palliative care.

    PubMed

    So, Chun-Yan; Chan, Kwok-Ying; Au, Ho-Yan; Chan, Man-Lui; Lai, Theresa

    2017-01-01

    Superior mesenteric artery (SMA) syndrome is an uncommon cause of intestinal obstruction and seldom been mentioned in palliative care. Hereby, we reported a case of SMA syndrome who presented with symptoms of upper intestinal obstruction in a 68-year-old patient; subsequent CT findings were classical of SMA syndrome. The patient's history of poliomyelitis and recent significant weight loss were the predisposing factors for SMA syndrome. It also highlights the importance of monitoring signs and symptoms of intestinal obstruction in such patients before considering switching to oral feeding.

  1. Shape Memory Alloy (SMA)-Based Launch Lock

    NASA Technical Reports Server (NTRS)

    Badescu, Mircea; Bao, Xiaoqi; Bar-Cohen, Yoseph

    2014-01-01

    Most NASA missions require the use of a launch lock for securing moving components during the launch or securing the payload before release. A launch lock is a device used to prevent unwanted motion and secure the controlled components. The current launch locks are based on pyrotechnic, electro mechanically or NiTi driven pin pullers and they are mostly one time use mechanisms that are usually bulky and involve a relatively high mass. Generally, the use of piezoelectric actuation provides high precession nanometer accuracy but it relies on friction to generate displacement. During launch, the generated vibrations can release the normal force between the actuator components allowing shaft's free motion which could result in damage to the actuated structures or instruments. This problem is common to other linear actuators that consist of a ball screw mechanism. The authors are exploring the development of a novel launch lock mechanism that is activated by a shape memory alloy (SMA) material ring, a rigid element and an SMA ring holding flexure. The proposed design and analytical model will be described and discussed in this paper.

  2. Structural design of morphing trailing edge actuated by SMA

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Xu, Zhiwei; Zhu, Qian

    2013-09-01

    In this paper, the morphing trailing edge is designed to achieve the up and down deflection under the aerodynamic load. After a detailed and accurate computational analysis to determine the SMA specifications and layout programs, a solid model is created in CATIA and the structures of the morphing wing trailing edge are produced by CNC machining. A set of DSP measurement and control system is designed to accomplish the controlling experiment of the morphing wing trailing edge. At last, via the force analysis, the trailing edge is fabricated with four sections of aluminum alloy, and the arrangement scheme of SMA wires is determined. Experiment of precise control integral has been performed to survey the control effect. The experiment consists of deflection angle tests of the third joint and the integral structure. Primarily, the ultimate deflection angle is tested in these two experiments. Therefore, the controlling experiment of different angles could be performed within this range. The results show that the deflection error is less than 4%and response time is less than 6.7 s, the precise controlling of the morphing trailing edge is preliminary realized.

  3. A Novel SMA-based Concept for Airfoil Structural Morphing

    NASA Astrophysics Data System (ADS)

    Barbarino, S.; Pecora, R.; Lecce, L.; Concilio, A.; Ameduri, S.; Calvi, E.

    2009-08-01

    The adaptive structures concept is of great interest in the aerospace field because of the several benefits which can be accomplished in the fields including noise reduction, load alleviation, weight reduction, etc., at a level in which they can be considered as compulsory in the design of future aircraft. Improvements in terms of the aerodynamic efficiency, aeroelastic behavior, stability, and manoeuvrability performance have already been proved through many international studies in the past. In the family of the Smart Materials, Shape Memory Alloys (SMA) seem to be a suitable solution for many static applications. Their high structural integrability in conjunction with actuation capabilities and a favorable performance per weight ratio, allows the development of original architectures. In this study, a morphing wing trailing edge concept is presented; morphing ability was introduced with the aim of replacing a conventional flap device. A compliant rib structure was designed, based on SMA actuators exhibiting structural potential (bearing external aerodynamic loads). Numerical results, achieved through a FE approach, are presented in terms of trailing edge induced displacement and morphed shape.

  4. Changes in keratins and alpha-smooth muscle actin during three-dimensional reconstitution of eccrine sweat glands.

    PubMed

    Li, Haihong; Li, Xuexue; Zhang, Bingna; Zhang, Mingjun; Chen, Wenlong; Tang, Shijie; Fu, Xiaobing

    2016-07-01

    We have examined the changes of keratins and alpha-SMA at various time points in order to investigate the development and differentiation of eccrine sweat gland cells during the course of three-dimensional (3D) reconstitution. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35, and 42 days post-implantation, Matrigel plugs were removed and immunostained. We found that during 3D reconstitution, keratin and alpha-SMA expression changed in a time-dependent manner. At day 1, all cells stained positively for keratin isoforms K5, K14, and K15, with the staining intensity of K15 being weak and K5 and K14 being strong, but none of the cells displayed K7, K8, or alpha-SMA. As time progressed, spheroid-like structures formed with the inner layer acquiring K7 and K8, but losing K5 and K14 expression, and the outer layer acquiring alpha-SMA expression, but losing K15 expression. K8 expression was first noted at day 14, and K7 and alpha-SMA at day 21. The loss of K15 expression was first noted at day 14, K14 at day 21, and K5 at day 28. At 28, 35, and 42 days, the spheroid-like structures could be distinguished, by immunohistochemistry, as having secretory coil-like and coiled duct-like structures. We conclude that the changes in expression of keratins and alpha-SMA in 3D-reconstituted eccrine sweat glands are similar to those of native eccrine sweat glands, indicating that the 3D reconstitution of sweat glands provides an excellent model for studying the development, cytodifferentiation, and regulation of eccrine sweat glands.

  5. Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones

    PubMed Central

    Garcia, Mikael; Leduc, Cécile; Lagardère, Matthieu; Argento, Amélie; Sibarita, Jean-Baptiste; Thoumine, Olivier

    2015-01-01

    Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin–coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration. PMID:26038554

  6. Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondiiD⃞

    PubMed Central

    Sahoo, Nivedita; Beatty, Wandy; Heuser, John; Sept, David; Sibley, L. David

    2006-01-01

    Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans. PMID:16319175

  7. Nuclear Actin in Development and Transcriptional Reprogramming

    PubMed Central

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin’s roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation. PMID:28326098

  8. Tissue inhibitor of matrix metalloproteinase-1 expression in colorectal cancer liver metastases is associated with vascular structures.

    PubMed

    Illemann, Martin; Eefsen, Rikke Helene Løvendahl; Bird, Nigel Charles; Majeed, Ali; Osterlind, Kell; Laerum, Ole Didrik; Alpízar-Alpízar, Warner; Lund, Ida Katrine; Høyer-Hansen, Gunilla

    2016-02-01

    Metastatic growth by colorectal cancer cells in the liver requires the ability of the cancer cells to interact with the new microenvironment. This interaction results in three histological growth patterns of liver metastases: desmoplastic, pushing, and replacement. In primary colorectal cancer several proteases, involved in the degradation of extracellular matrix components, are up-regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a strong prognostic marker in plasma from colorectal cancer patients, with significant higher levels in patients with metastatic disease. We therefore wanted to determine the expression pattern of TIMP-1 in primary colorectal cancers and their matching liver metastases. TIMP-1 mRNA was primarily seen in α-smooth-muscle actin (α-SMA)-positive cells. In all primary tumors and liver metastases with desmoplastic growth pattern, TIMP-1 mRNA was primarily found in α-SMA-positive myofibroblasts located at the invasive front. Some α-SMA-positive cells with TIMP-1 mRNA were located adjacent to CD34-positive endothelial cells, identifying them as pericytes. This indicates that TIMP-1 in primary tumors and liver metastases with desmoplastic growth pattern has dual functions; being an MMP-inhibitor at the cancer periphery and involved in tumor-induced angiogenesis in the pericytes. In the liver metastases with pushing or replacement growth patterns, TIMP-1 was primarily expressed by activated hepatic stellate cells at the metastasis/liver parenchyma interface. These cells were located adjacent to CD34-positive endothelial cells, suggesting a function in tumor-induced angiogenesis. We therefore conclude that TIMP-1 expression is growth pattern dependent in colorectal cancer liver metastases.

  9. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  10. KIF17 regulates RhoA-dependent actin remodeling at epithelial cell–cell adhesions

    PubMed Central

    Acharya, Bipul R.; Espenel, Cedric; Libanje, Fotine; Raingeaud, Joel; Morgan, Jessica; Jaulin, Fanny; Kreitzer, Geri

    2016-01-01

    ABSTRACT The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell–cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP–actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell–cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilinS3A inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA–GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells. PMID:26759174

  11. Long non-coding RNA UC001kfo promotes hepatocellular carcinoma proliferation and metastasis by targeting α-SMA.

    PubMed

    Pan, Yanfeng; Qin, Tao; Yin, Shenglu; Zhang, Xianqiang; Gao, Xiaojuan; Mu, Lifen

    2017-03-01

    Several long non-coding RNAs (lncRNAs) have been investigated and found to be correlated with the behaviours and prognosis of hepatocellular carcinoma (HCC); Specifically, we revealed that the lncRNA UC001kfo was differentially expressed in HCC tissues compared with normal liver tissues using lncRNA microarrays, but its functional role in cancers, including HCC, has not yet been elucidated. The present study found that the expression of UC001kfo was upregulated in HCC tissues and cell lines in comparison with tumour-adjacent tissues and normal hepatocytes, respectively. In addition, a high UC001kfo level was determined to be correlated with macro-vascular invasion and TNM stage of HCC. Specifically, patients with high UC001kfo expression displayed a significantly lower overall survival rate and progression-free survival rate. Moreover, both univariate and multivariate COX regression analyses identified TNM stage and high UC001kfo expression as risk factors for poor prognosis in HCC patients. In addition, UC001kfo was verified to promote the proliferation, metastasis and epithelial-mesenchymal transition (EMT) in HCC cells in both in vitro and in vivo assays. Mechanistically, α-SMA was indicated as a potential target gene of UC001kfo in mediating HCC metastasis. In conclusion, UC001kfo promotes HCC proliferation and metastasis by targeting α-SMA, and UC001kfo may potentially serve as a prognostic marker and a therapeutic target for treatment of HCC.

  12. Heuristics for connectivity-based brain parcellation of SMA/pre-SMA through force-directed graph layout.

    PubMed

    Crippa, Alessandro; Cerliani, Leonardo; Nanetti, Luca; Roerdink, Jos B T M

    2011-02-01

    We propose the use of force-directed graph layout as an explorative tool for connectivity-based brain parcellation studies. The method can be used as a heuristic to find the number of clusters intrinsically present in the data (if any) and to investigate their organisation. It provides an intuitive representation of the structure of the data and facilitates interactive exploration of properties of single seed voxels as well as relations among (groups of) voxels. We validate the method on synthetic data sets and we investigate the changes in connectivity in the supplementary motor cortex, a brain region whose parcellation has been previously investigated via connectivity studies. This region is supposed to present two easily distinguishable connectivity patterns, putatively denoted by SMA (supplementary motor area) and pre-SMA. Our method provides insights with respect to the connectivity patterns of the premotor cortex. These present a substantial variation among subjects, and their subdivision into two well-separated clusters is not always straightforward.

  13. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    PubMed

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  14. Cloning and sequence analysis of an actin gene in aloe.

    PubMed

    Wen, S S; He, D W; Liao, C M; Li, J; Wen, G Q; Liu, X H

    2014-07-04

    Aloe (Aloe spp), containing abundant polysaccharides and numerous bioactive ingredients, has remarkable medical, ornamental, calleidic, and edible values. In the present study, the total RNA was extracted from aloe leaf tissue. The isolated high-quality RNA was further used to clone actin gene by using reverse transcription-polymerase chain reaction (RT-PCR). The result of sequence analysis for the amplified fragment revealed that the cloned actin gene was 1012 bp in length (GenBank accession No. KC751541.1) and contained a 924-bp coding region and encoded a protein consisting of 307 amino acids. Homologous alignment showed that it shared over 80 and 96% identity with the nucleotide and amino acid sequences of actin from other plants, respectively. In addition, the cloned gene was used for phylogenetic analyses based on the deduced amino acid sequences, and the results suggested that the actin gene is highly conserved in evolution. The findings of this study will be useful for investigating the expression patterns of other genes in Aloe.

  15. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

    PubMed Central

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies. PMID:27124114

  16. Spontaneous Breathing Pattern as Respiratory Functional Outcome in Children with Spinal Muscular Atrophy (SMA)

    PubMed Central

    LoMauro, A.; Aliverti, A.; Mastella, C.; Arnoldi, M. T.; Banfi, P.; Baranello, G.

    2016-01-01

    Introduction SMA is characterised by progressive motor and respiratory muscle weakness. We aimed to verify if in SMA children 1)each form is characterized by specific ventilatory and thoraco-abdominal pattern(VTAp) during quiet breathing(QB); 2)VTAp is affected by salbutamol therapy, currently suggested as standard treatment, or by the natural history(NH) of SMA; 3)the severity of global motor impairment linearly correlates with VTAp. Materials and methods VTAp was analysed on 32 SMA type I (SMA1,the most severe form), 51 type II (SMA2,the moderate), 8 type III (SMA3,the mildest) and 20 healthy (HC) using opto-electronic plethysmography. Spirometry, cough and motor function were measured in a subgroup of patients. Results In SMA1, a normal ventilation is obtained in supine position by rapid and shallow breathing with paradoxical ribcage motion. In SMA2, ventilation is within a normal range in seated position due to an increased respiratory rate(p<0.05) with reduced tidal volume(p<0.05) secondary to a poor contribution of pulmonary ribcage(%ΔVRC,P, p<0.001). Salbutamol therapy had no effect on VTAp during QB(p>0.05) while tachypnea occurred in type I NH. A linear correlation(p<0.001) was found between motor function scales and VTAp. Conclusion A negative or reduced %ΔVRC,P, indicative of ribcage muscle weakness, is a distinctive feature of SMA1 and SMA2 since infancy. Its quantitative assessment represents a non-invasive, non-volitional index that can be obtained in all children, even uncollaborative, and provides useful information on the action of ribcage muscles that are known to be affected by the disease.Low values of motor function scales indicate impairment of motor but also of respiratory function. PMID:27820869

  17. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides.

    PubMed

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies.

  18. Actin-Binding Protein Requirement for Cortical Stability and Efficient Locomotion

    NASA Astrophysics Data System (ADS)

    Cunningham, C. Casey; Gorlin, Jed B.; Kwiatkowski, David J.; Hartwig, John H.; Janmey, Paul A.; Randolph Byers, H.; Stossel, Thomas P.

    1992-01-01

    Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

  19. Actin dynamics in mouse fibroblasts in microgravity

    NASA Astrophysics Data System (ADS)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  20. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  1. Polymerization of actin by positively charged liposomes

    PubMed Central

    1988-01-01

    By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N- pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts. PMID:3360852

  2. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    PubMed

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.

  3. SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

    PubMed Central

    Tiziano, Francesco Danilo; Pinto, Anna Maria; Fiori, Stefania; Lomastro, Rosa; Messina, Sonia; Bruno, Claudio; Pini, Antonella; Pane, Marika; D'Amico, Adele; Ghezzo, Alessandro; Bertini, Enrico; Mercuri, Eugenio; Neri, Giovanni; Brahe, Christina

    2010-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials. PMID:19603064

  4. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  5. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  6. Modeling actin waves in dictyostelium cells

    NASA Astrophysics Data System (ADS)

    Wasnik, Vaibhav; Mukhopadhyay, Ranjan

    2011-03-01

    Actin networks in living cells demonstrate a high capacity for self-organization and are responsible for the formation of a variety of structures such as lamellopodia, phagocytic cups, and cleavage furrows. Recent experiments have studied actin waves formed on the surface of dictyostelium cells that have been treated with a depolymerizing agent. These waves are believed to be physiologically important, for example, for the formation of phagocytic cups. We propose and study a minimal model, based on the dendritic nucleation of actin polymers, to explain the formation of these waves. This model can be extended to study the dynamics of the coupled actin-membrane system.

  7. GPCRs and actin-cytoskeleton dynamics.

    PubMed

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

  8. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-07

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

  9. Bioinformatics study of the mangrove actin genes

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  10. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

  11. Photodynamic therapy for actinic keratoses.

    PubMed

    Kalisiak, Michal S; Rao, Jaggi

    2007-01-01

    Actinic keratoses (AKs) are one of the most common conditions that are treated by dermatologists and they have the potential to progress to squamous cell carcinoma if left untreated. Photodynamic therapy (PDT) has emerged as a novel and versatile method of treating those lesions. Topical preparations of aminolevulinic acid and methyl aminolevulinate are commercially available photosensitizers, and numerous light sources may be used for photoactivation. This article focuses on practical aspects of PDT in the treatment of AKs, outcomes of relevant clinical trials, and special applications of PDT in transplant recipients and other who are predisposed to AK formation. Step-by-step descriptions of PDT sessions are presented.

  12. Experimental Validation of a Thermoelastic Model for SMA Hybrid Composites

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.

    2001-01-01

    This study presents results from experimental validation of a recently developed model for predicting the thermomechanical behavior of shape memory alloy hybrid composite (SMAHC) structures, composite structures with an embedded SMA constituent. The model captures the material nonlinearity of the material system with temperature and is capable of modeling constrained, restrained, or free recovery behavior from experimental measurement of fundamental engineering properties. A brief description of the model and analysis procedures is given, followed by an overview of a parallel effort to fabricate and characterize the material system of SMAHC specimens. Static and dynamic experimental configurations for the SMAHC specimens are described and experimental results for thermal post-buckling and random response are presented. Excellent agreement is achieved between the measured and predicted results, fully validating the theoretical model for constrained recovery behavior of SMAHC structures.

  13. Nonlinear Thermoelastic Model for SMAs and SMA Hybrid Composites

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.

    2004-01-01

    A constitutive mathematical model has been developed that predicts the nonlinear thermomechanical behaviors of shape-memory-alloys (SMAs) and of shape-memory-alloy hybrid composite (SMAHC) structures, which are composite-material structures that contain embedded SMA actuators. SMAHC structures have been investigated for their potential utility in a variety of applications in which there are requirements for static or dynamic control of the shapes of structures, control of the thermoelastic responses of structures, or control of noise and vibrations. The present model overcomes deficiencies of prior, overly simplistic or qualitative models that have proven ineffective or intractable for engineering of SMAHC structures. The model is sophisticated enough to capture the essential features of the mechanics of SMAHC structures yet simple enough to accommodate input from fundamental engineering measurements and is in a form that is amenable to implementation in general-purpose structural analysis environments.

  14. Mixing temperature design and properties evaluation for SMA-13 mixture

    NASA Astrophysics Data System (ADS)

    Huang, X. Y.; Li, B. Y.

    2017-01-01

    The mixing temperature of hot asphalt mixture could be reduced by adding WMA additive Sasobit, as well as reducing smoke emissions and energy construction during the mixing construction and paving. The reasonable mixing temperature were investigated in this paper. In addition, high temperature stability, water stability and low-temperature performance of warm asphalt mixture were evaluaterd. The test results indicate that the mixing temperature of SMA-13 with WMA additive Sasobit may reduce 15-20°C at the same energy (compaction times). the dynamic stability were improved after adding WMA additive Sasobit, and the Water stability and low-temperature performance of mixture decreased, while all kinds of asphalt mixture properties can meet the requirements.

  15. Development of damage suppression system using embedded SMA foil in CFRP laminates

    NASA Astrophysics Data System (ADS)

    Ogisu, Toshimichi; Nomura, Masato; Ando, Norio; Takaki, Junji; Kobayashi, Masakazu; Okabe, Tomonaga; Takeda, Nobuo

    2001-07-01

    Some recent studies have suggested possible applications of Shape Memory Alloy (SMA) for a smart health monitoring and suppression of damage growth. The authors have been conducting research and development studies on applications of embedded SMA foil actuators in CFRP laminates as the basic research for next generation aircrafts. First the effective surface treatment for improvement of bonding properties between SMA and CFRP was studied. It was certified that the anodic oxide treatment by 10% NaOH solution was the most effective treatment from the results of peel resistance test and shear strength test. Then, CFRP laminates with embedded SMA foils were successfully fabricated using this effective surface treatment. The damage behavior of quasi-isotropic CFRP laminates with embedded SMA foils was characterized in both quasi-static load-unload and fatigue tests. The relationship between crack density and applied strain was obtained. The recovery stress generated by embedded SMA foils could increase the onset strain of transverse cracking by 0.2%. The onset strain of delmination in CFRP laminates was also increased accordingly. The shear-lag analysis was also conducted to predict the damage evolution in CFRP laminates with embedded SMA foils. The adhesive layers on both sides of SMA foils were treated as shear elements. The theoretical analysis successfully predicted the experimental results.

  16. Genome Sequence of Methanosarcina soligelidi SMA-21, Isolated from Siberian Permafrost-Affected Soil

    PubMed Central

    Shapiro, Nicole; Woyke, Tanja; Horn, Fabian; Bakermans, Corien; Wagner, Dirk

    2015-01-01

    Here, we announce the genome sequence of Methanosarcina soligelidi SMA-21, an anaerobic methanogenic archaeon that was previously isolated from Siberian permafrost-affected soil. The sequencing of strain SMA-21 yielded a 4.06-Mb genome with 41.5% G+C content, containing a total of 2,647 open reading frames. PMID:25908125

  17. Reconstitution of a Minimal Actin Cortex by Coupling Actin Filaments to Reconstituted Membranes.

    PubMed

    Vogel, Sven K

    2016-01-01

    A thin layer of actin filaments in many eukaryotic cell types drives pivotal aspects of cell morphogenesis and is generally cited as the actin cortex. Myosin driven contractility and actin cytoskeleton membrane interactions form the basis of fundamental cellular processes such as cytokinesis, cell migration, and cortical flows. How the interplay between the actin cytoskeleton, the membrane, and actin binding proteins drives these processes is far from being understood. The complexity of the actin cortex in living cells and the hardly feasible manipulation of the omnipotent cellular key players, namely actin, myosin, and the membrane, are challenging in order to gain detailed insights about the underlying mechanisms. Recent progress in developing bottom-up in vitro systems where the actin cytoskeleton is combined with reconstituted membranes may provide a complementary route to reveal general principles underlying actin cortex properties. In this chapter the reconstitution of a minimal actin cortex by coupling actin filaments to a supported membrane is described. This minimal system may be very well suited to study for example protein interactions on membrane bound actin filaments in a very controlled and quantitative manner as it may be difficult to perform in living systems.

  18. Revised Hammersmith Scale for spinal muscular atrophy: A SMA specific clinical outcome assessment tool

    PubMed Central

    Scoto, Mariacristina; Mayhew, Anna; Main, Marion; Mazzone, Elena S.; Montes, Jacqueline; de Sanctis, Roberto; Dunaway Young, Sally; Salazar, Rachel; Glanzman, Allan M.; Pasternak, Amy; Quigley, Janet; Mirek, Elizabeth; Duong, Tina; Gee, Richard; Civitello, Matthew; Tennekoon, Gihan; Pane, Marika; Pera, Maria Carmela; Bushby, Kate; Day, John; Darras, Basil T.; De Vivo, Darryl; Finkel, Richard; Mercuri, Eugenio; Muntoni, Francesco

    2017-01-01

    Recent translational research developments in Spinal Muscular Atrophy (SMA), outcome measure design and demands from regulatory authorities require that clinical outcome assessments are ‘fit for purpose’. An international collaboration (SMA REACH UK, Italian SMA Network and PNCRN USA) undertook an iterative process to address discontinuity in the recorded performance of the Hammersmith Functional Motor Scale Expanded and developed a revised functional scale using Rasch analysis, traditional psychometric techniques and the application of clinical sensibility via expert panels. Specifically, we intended to develop a psychometrically and clinically robust functional clinician rated outcome measure to assess physical abilities in weak SMA type 2 through to strong ambulant SMA type 3 patients. The final scale, the Revised Hammersmith Scale (RHS) for SMA, consisting of 36 items and two timed tests, was piloted in 138 patients with type 2 and 3 SMA in an observational cross-sectional multi-centre study across the three national networks. Rasch analysis demonstrated very good fit of all 36 items to the construct of motor performance, good reliability with a high Person Separation Index PSI 0.98, logical and hierarchical scoring in 27/36 items and excellent targeting with minimal ceiling. The RHS differentiated between clinically different groups: SMA type, World Health Organisation (WHO) categories, ambulatory status, and SMA type combined with ambulatory status (all p < 0.001). Construct and concurrent validity was also confirmed with a strong significant positive correlation with the WHO motor milestones rs = 0.860, p < 0.001. We conclude that the RHS is a psychometrically sound and versatile clinical outcome assessment to test the broad range of physical abilities of patients with type 2 and 3 SMA. Further longitudinal testing of the scale with regards change in scores over 6 and 12 months are required prior to its adoption in clinical trials. PMID:28222119

  19. SMA-SH: Modified Styrene-Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs.

    PubMed

    Lindhoud, Simon; Carvalho, Vanessa; Pronk, Joachim W; Aubin-Tam, Marie-Eve

    2016-04-11

    Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.

  20. Revised Hammersmith Scale for spinal muscular atrophy: A SMA specific clinical outcome assessment tool.

    PubMed

    Ramsey, Danielle; Scoto, Mariacristina; Mayhew, Anna; Main, Marion; Mazzone, Elena S; Montes, Jacqueline; de Sanctis, Roberto; Dunaway Young, Sally; Salazar, Rachel; Glanzman, Allan M; Pasternak, Amy; Quigley, Janet; Mirek, Elizabeth; Duong, Tina; Gee, Richard; Civitello, Matthew; Tennekoon, Gihan; Pane, Marika; Pera, Maria Carmela; Bushby, Kate; Day, John; Darras, Basil T; De Vivo, Darryl; Finkel, Richard; Mercuri, Eugenio; Muntoni, Francesco

    2017-01-01

    Recent translational research developments in Spinal Muscular Atrophy (SMA), outcome measure design and demands from regulatory authorities require that clinical outcome assessments are 'fit for purpose'. An international collaboration (SMA REACH UK, Italian SMA Network and PNCRN USA) undertook an iterative process to address discontinuity in the recorded performance of the Hammersmith Functional Motor Scale Expanded and developed a revised functional scale using Rasch analysis, traditional psychometric techniques and the application of clinical sensibility via expert panels. Specifically, we intended to develop a psychometrically and clinically robust functional clinician rated outcome measure to assess physical abilities in weak SMA type 2 through to strong ambulant SMA type 3 patients. The final scale, the Revised Hammersmith Scale (RHS) for SMA, consisting of 36 items and two timed tests, was piloted in 138 patients with type 2 and 3 SMA in an observational cross-sectional multi-centre study across the three national networks. Rasch analysis demonstrated very good fit of all 36 items to the construct of motor performance, good reliability with a high Person Separation Index PSI 0.98, logical and hierarchical scoring in 27/36 items and excellent targeting with minimal ceiling. The RHS differentiated between clinically different groups: SMA type, World Health Organisation (WHO) categories, ambulatory status, and SMA type combined with ambulatory status (all p < 0.001). Construct and concurrent validity was also confirmed with a strong significant positive correlation with the WHO motor milestones rs = 0.860, p < 0.001. We conclude that the RHS is a psychometrically sound and versatile clinical outcome assessment to test the broad range of physical abilities of patients with type 2 and 3 SMA. Further longitudinal testing of the scale with regards change in scores over 6 and 12 months are required prior to its adoption in clinical trials.

  1. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  2. Actin-Regulator Feedback Interactions during Endocytosis

    PubMed Central

    Wang, Xinxin; Galletta, Brian J.; Cooper, John A.; Carlsson, Anders E.

    2016-01-01

    Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others. PMID:27028652

  3. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

    PubMed Central

    Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

    2014-01-01

    Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

  4. Mammalian verprolin CR16 acts as a modulator of ITSN scaffold proteins association with actin.

    PubMed

    Kropyvko, Sergii; Gryaznova, Tetyana; Morderer, Dmytro; Rynditch, Alla

    2017-03-18

    Actin cytoskeleton rearrangements are required for normal cell functioning, and their deregulation leads to various pathologies. Members of two mammalian protein families - ITSNs (ITSN1 and ITSN2) and verprolins (WIP, CR16 and WIRE) are involved in Cdc42/N-WASP/Arp2/3 signaling pathway-mediated remodeling of the actin cytoskeleton. Recently we demonstrated that ITSNs interact with the actin-regulating protein WIP. Here, we show that other member of verprolin family, CR16, also forms complexes with ITSN1 and ITSN2 in human cell lines. The actin-binding protein CR16 modulates ITSN/β-actin association. Moreover, overexpressed CR16 promoted co-localization of ITSN1 with F-actin in MCF-7 breast cancer cells. Our data demonstrated that CR16 mRNA is expressed in glioblastoma and breast tumors. These findings provide the basis for further functional investigations of the ITSN/CR16 complex that may play an important role in actin remodeling and cellular invasion.

  5. Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration

    PubMed Central

    Hong, Ji-Ho; Kwak, Yongdo; Woo, Youngsik; Park, Cana; Lee, Seol-Ae; Lee, Haeryun; Park, Sung Jin; Suh, Yeongjun; Suh, Bo Kyoung; Goo, Bon Seong; Mun, Dong Jin; Sanada, Kamon; Nguyen, Minh Dang; Park, Sang Ki

    2016-01-01

    Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement. PMID:27546710

  6. ZNF185, an actin-cytoskeleton-associated growth inhibitory LIM protein in prostate cancer.

    PubMed

    Zhang, J-S; Gong, A; Young, C Y F

    2007-01-04

    We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.

  7. The chloroplast outer membrane protein CHUP1 interacts with actin and profilin.

    PubMed

    Schmidt von Braun, Serena; Schleiff, Enrico

    2008-04-01

    Chloroplasts accumulate in response to low light, whereas high light induces an actin-dependent avoidance movement. This is a long known process, but its molecular base is barely understood. Only recently first components of the blue light perceiving signal cascade initiating this process were described. Among these, a protein was identified by the analysis of a deletion mutant in the corresponding gene resulting in a chloroplast unusual positioning phenotype. The protein was termed CHUP1 and initial results suggested chloroplast localization. We demonstrate that the protein is indeed exclusively and directly targeted to the chloroplast surface. The analysis of the deletion mutant of CHUP1 using microarray analysis shows an influence on the expression of genes found to be up-regulated, but not on genes found to be down-regulated upon high light exposure in wild-type. Analyzing a putative role of CHUP1 as a linker between chloroplasts and the cytoskeleton, we demonstrate an interaction with actin, which is independent on the filamentation status of actin. Moreover, binding of CHUP1 to profilin -- an actin modifying protein -- could be shown and an enhancing effect of CHUP1 on the interaction of profilin to actin is demonstrated. Therefore, a role of CHUP1 in bridging chloroplasts to actin filaments and a regulatory function in actin polymerization can be discussed.

  8. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.

    PubMed

    Yeyati, Patricia L; Schiller, Rachel; Mali, Girish; Kasioulis, Ioannis; Kawamura, Akane; Adams, Ian R; Playfoot, Christopher; Gilbert, Nick; van Heyningen, Veronica; Wills, Jimi; von Kriegsheim, Alex; Finch, Andrew; Sakai, Juro; Schofield, Christopher J; Jackson, Ian J; Mill, Pleasantine

    2017-02-28

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.

  9. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

    PubMed Central

    Szabó, Eszter C.; Manguinhas, Rita; Fonseca, Rosalina

    2016-01-01

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture. PMID:27650071

  10. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability

    PubMed Central

    Schiller, Rachel; Kawamura, Akane; Gilbert, Nick; Wills, Jimi; von Kriegsheim, Alex

    2017-01-01

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive “actin gate” that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics. PMID:28246120

  11. Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth.

    PubMed

    Daher, Firas Bou; Geitmann, Anja

    2012-07-01

    An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.

  12. The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p.

    PubMed

    Yu, Xianwen; Cai, Mingjie

    2004-08-01

    Recent studies have suggested that the function of the large GTPase dynamin in endocytosis in mammalian cells may comprise a modulation of actin cytoskeleton. The role of dynamin in actin cytoskeleton organization in the yeast Saccharomyces cerevisiae has remained undefined. In this report, we found that one of the yeast dynamin-related proteins, Vps1p, is required for normal actin cytoskeleton organization. At both permissive and non-permissive temperatures, the vps1 mutants exhibited various degrees of phenotypes commonly associated with actin cytoskeleton defects: depolarized and aggregated actin structures, hypersensitivity to the actin cytoskeleton toxin latrunculin-A, randomized bud site selection and chitin deposition, and impaired efficiency in the internalization of membrane receptors. Over-expression of the GTPase mutants of vps1 also led to actin abnormalities. Consistent with these actin-related defects, Vps1p was found to interact physically, and partially co-localize, with the actin-regulatory protein Sla1p. The normal cellular localization of Sla1p required Vps1p and could be altered by over-expression of a region of Vps1p that was involved in the interaction with Sla1p. The same region also promoted mis-sorting of the vacuolar protein carboxypeptidase Y upon over-expression. These findings suggest that the functions of the dynamin-related protein Vps1p in actin cytoskeleton dynamics and vacuolar protein sorting are probably related to each other.

  13. Comparative transcriptome analysis reveals that lactose acts as an inducer and provides proper carbon sources for enhancing exopolysaccharide yield in the deep-sea bacterium Zunongwangia profunda SM-A87.

    PubMed

    Qin, Qi-Long; Li, Yi; Sun, Mei-Ling; Rong, Jin-Cheng; Liu, Sheng-Bo; Chen, Xiu-Lan; Su, Hai-Nan; Zhou, Bai-Cheng; Xie, Bin-Bin; Zhang, Yu-Zhong; Zhang, Xi-Ying

    2015-01-01

    Many marine bacteria secrete exopolysaccharides (EPSs) that have important ecological and physiological functions. Numerous nutritional and environmental factors influence bacterial EPS production. However, the regulatory mechanisms of EPS production are poorly understood. The deep-sea Bacteroidetes bacterium Zunongwangia profunda SM-A87 can produce high quantities of EPS, and its EPS production is enhanced significantly by lactose. Here, we studied the reasons behind the significant advantage that lactose has over other carbon sources in EPS production in SM-A87. RNA-seq technologies were used to study lactose-regulated genes in SM-A87. The expression level of genes within the EPS gene cluster was up-regulated when lactose was added. Supplement of lactose also influenced the expression of genes located outside the EPS gene cluster that are also involved in EPS biosynthesis. The major glycosyl components of SM-A87 EPS are mannose, glucose and galactose. Genomic metabolic pathway analyses showed that the EPS precursor GDP-mannose can be synthesized from glucose, while the precursor UDP-glucose must be synthesized from galactose. Lactose can provide glucose and galactose simultaneously and prevent glucose inhibition. Lactose can also greatly stimulate the growth of SM-A87. Taken together, lactose acts not only as an inducer but also as a carbohydrate source for EPS production. This research broadens our knowledge of the regulation of EPS production in marine bacteria.

  14. Comparative Transcriptome Analysis Reveals That Lactose Acts as an Inducer and Provides Proper Carbon Sources for Enhancing Exopolysaccharide Yield in the Deep-Sea Bacterium Zunongwangia profunda SM-A87

    PubMed Central

    Qin, Qi-Long; Li, Yi; Sun, Mei-Ling; Rong, Jin-Cheng; Liu, Sheng-Bo; Chen, Xiu-Lan; Su, Hai-Nan; Zhou, Bai-Cheng; Xie, Bin-Bin; Zhang, Yu-Zhong; Zhang, Xi-Ying

    2015-01-01

    Many marine bacteria secrete exopolysaccharides (EPSs) that have important ecological and physiological functions. Numerous nutritional and environmental factors influence bacterial EPS production. However, the regulatory mechanisms of EPS production are poorly understood. The deep-sea Bacteroidetes bacterium Zunongwangia profunda SM-A87 can produce high quantities of EPS, and its EPS production is enhanced significantly by lactose. Here, we studied the reasons behind the significant advantage that lactose has over other carbon sources in EPS production in SM-A87. RNA-seq technologies were used to study lactose-regulated genes in SM-A87. The expression level of genes within the EPS gene cluster was up-regulated when lactose was added. Supplement of lactose also influenced the expression of genes located outside the EPS gene cluster that are also involved in EPS biosynthesis. The major glycosyl components of SM-A87 EPS are mannose, glucose and galactose. Genomic metabolic pathway analyses showed that the EPS precursor GDP-mannose can be synthesized from glucose, while the precursor UDP-glucose must be synthesized from galactose. Lactose can provide glucose and galactose simultaneously and prevent glucose inhibition. Lactose can also greatly stimulate the growth of SM-A87. Taken together, lactose acts not only as an inducer but also as a carbohydrate source for EPS production. This research broadens our knowledge of the regulation of EPS production in marine bacteria. PMID:25679965

  15. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    DOEpatents

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  16. A Dichotomy in Cortical Actin and Chemotactic Actin Activity between Human Memory and Naive T Cells Contributes to Their Differential Susceptibility to HIV-1 Infection*

    PubMed Central

    Wang, Weifeng; Guo, Jia; Yu, Dongyang; Vorster, Paul J.; Chen, WanJun; Wu, Yuntao

    2012-01-01

    Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO+Actinhigh. In contrast, CD45RA T cells are phenotypically CD45RA+Actinlow. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets. PMID:22879601

  17. Growing an actin gel on spherical surfaces.

    PubMed Central

    Noireaux, V; Golsteyn, R M; Friederich, E; Prost, J; Antony, C; Louvard, D; Sykes, C

    2000-01-01

    Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails. PMID:10692348

  18. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  19. Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

    PubMed Central

    Miyata, H; Hotani, H

    1992-01-01

    Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes. Images PMID:1454846

  20. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins.

    PubMed

    Higashida, Chiharu; Kiuchi, Tai; Akiba, Yushi; Mizuno, Hiroaki; Maruoka, Masahiro; Narumiya, Shuh; Mizuno, Kensaku; Watanabe, Naoki

    2013-04-01

    Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca(2+) channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca(2+) nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

  1. Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

    PubMed Central

    Hsia, Lin-ting; Ashley, Neil; Ouaret, Djamila; Wang, Lai Mun; Wilding, Jennifer; Bodmer, Walter F.

    2016-01-01

    Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actinSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts. PMID:27036009

  2. Lifeact: a versatile marker to visualize F-actin.

    PubMed

    Riedl, Julia; Crevenna, Alvaro H; Kessenbrock, Kai; Yu, Jerry Haochen; Neukirchen, Dorothee; Bista, Michal; Bradke, Frank; Jenne, Dieter; Holak, Tad A; Werb, Zena; Sixt, Michael; Wedlich-Soldner, Roland

    2008-07-01

    Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

  3. Synthetic mimetics of actin-binding macrolides: rational design of actin-targeted drugs.

    PubMed

    Perrins, Richard D; Cecere, Giuseppe; Paterson, Ian; Marriott, Gerard

    2008-03-01

    Actin polymerization and dynamics are involved in a wide range of cellular processes such as cell division and migration of tumor cells. At sites of cell lysis, such as those occurring during a stroke or inflammatory lung diseases, actin is released into the serum where it polymerizes, leading to problems with clot dissolution and sputum viscosity. Therefore, drugs that target these actin-mediated processes may provide one mechanism to treat these conditions. Marine-organism-derived macrolides, such as reidispongiolide A, can bind to, sever, and inhibit polymerization of actin. Our studies show that the function of these complex macrolides resides in their tail region, whereas the head group stabilizes the actin-drug complex. Synthetic compounds derived from this tail region could therefore be used as a mimetic of the natural product, providing a range of designer compounds to treat actin-associated diseases or as probes to study actin polymerization.

  4. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  5. Signalling Pathways Controlling Cellular Actin Organization.

    PubMed

    Steffen, Anika; Stradal, Theresia E B; Rottner, Klemens

    2017-01-01

    The actin cytoskeleton is essential for morphogenesis and virtually all types of cell shape changes. Reorganization is per definition driven by continuous disassembly and re-assembly of actin filaments, controlled by major, ubiquitously operating machines. These are specifically employed by the cell to tune its activities in accordance with respective environmental conditions or to satisfy specific needs.Here we sketch some fundamental signalling pathways established to contribute to the reorganization of specific actin structures at the plasma membrane. Rho-family GTPases are at the core of these pathways, and dissection of their precise contributions to actin reorganization in different cell types and tissues will thus continue to improve our understanding of these important signalling nodes. Furthermore, we will draw your attention to the emerging theme of actin reorganization on intracellular membranes, its functional relation to Rho-GTPase signalling, and its relevance for the exciting phenomenon autophagy.

  6. Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes.

    PubMed

    Nicchia, Grazia Paola; Rossi, Andrea; Mola, Maria Grazia; Procino, Giuseppe; Frigeri, Antonio; Svelto, Maria

    2008-12-01

    Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.

  7. Apoptosis and apoptotic pathway in actinic prurigo by immunohistochemistry

    PubMed Central

    Cuevas-González, Juan-Carlos; García-Vázquez, Francisco-Javier; Rodríguez-Lobato, Erika; Farfán-Morales, José-Eduardo

    2016-01-01

    Background Actinic prurigo (AP) is an idiopathic photodermatosis, this entity requires exposure to UV-B and -A to develop lesions. Apoptosis is a physiological death program that can be initiated by a permanently active mechanism (extrinsic pathway) or irreparable damage (intrinsic pathway). Material and Methods Descriptive study, the sample size comprised 64 paraffin blocks of tissue with a diagnosis of AP. In H&E-stained slides, the diagnosis of AP was corroborated, and 1-µm-thick sections were processed for immunohistochemistry (IHC). A database was constructed with SPSS version 20, Inc., Chicago, IL, USA, and descriptive statistics were analyzed by X2 test and comparison of means. Results A total of 64 cases were processed, of which 40 (62.5%) were cheilitis AP and 24 (37.5%) were AP in the skin. Of the 40 cheilitis samples, 27 were positive for Bcl-2 and caspase 3 (67.5%), p53 was expressed in 30 (75%). Of the skin lesions,p53 and caspase 3 were expressed in 18 of 24 cases (75%), and 13 were positive for Bcl-2 (54%). Conclusions We propose that apoptosis is the last step in the type IV subtype a-b hypersensitivity response-activation of the intrinsic pathway indicates that external factors, such as UV-A and -B are the trigger. Key words:Apoptosis, actinic prurigo, cheilitis actinic prurigo. PMID:26615506

  8. Mechanics of biomimetic systems propelled by actin comet tails

    NASA Astrophysics Data System (ADS)

    Kang, Hyeran; Tambe, Dhananjay; Shenoy, Vivek; Tang, Jay

    2009-03-01

    The motility of intracellular bacterial pathogens such as Listeria monocytogenes is driven by filamentous actin comet tails in a variety of trajectories. Here, we present the in vitro study on the actin-based movements using spherical beads of different sizes coated with VCA protein, a partial domain of N-Wasp, in platelet extracts. Long term two-dimensional trajectories of the spherical beads motility show characteristic difference than those observed for bacteria, which have both elongated shape and asymmetric expression of the polymerization inducing enzyme. The trajectories also vary sensitively with the bead size and shape. These results provide a useful test to our new analytical model including the rotation of the bead relative to the tail.

  9. Cervical Spinal Cord Atrophy Profile in Adult SMN1-Linked SMA

    PubMed Central

    El Mendili, Mohamed-Mounir; Lenglet, Timothée; Stojkovic, Tanya; Behin, Anthony; Guimarães-Costa, Raquel; Salachas, François; Meininger, Vincent; Bruneteau, Gaelle; Le Forestier, Nadine; Laforêt, Pascal; Lehéricy, Stéphane; Benali, Habib; Pradat, Pierre-François

    2016-01-01

    Purpose The mechanisms underlying the topography of motor deficits in spinal muscular atrophy (SMA) remain unknown. We investigated the profile of spinal cord atrophy (SCA) in SMN1-linked SMA, and its correlation with the topography of muscle weakness. Materials and Methods Eighteen SMN1-linked SMA patients type III/V and 18 age/gender-matched healthy volunteers were included. Patients were scored on manual muscle testing and functional scales. Spinal cord was imaged using 3T MRI system. Radial distance (RD) and cord cross-sectional area (CSA) measurements in SMA patients were compared to those in controls and correlated with strength and disability scores. Results CSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10−5). There were no correlations between atrophy measurements, strength and disability scores. Conclusions Spinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in more complex mechanisms underlying weakness in these patients. PMID:27089520

  10. Review of Spinal Muscular Atrophy (SMA) for Prenatal and Pediatric Genetic Counselors.

    PubMed

    Carré, Amanda; Empey, Candice

    2016-02-01

    Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular condition with degeneration of the anterior horn cells in the spinal column. Five SMA subtypes exist with classification dependent upon the motor milestones achieved. Study of the SMN1 (survival motor neuron) and SMN2 genes as well as the concepts of the "2 + 0" carriers, gene conversion, de novo mutations and intragenic mutations allow for a better understanding of SMA. Detailing the carrier and diagnostic testing options further deepens the genetic counselor's knowledge of SMA. A review of care guidelines and research options is included as this information gives a patient a well-rounded view of SMA. Although SMA is most commonly associated with the SMN1 gene, a number of spinal muscular atrophies not caused by genetic changes in this gene may be included as differential diagnoses until confirmatory testing can be completed. SMA is a complex condition requiring a detailed knowledge on the genetic counselor's part in order to explain the disorder to the patient with clarity thus facilitating increased communication and decision making guidance with the patient.

  11. Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models.

    PubMed

    Osborne, Melissa; Gomez, Daniel; Feng, Zhihua; McEwen, Corissa; Beltran, Jose; Cirillo, Kim; El-Khodor, Bassem; Lin, Ming-Yi; Li, Yun; Knowlton, Wendy M; McKemy, David D; Bogdanik, Laurent; Butts-Dehm, Katherine; Martens, Kimberly; Davis, Crystal; Doty, Rosalinda; Wardwell, Keegan; Ghavami, Afshin; Kobayashi, Dione; Ko, Chien-Ping; Ramboz, Sylvie; Lutz, Cathleen

    2012-10-15

    A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

  12. Isolation of cDNA clones from within the spinal muscular atrophy (SMA) disease gene region

    SciTech Connect

    McLean, M.; Roy, N.; Tamai, K.

    1994-09-01

    Spinal muscular atrophy (SMA) is a recessive neuromuscular disease characterized by death of spinal cord {alpha} motor neurons, resulting in skeletal muscle atrophy. The critical SMA disease gene region on 5q13.1 contains families of microsatellite repeat sequences which exist at multiple subloci that are dispersed over a 100 to 200 kbp region. We have detected significant linkage disequilibrium between SMA type 1, the most severe form of the disorder, and two subloci of one such microsatellite, the CATT-1 family of microsatellites. Furthermore, a recombination event in a chromosome of an individual with SMA type 1 mapping between the members of two other extended microsatellite families, including CMS-1, has been observed. Combining this with previously reported recombinants refines the critical SMA region to approximately 300 kbp. P1 artificial chromosome (PAC), YAC and cosmid clones which possess both CMS-1 alleles which bracket this recombination event, as well as CATT-1 alleles showing linkage disequilibrium with SMA, have been used to probe cDNA libraries from human and other mammalian sources in search of genes within this interval; three of these cDNAs are currently being tested as candidates for the SMA gene.

  13. alpha-Smooth muscle actin immunoreactivity may change in nature in interlobular fibrosis of the pancreas in patients with congenital biliary dilatation.

    PubMed

    Matsubara, Kenro; Suda, Koichi; Suzuki, Fujihiko; Kumasaka, Toshio; Shiotsu, Hidetoshi; Miyano, Takeshi

    2004-07-01

    Pancreatic fibrosis in patients with congenital biliary dilatation (CBD) or choledochal cyst was studied to determine why biliary pancreatitis seldom progresses to chronic pancreatitis/more progressive state. Pancreatic collagenization in eight patients (three adults with pancreatoduodenectomy and five children with biopsy of the pancreas performed when excising the cyst) with CBD was evaluated histopathologically and immunohistochemically. Interlobular and periductal fibrosis with both collagen Type I and Type III immunoreactivities was found in six out of eight cases and in all four cases in which the pancreatic duct was included, respectively. The interlobular area was seldom immunoreactive for alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, but was usually positive for CD34, a human progenitor cell antigen. In contrast, the periductal area was usually immunoreactive for alpha-SMA, but usually negative for CD34 and immunopositive for bcl-2, indicating a continuously progressive state of fibrosis, in which 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature and lead to CD34-positive fibrosis or apoptosis. In conclusion, biliary pancreatitis is not likely to evolve into chronic pancreatitis/more progressive state because 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature.

  14. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  15. F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

    PubMed

    Pérez-Martínez, Manuel; Gordón-Alonso, Mónica; Cabrero, José Román; Barrero-Villar, Marta; Rey, Mercedes; Mittelbrunn, María; Lamana, Amalia; Morlino, Giulia; Calabia, Carmen; Yamazaki, Hiroyuki; Shirao, Tomoaki; Vázquez, Jesús; González-Amaro, Roberto; Veiga, Esteban; Sánchez-Madrid, Francisco

    2010-04-01

    The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

  16. NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1

    PubMed Central

    Zhang, Cheng; Zhang, Wen; Lu, Yi; Yan, Xiaoyi; Yan, Xiumin; Zhu, Xueliang; Liu, Wei; Yang, Yuehong; Zhou, Tianhua

    2016-01-01

    Emerging data indicate that actin dynamics is associated with ciliogenesis. However, the underlying mechanism remains unclear. Here we find that nuclear distribution gene C (NudC), an Hsp90 co-chaperone, is required for actin organization and dynamics. Depletion of NudC promotes cilia elongation and increases the percentage of ciliated cells. Further results show that NudC binds to and stabilizes cofilin 1, a key regulator of actin dynamics. Knockdown of cofilin 1 also facilitates ciliogenesis. Moreover, depletion of either NudC or cofilin 1 causes similar ciliary defects in zebrafish, including curved body, pericardial edema and defective left-right asymmetry. Ectopic expression of cofilin 1 significantly reverses the phenotypes induced by NudC depletion in both cultured cells and zebrafish. Thus, our data suggest that NudC regulates actin cytoskeleton and ciliogenesis by stabilizing cofilin 1. PMID:26704451

  17. Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell PhagosomesV⃞

    PubMed Central

    Yam, Patricia T.; Theriot, Julie A.

    2004-01-01

    We have found