Alarcon-Martinez, Luis; Yilmaz-Ozcan, Sinem; Yemisci, Muge; Schallek, Jesse; Kılıç, Kıvılcım; Can, Alp; Di Polo, Adriana; Dalkara, Turgay
2018-03-21
Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling. © 2018, Alarcon-Martinez et al.
Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord
2018-06-15
Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 ( col1a1 ). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFβ-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
[Expression of FAP and alpha-SMA during the incised wound healing in mice skin].
Gao, Yang; Peng, Xue; Jin, Zhan-Fen; Fu, Zhi-Jun
2009-12-01
OBJECTIVE To investigate the time-dependent expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin(alpha-SMA) during the incised wound healing of the skin in mice. The expression of FAP and alpha-SMA in incised wound of mice skin was detected by immunohistochemistry and Western blot. By immunohistochemistry, the expression of FAP and alpha-SMA in the normal skin and the skin 1 h after injury maintained at a very low level, but the positive cells expressing FAP and alpha-SMA started to elevate 6 h after injury and reached its peak on 5 d for FAP and on 3 d for alpha-SMA, then gradually decreased to the normal level on 14 d. The expression of FAP and alpha-SMA was observed throughout the wound healing stages 1 d after injuries by Western blot as well with a peak expression occurring on 5 d for FAP and on 3 d for alpha-SMA after injury. FAP may be a potentially useful marker for wound age determination and alpha-SMA may be used as an effective indicator for the mid- and late stage incised wound of mice skin. The combination use of FAP and alpha-SMA may be potentially effective indicators for wound age determination.
Sun, Yan; Yang, Zhan; Zheng, Bin; Zhang, Xin-Hua; Zhang, Man-Li; Zhao, Xue-Shan; Zhao, Hong-Ye; Suzuki, Toru; Wen, Jin-Kun
2017-09-01
Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor-like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-β1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-β1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3' untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular
Anggorowati, Nungki; Ratna Kurniasari, Chatarina; Damayanti, Karina; Cahyanti, Titik; Widodo, Irianiwati; Ghozali, Ahmad; Romi, Muhammad Mansyur; Sari, Dwi Cahyani Ratna; Arfian, Nur
2017-03-01
Background: Alpha-smooth muscle actin (α-SMA) is an isoform of actin, positive in myofibroblasts and is an epithelial to mesenchymal transition (EMT) marker. EMT is a process by which tumor cells develop to be more hostile and able to metastasize. Progression of tumor cells is always followed by cell composition and extracellular matrix component alteration. Increased α-SMA expression and collagen alteration may predict the progressivity of ovarian neoplasms. Objective: The aim of this research was to analyse the characteristic of α-SMA and collagen in tumor cells and stroma of ovarian neoplasms. In this study, PCNA (proliferating cell nuclear antigen) expression was also investigated. Methods: Thirty samples were collected including serous, mucinous, endometrioid, and clear cell subtypes. The expression of α-SMA and PCNA were calculated in cells and stroma of ovarian tumors. Collagen was detected using Sirius Red staining and presented as area fraction. Results: The overexpressions of α-SMA in tumor cells were only detected in serous and clear cell ovarian carcinoma. The histoscore of α-SMA was higher in malignant than in benign or borderline ovarian epithelial neoplasms (105.3±129.9 vs. 17.3±17.1, P=0.011; mean±SD). Oppositely, stromal α-SMA and collagen area fractions were higher in benign than in malignant tumors (27.2±6.6 vs 20.5±8.4, P=0.028; 31.0±5.6 vs. 23.7±6.4, P=0.04). The percentages of epithelial and stromal PCNA expressions were not significantly different between benign and malignant tumors. Conclusion: Tumor cells of serous and clear cell ovarian carcinoma exhibit mesenchymal characteristic as shown by α-SMA positive expression. This expression might indicate that these subtypes were more aggressive. This research showed that collagen and α-SMA area fractions in stroma were higher in benign than in malignant neoplasms. 10.22034/APJCP.2017.18.3.667
DOE Office of Scientific and Technical Information (OSTI.GOV)
Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.
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 solublemore » 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}.« less
Todros, T; Marzioni, D; Lorenzi, T; Piccoli, E; Capparuccia, L; Perugini, V; Cardaropoli, S; Romagnoli, R; Gesuita, R; Rolfo, A; Paulesu, L; Castellucci, M
2007-01-01
There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.
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-β1 (TGF-β1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β1 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. Copyright © 2015. Published by Elsevier Inc.
Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)
Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José
2013-01-01
Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980
Murphy-Marshman, Hannah; Quensel, Katherine; Shi-wen, Xu; Barnfield, Rebecca; Kelly, Jacalyn; Peidl, Alex; Stratton, Richard J.
2017-01-01
TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents. PMID:29049376
Renal alpha-smooth muscle actin: a new prognostic factor for lupus nephritis.
Makni, Kaouthar; Jarraya, Faïçal; Khabir, Abdelmajid; Hentati, Basma; Hmida, Mohamed Ben; Makni, Hafedh; Boudawara, Tahia; Jlidi, Rchid; Hachicha, Jamil; Ayadi, Hammadi
2009-08-01
Systemic lupus erythematosus (SLE) is the prototype of autoimmune disease where renal involvement is frequent and always severe. Histological prognostic factors proposed for lupus nephritis (LN) including the World Health Organization and International Society of Nephrology/Renal Pathology Society--Working Group on the Classification classifications, active (AI) and chronicity (CI) indices may not predict response to treatment. The aim of this study was to correlate alpha-smooth muscle actin (alpha-SMA) expression, an early marker of glomerular and interstitial response to injury, to AI and CI, renal scarring progression and response to treatment. Fifty-seven kidney biopsy specimens obtained from 32 patients suffering from LN were studied. Twenty patients with class IV LN at first biopsy were identified to study renal progression to chronic renal failure until the use of immunosuppressive treatment. Interstitial alpha-SMA (I-alpha-SMA) was correlated only with CI (P < 0.001) whereas mesangial alpha-SMA (M-alpha-SMA) was correlated with neither LN activity (P = 0.126) nor sclerosis (P = 0.297). Only I-alpha-SMA was correlated with renal failure (P = 0.01). We divided patients with class IV LN into progressors and non-progressors based on the slope of serum creatinine. At first biopsy, M-alpha-SMA and I-alpha-SMA, but not AI and CI, were correlated with renal failure progression (M-alpha-SMA, 9.7b1.1 vs 7.8b1.4, P = 0.004; and I-alpha-SMA, 9.3b1.1 vs 6.5b3.2, P = 0.011). The study data highlight that I-alpha-SMA immunostain in class IV LN patients was correlated with chronicity indices. Moreover, M-alpha-SMA and I-alpha-SMA expression in first biopsy predicted renal progression modality. alpha-SMA expression may therefore be a useful marker to predict renal prognosis in LN.
Ham, Sun Ah; Hwang, Jung Seok; Yoo, Taesik; Lee, Won Jin; Paek, Kyung Shin; Oh, Jae-Wook; Park, Chan-Kyu; Kim, Jin-Hoi; Do, Jung Tae; Kim, Jae-Hwan; Seo, Han Geuk
2015-12-01
The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. PPARδ plays pivotal roles in wound healing by promoting
Actin expression in some Platyhelminthe species.
Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R
1994-10-01
Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.
Zhou, C T; Zhang, S L; Ding, R Y; Hua, L; Zhong, W J
2000-06-01
To observe dynamically that Erigeron Breviscapus (Vant) Hand-Mazz (HEr) affects the expression of alpha-smooth muscle actin (alpha-SMA). To discuss the probable mechanism of obstructing leukoplakia carcinogenesis of this medicine. 120 golden hamsters were randomly divided into model group (48), HEr group (48) and control group (6). HEr was applied to obstruct the evolution of carcinogenesis of golden hamster cheek pouch. Immunohistochemistry was used to detect the expression level of alpha-SMA with cheek pouch specimen that besmears DMBA in 4-9 weeks. Results were compared with model group. Vessel density dyed with alpha-SMA continuously of HEr group was 65.76 significantly higher than that of model group 42.12 (P<0.001). High classification cases in HEr group were much more than model group when cases were divided into five groups as follow: 100%, 50%, 20%, 10%, 3% (P<0.01). HEr can raise the expression level of alpha-SMA exactly during the evolution of leukoplakia carcinogenesis of golden hamster, which shows that this medicine obstructs carcinogenesis by keeping the normal physiological function of vascular myoepithelial cell and integrity of vascular basement membrane.
Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.
Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai
2018-04-01
Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.
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.
2009-01-01
We previously found that vascular smooth muscle actin (SMA) is reduced in the brains of patients with late stage Alzheimer disease (AD) compared to brains of non-demented, neuropathologically normal subjects. To assess the pathogenetic significance and disease specificity of this finding, we studied 3 additional patient groups: non-demented subjects without significant AD type pathology (“Normal”, n = 20); non-demented subjects with frequent senile plaques at autopsy (“Preclinical AD”, n = 20); and subjects with frontotemporal dementia, (“FTD”, n = 10). The groups were matched for gender 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. SMA 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 non-demented subjects suggest that increased SMA expression might represent a physiologic response to neurodegeneration that could prevent or delay overt expression dementia in AD. PMID:19287310
From Cytoskeleton to Gene Expression: Actin in the Nucleus.
Viita, Tiina; Vartiainen, Maria K
2017-01-01
Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.
Changes in beta-actin mRNA expression in remodeling canine myocardium.
Carlyle, W C; Toher, C A; Vandervelde, J R; McDonald, K M; Homans, D C; Cohn, J N
1996-01-01
Beta-actin, a cytoskeletal protein important in the maintenance of cytoarchitecture, has long been thought to be expressed constitutively in myocardial tissue. As such, beta-actin mRNA has been used as a control gene in a wide range of experiments. However, we have uncovered consistent changes in beta-actin mRNA expression in canine myocardium remodeling as a result of insult to the left ventricle. The experimental canine models used were either DC shock damage to the left ventricle or volume overload resulting from severe mitral regurgitation. The remodeling process in both canine models is characterized by an increase in left ventricular mass. PCR amplification using primers designed to selectively amplify the 3' end and a portion of the 3' untranslated region of beta-actin mRNA resulted in the generation of a 297 base pair product predominant only in normal canine myocardium and a 472 base pair product that became increasingly prominent from 1 to 30 days after DC shock damage to the left ventricle and from 10 to 90 days after creation of mitral regurgitation. Northern analysis showed a three-fold increase in beta-actin mRNA after either DC shock or creation of mitral regurgitation. Western analysis revealed an early increase in beta-actin protein followed by an apparent decrease to below baseline levels. These observations suggest that changes in beta-actin mRNA expression accompany the structural alterations that occur in response to myocardial damage. Whether or not the changes in beta-actin mRNA expression play a role in mediating these structural alterations remains to be determined.
Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.
Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J
1996-01-01
In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008
Uchida, Yasuto; Uchida, Yasumi; Maezawa, Yoshiro; Maezawa, Yuko; Tabata, Tsuyoshi
2012-01-01
It was previously thought that arteriogenesis and venogenesis are induced not only by proliferation of vessel-resident smooth muscle cells (SMCs) and endothelial cells (ECs) but also by migration of their precursors. However, it is not well understood through what route(s) the precursors migrate into the existing vessels.We examined through what route or routes circulating mononuclear cells expressing β-actin (β-MNCs), which we identified in canine coronary vessels, migrate into coronary vessel walls and cause arteriogenesis and venogenesis at 1, 2, 4 and 8 weeks after induction of myocardial infarction.The following changes were observed: (1) The β-MNCs migrated via coronary microvessels to the interstitial space at one week; (2) β-MNCs traversed the adventitia into the media and settled in parallel with pre-existing smooth muscle cells (SMCs) in arterioles and arteries and lost β-actin and acquired α-smooth muscle actin (α-SMA) to become mature SMCs at 2-4 weeks; (3) at the same time, other β-MNCs migrated across the adventitia and media into the intima and settled in parallel with pre-existing endothelial cells (ECs) and lost β-actin, while acquiring CD(31), to become mature ECs, resulting in arteriogenesis; (4) Similarly, β-MNCs migrated into venular and venous walls and became SMCs or ECs, resulting in venogenesis.β-MNCs in the interstitial space expressed CD(34) but not other major vascular cell markers.β-MNCs, possibly a vascular progenitor, migrate not from the lumen but across the adventitia into the media or intima of coronary vessels and transit to SMCs or ECs, and participate in arteriogenesis and venogenesis in ischemic myocardium.
p53 AND MDM2 PROTEIN EXPRESSION IN ACTINIC CHEILITIS
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. PMID:19082401
p53 and MDM2 protein expression in actinic cheilitis.
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.
Development of wound healing models to study TGFβ3's effect on SMA.
Sriram, Sriniwas; Tran, Jennifer A; Guo, Xiaoqing; Hutcheon, Audrey E K; Kazlauskas, Andrius; Zieske, James D
2017-08-01
The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy. Copyright © 2017. Published by Elsevier Ltd.
Expression of matrix metalloproteinases 9 and 12 in actinic cheilitis
Poulopoulos, Athanasios K; Andreadis, Dimitrios; Markopoulos, Anastasios K
2013-01-01
AIM: To investigate the role of matrix-degrading metalloproteinases 9, 12 (MMPs), as mediators of functional connective tissue damage in actinic cheilitis. METHODS: Thirty five formalin-fixed, paraffin embedded specimens of actinic cheilitis, and twelve specimens of normal lower lip vermillion, which were obtained by the archives of the Department of Oral Medicine and Maxillofacial Pathology, were examined. From each block, 5 μm thick sections were cut and routinely stained with Hematoxylin and Eosin. Immunohistochemical studies were performed on 4-μm thick sections of formalin-fixed paraffin embedded actinic cheilitis lesions and of normal lower lip vermillion, for MMP-9 and MMP-12 in serial sections of our specimens. Appropriate positive and negative controls were performed to confirm the specificity of the staining reaction. MMP immunohistochemistry was evaluated using a semiquantitative immunoreactive score. RESULTS: Haematoxylin and eosin staining revealed in actinic cheilitis lesions atrophic stratified squamous cell epithelium, or focally and irregularly hyperplastic of variable thickness, in some areas was observed marked keratin production. Varying degrees of epithelial dysplasia were noticed with a wide spectrum of change within the same specimen. Characteristic was the appearance of chronic inflammatory infiltration, and a band of amorphous acellular, basophilic change like solar elastosis (elastin replacement of collagen). In normal lower lip specimens weak and scanty positive expression of MMP-9 and MMP-12 was observed. Anti-MMP-9 antibody showed a weak reaction, in actinic cheilitis lesions, focal in the elastotic material, in chronic inflammatory cells and mostly in macrophages and neutrophils. Strong and in some cases diffused immunohistochemical expression of MMP-12 was detected in actinic cheilitis lesions in the areas of the fragmented, distorted and thickened elastic fibers. MMP-12 was also expressed in chronic inflammatory cells and mostly
Leduc, Chloe; Sobilo, Lauren; Toumi, Hechmi; Mondon, Philippe; Lespessailles, Eric; Ossant, Fédéric; Kurfurst, Robin; Pichon, Chantal
2016-06-01
Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-β. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK). TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65±5%) and exposure to ultraviolet further increased this effect (47±3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed. TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. This study enlightens the role of TIEG-1 role in skin biology. Copyright © 2016 Elsevier B.V. All rights reserved.
Chaudhary, Minal; Gadbail, Amol Ramchandra; Vidhale, Gaurav; Mankar Gadbail, Mugdha P; Gondivkar, Shailesh M; Gawande, Madhuri; Patil, Swati
2012-09-01
The aim was to evaluate and compare the presence of myofibroblasts in oral squamous cell carcinoma (OSCC), verrucous carcinoma (VC), high-risk epithelial dysplasia (HRED), low-risk epithelial dysplasia (LRED), and normal oral mucosa (NOM). The study consisted of 37 OSCC, 15 VC, 15 HRED, 15 LRED and 15 NOM. α-smooth muscle actin (α-SMA) antibody was used to identify myofibroblasts. The α-SMA expression was not observed in NOM and LRED. The α-SMA was expressed in 97.29% of OSCC, 86.66% of VC, 46.66 % of HRED. The α-SMA expression was significantly higher in OSCC than VC (p = 0.023) and HRED (p < 0.000). The α-SMA expression was significantly higher in VC than HRED (p = 0.043). Myofibroblastic expression, as highlighted by α-SMA, is undetectable in NOM and LRED but increases as the disease progresses from potentially malignant disorders, as HRED to VC to invasive OSCC. Thus, proliferation of myofibroblasts may be used as a stromal marker of oral premalignancy and malignancy.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang, Xiang; Zhao, Fang
Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit themore » expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.« less
Role of Alpha-Smooth Muscle Actin and Fibroblast Activation Protein Alpha in Ovarian Neoplasms.
da Silva, Ana Carolinne; Jammal, Millena Prata; Etchebehere, Renata Margarida; Murta, Eddie Fernando Candido; Nomelini, Rosekeila Simões
2018-04-05
Studies show that tumor growth is not just determined by the presence of malignant cells, since interactions between cancer cells and stromal microenvironment have important impacts on the cancer growth and progression. Cancer-associated fibroblasts play a prominent role in this process. The aims of the study were to investigate 2 cancer-associated fibroblasts markers, alpha-smooth muscle actin (α-SMA), and fibroblast activation protein alpha (FAP) in the stromal microenvironment of benign and malignant ovarian epithelial neoplasms, and to relate their tissue expression with prognostic factors in ovarian cancer. α-SMA and FAP were evaluated by immunohistochemistry in malignant (n = 28) and benign (n = 28) ovarian neoplasms. Fisher's exact test was used with a significance level lower than 0.05. FAP immunostaining was stronger in ovarian cancer when compared to benign neoplasms (p = 0.0366). There was no significant difference in relation to α-SMA expression between malignant and benign ovarian neoplasms as well as prognostic factors. In ovarian cancer, FAP stainings 2/3 was significantly related to histological grades 2 and 3 (p = 0.0183). FAP immunostaining is more intense in malignant neoplasms than in benign ovarian neoplasms, as well as in moderately differentiated and undifferentiated ovarian carcinomas compared to well-differentiated neoplasms, thus indicating that it can be used as a marker of worse prognosis. © 2018 S. Karger AG, Basel.
Strong fascin expression promotes metastasis independent of its F-actin bundling activity.
Heinz, Lisa S; Muhs, Stefanie; Schiewek, Johanna; Grüb, Saskia; Nalaskowski, Marcus; Lin, Yuan-Na; Wikman, Harriet; Oliveira-Ferrer, Leticia; Lange, Tobias; Wellbrock, Jasmin; Konietzny, Anja; Mikhaylova, Marina; Windhorst, Sabine
2017-12-15
High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo , showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules ( MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.
Low-intensity infrared lasers alter actin gene expression in skin and muscle tissue
NASA Astrophysics Data System (ADS)
Fonseca, A. S.; Mencalha, A. L.; Campos, V. M. A.; Ferreira-Machado, S. C.; Peregrino, A. A. F.; Magalhães, L. A. G.; Geller, M.; Paoli, F.
2013-02-01
The biostimulative effect of low-intensity lasers is the basis for treatment of diseases in soft tissues. However, data about the influence of biostimulative lasers on gene expression are still scarce. The aim of this work was to evaluate the effects of low-intensity infrared lasers on the expression of actin mRNA in skin and muscle tissue. Skin and muscle tissue of Wistar rats was exposed to low-intensity infrared laser radiation at different fluences and frequencies. One and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis and evaluation of actin gene expression by quantitative polymerase chain reaction. The data obtained show that laser radiation alters the expression of actin mRNA differently in skin and muscle tissue of Wistar rats depending of the fluence, frequency and time after exposure. The results could be useful for laser dosimetry, as well as to justify the therapeutic protocols for treatment of diseases of skin and muscle tissues based on low-intensity infrared laser radiation.
Heterogeneity of keratin expression and actin distribution in benign and malignant mammary diseases.
Wada, T; Yasutomi, M; Yamada, K; Hashimura, K; Kunikata, M; Tanaka, T; Huang, J W; Mori, M
1991-01-01
Immunoreactivity of monoclonal anti-cytokeratin KL1, PKK1, K8.12 and anti-actin antibodies in 101 cases of diseased human breast lesions showed irregular keratin distribution in luminal cells of terminal ductal-lobular unit and basal layer cells of the interlobular and main duct. Actin staining was confined to myoepithelial cells. Benign lesions showed great heterogeneity in luminal cells of the terminal ductal-lobular units. Breast carcinoma showed a reduced staining for keratins, heterogeneity of keratin expression was found in solid tubular carcinoma, and actin was usually absent: however, papillo-ductal or comedo type had actin positive myoepithelial cells around carcinoma foci.
Tsapara, Anna; Luthert, Phillip; Greenwood, John; Hill, Caroline S.
2010-01-01
Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases. PMID:20089843
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
Lopes, Maria Luiza Diniz de Sousa; Nonaka, Cassiano Francisco Weege; Queiroz, Lélia Maria Guedes; de Souza, Lélia Batista; Miguel, Márcia Cristina da Costa; da Silveira, Éricka Janine Dantas
2016-09-01
Actinic cheilitis (AC) is a chronic inflammatory lesion that in some situations can turn into squamous cell carcinoma of the lip. The molecular mechanisms involved in this process are not yet completely understood. This study aimed to investigate the expression pattern of galectins in actinic cheilitis according to the histopathological grading. Immunoexpression of galectin-1, galectin-3, galectin-7, and galectin-9 was semiquantitatively analyzed in 65 cases of actinic cheilitis graded as low risk (n = 40) or high risk (n = 25) of malignant transformation. Association between the location of the galectins in the cellular compartments and histopathological grading was analyzed. Galectin-1 was mainly observed in the cell cytoplasm, and was elevated (score 3) in 60% of cases, regardless of the histopathological grade (P > 0.05). Galectin-3 expression was higher in high-risk group than in the low-risk group (P < 0.05), with a predominant expression in the cytoplasm and nucleus of low-risk (67.5%), and only in the cytoplasm of high-risk cases (60%) (P < 0.05). Galectin-7 expression did not show significant differences between low-risk and high-risk groups (P > 0.05). With respect to galectin-9, 89.2% of cases were positive, showing decrease in median of scores as there was an increase in histological grade (P < 0.001), with predominant expression in the nucleus and cytoplasm. This study is the first indication of galectins involvement in the pathogenesis and morphologic progression of actinic cheilitis, particularly galectin-3 and galectin-9. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.
Sharma, Varun; Ling, Tina W; Rewell, Sarah S; Hare, David L; Howells, David W; Kourakis, Angela; Wookey, Peter J
2012-11-01
In a rat model of stroke, the spatio-temporal distribution of α-smooth muscle actin-positive, (αSMA+) cells was investigated in the infarcted hemisphere (ipsilateral) and compared with the contralateral hemisphere. At day 3 postischemia, αSMA+ cells were concentrated in two main loci within the ipsilateral hemisphere (Area A) in the medial corpus callosum and (Area B) midway through the striatum adjacent to the lateral ventricle. By day 7 and further by day 14, fewer αSMA+ cells remained in Areas A and B but a steady increase in the peri-infarct was observed. αSMA+ cells also expressed glial acidic fibrillary protein [GFAP: αSMA+/GFAP+ (29%); αSMA+/GFAP- (71%) phenotypes] and feline leukemia virus C receptor 2 (FLVCR2), but not ED1(microglia) and established markers of pericytes normally located in vascular wall. αSMA+ cells were also located close to the subventricular zones (SVZ) adjacent to Areas A and B. In conclusion, αSMA+ cells have been identified in a spatial and temporal sequence from the SVZ, at intermediate loci and in the vicinity of the peri-infarct. It is hypothesized that novel populations of αSMA+ precursors of pericytes are born on the SVZ, migrate into the peri-infarct region and are incorporated into new vessels of the peri-infarct regions.
Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis
Ju, Wang; Zhihong, Yu; Zhiyou, Zhou; Qin, Huang; Dingding, Wang; Li, Sun; Baowei, Zhu; Xing, Wei; Ying, He; An, Hong
2012-01-01
The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist. PMID:22451267
Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar.
Bushman, F D; Crain, W R
1983-08-01
An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.
Yu, Sheng-ji; Qiu, Gui-xing; Burton, Yang; Sandra, Roth; Cari, Whyne; Albert, Yee
2005-12-15
To investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading. The expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.
Actin stress in cell reprogramming
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
Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.
Zepeta-Flores, Nahum; Valverde, Mahara; Lopez-Saavedra, Alejandro; Rojas, Emilio
2018-06-04
The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.
Lineaweaver, William; Bush, Katie; James, Kenneth
2015-01-01
Abstract The suppression of elements associated with wound contracture and unfavorable scarring is a potentially important strategy in clinical wound management. In this study, the presence of α smooth muscle actin (αSMA), a protein involved in wound contraction, was analyzed in a series of wounds in which bovine fetal collagen (BFC) acellular dermal matrix (PriMatrix) was used in staged split thickness skin graft procedures. The results obtained through histological and quantitative image analyses of incidental biopsies from these wounds demonstrated a suppression of αSMA in the wound regions occupied by assimilated BFC relative to increased levels of αSMA found in other areas of the wound. The αSMA levels found in assimilated BFC were similar to αSMA levels in uninjured human dermis. These findings suggest a mechanism by which application of BFC could decrease contraction of full thickness skin wounds. PMID:25695450
Lineaweaver, William; Bush, Katie; James, Kenneth
2015-06-01
The suppression of elements associated with wound contracture and unfavorable scarring is a potentially important strategy in clinical wound management. In this study, the presence of α smooth muscle actin (αSMA), a protein involved in wound contraction, was analyzed in a series of wounds in which bovine fetal collagen (BFC) acellular dermal matrix (PriMatrix) was used in staged split thickness skin graft procedures. The results obtained through histological and quantitative image analyses of incidental biopsies from these wounds demonstrated a suppression of αSMA in the wound regions occupied by assimilated BFC relative to increased levels of αSMA found in other areas of the wound. The αSMA levels found in assimilated BFC were similar to αSMA levels in uninjured human dermis. These findings suggest a mechanism by which application of BFC could decrease contraction of full thickness skin wounds.
Chen, Daxin; Shrivastava, Seema; Ma, Liang; Tham, El-Li; Abrahams, Joel; Coe, J David; Scott, Diane; Lechler, Robert I; McVey, John H; Dorling, Anthony
2012-01-01
The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.
Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R
2006-01-01
Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.
Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.
Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J
2003-01-01
Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the
Li, Ke Yue; Shi, Cheng Xian; Huang, Jian Zhao; Tang, Ke Li
2016-10-01
To investigate the effects of tetramethylpyrazine (TMP) on transforming growth factor-β1 (TGF- β1), α-smooth muscle actin (α-SMA), and neuronal regeneration related protein (P311) in benign biliary stricture fibroblasts of rabbit. An experimental study. Guizhou Medical University, Guiyang, Guizhou, China, from April to December 2015. Fibroblasts isolated from rabbits following benign biliary stricture were cultured and treated with different concentrations of TMP(0.08, 0.4, and 2.0 mg/ml). TMP-treated cells and non-treated control groups were incubated for 48-hours, and proliferation was assessed using the cell counting kit-8 assay. The mRNAexpressions of TGF-β1, α-SMA, and P311 were assessed by quantitative RT-PCR. Protein expressions of TGF-β1 and α-SMAwere investigated by Western blotting. Treatment with TMPsignificantly reduced the proliferation of benign biliary stricture fibroblasts, and significantly attenuated both the mRNAand protein expressions of TGF-β1, α-SMA, and P311 (p < 0.05) in a dose-dependent manner. TMPsignificantly reduced the proliferation of benign biliary stricture fibroblasts, and significantly downregulated the mRNA/protein expression of TGF-β1, α-SMA, and P311. Therefore, TMPmay be a therapeutic option for the prevention of benign biliary stricture.
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
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.
Kim, Bo Kwang; Kim, Kyoung Sun; Oh, Chul-Woong; Mykles, Donald L; Lee, Sung Gu; Kim, Hak Jun; Kim, Hyun-Woo
2009-06-01
Lobster muscles express a diverse array of myofibrillar protein isoforms. Three fiber types (fast, slow-twitch or S1, and slow-tonic or S2) differ qualitatively and quantitatively in myosin heavy and light chains, troponin-T, -I, and -C, paramyosin, and tropomyosin variants. However, little is known about the diversity of actin isoforms present in crustacean tissues. In this report we characterized cDNAs that encode twelve actin isoforms in the American lobster, Homarus americanus: eight from skeletal muscle (Ha-ActinSK1-8), one from heart (Ha-ActinHT1), and three cytoplasmic type actins from hepatopancreas (Ha-ActinCT1-3). All twelve cDNAs were products of distinct genes, as indicated by differences in the 3'-untranslated regions (UTRs). The open reading frames specified polypeptides 376 or 377 amino acids in length. Although key amino residues are conserved in the lobster actins, variations in nearby sequences may affect actin polymerization and/or interactions with other myofibrillar proteins. Quantitative reverse transcription-polymerase chain reaction showed muscle fiber type- and tissue-specific expression patterns. Ha-Actin-HT1 was expressed exclusively in heart (87% of the total; 12% of the total was Ha-ActinCT1). Ha-ActinCT1 was expressed in all tissues, while CT2 and CT3 were expressed only in hepatopancreas, with Ha-ActinCT2 as the major isoform (93% of the total). Ha-ActinSK1 and SK2 were the major isoforms (88% and 12% of the total, respectively) in the S1 fibers of crusher claw closer muscle. Fast fibers in the cutter claw closer and deep abdominal muscles differed in SK isoforms. Ha-ActinSK3, SK4, and SK5 were the major isoforms in cutter claw closer muscle (12%, 48%, and 37% of the total, respectively). Ha-ActinSK5 and SK8 were the major isoforms in deep abdominal flexor (31% and 65% of the total, respectively) and extensor (46% and 53% of the total, respectively) muscles, with SK6 and SK7 expressed at low levels. These data indicate that fast
Nagamine, E; Hirayama, K; Matsuda, K; Okamoto, M; Ohmachi, T; Kadosawa, T; Taniyama, H
2015-09-01
Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins. © The Author(s) 2015.
Increased IGF-1 in muscle modulates the phenotype of severe SMA mice
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
Comparative Life Cycle Assessment between Warm SMA and Conventional SMA
DOT National Transportation Integrated Search
2011-09-01
This report presents the comparative life cycle assessment (LCA) between warm stone mastic asphalt (SMA) and conventional : SMA. Specifically, the study evaluated and compared the life cycle environmental and economic performances of two mixtures: a ...
Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia
Kakugawa, Tomoyuki; Mukae, Hiroshi; Hayashi, Tomayoshi; Ishii, Hiroshi; Nakayama, Seiko; Sakamoto, Noriho; Yoshioka, Sumako; Sugiyama, Kanako; Mine, Mariko; Mizuta, Yohei; Kohno, Shigeru
2005-01-01
Background Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP). Methods We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining). Results The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP. Conclusion Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen. PMID:15955241
RickA Expression Is Not Sufficient to Promote Actin-Based Motility of Rickettsia raoultii
Balraj, Premanand; Karkouri, Khalid El; Vestris, Guy; Espinosa, Leon; Raoult, Didier; Renesto, Patricia
2008-01-01
Background Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG). The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species. Methodology/Principal Findings Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading) of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA. Conclusion/Significance These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsia genus. PMID:18612416
Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong
2010-01-01
In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.
Hensel, Niko; Stockbrügger, Inga; Rademacher, Sebastian; Broughton, Natasha; Brinkmann, Hella; Grothe, Claudia; Claus, Peter
2014-03-01
Rho-kinase (ROCK) as well as extracellular signal regulated kinase (ERK) control actin cytoskeletal organization thereby regulating dynamic changes of cellular morphology. In neurons, motility processes such as axonal guidance and neurite outgrowth demand a fine regulation of upstream pathways. Here we demonstrate a bilateral ROCK-ERK information flow in neurons. This process is shifted towards an unidirectional crosstalk in a model of the neurodegenerative disease Spinal Muscular Atrophy (SMA), ultimately leading to neurite outgrowth dysregulations. As both pathways are of therapeutic relevance for SMA, our results argue for a combinatorial ROCK/ERK-targeting as a future treatment strategy. Copyright © 2013 Elsevier Inc. All rights reserved.
The DcpS inhibitor RG3039 improves motor function in SMA mice
Van Meerbeke, James P.; Gibbs, Rebecca M.; Plasterer, Heather L.; Miao, Wenyan; Feng, Zhihua; Lin, Ming-Yi; Rucki, Agnieszka A.; Wee, Claribel D.; Xia, Bing; Sharma, Shefali; Jacques, Vincent; Li, Darrick K.; Pellizzoni, Livio; Rusche, James R.; Ko, Chien-Ping; Sumner, Charlotte J.
2013-01-01
Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 (SMN1) gene, retention of the survival motor neuron 2 (SMN2) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy. PMID:23727836
Aupperle, H; März, I; Thielebein, J; Schoon, H-A
2008-01-01
The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.
Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R
1999-04-01
The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.
Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn
2012-01-01
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
Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny
Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe
2014-01-01
The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333
Li, Gang; Liang, Wanqi; Zhang, Xiaoqing; Ren, Haiyun; Hu, Jianping; Bennett, Malcolm J; Zhang, Dabing
2014-07-15
The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.
Actin, actin-binding proteins, and actin-related proteins in the nucleus.
Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter
2016-04-01
Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.
Maselli, Andrew; Furukawa, Ruth; Thomson, Susanne A. M.; Davis, Richard C.; Fechheimer, Marcus
2003-01-01
Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. PMID:12912897
Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.
Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O
2013-02-01
The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.
Xie, Qiang; Wang, Hongbei; Heilman, Edward R; Walsh, Michael G; Haseeb, M A; Gupta, Raavi
2014-01-01
Enhancer of Zeste Homolog 2 (EZH2) is a polycomb group protein that has been shown to be involved in the progression of multiple human cancers including melanoma. The expression of EZH2 in normal skin and in pre-malignant and malignant cutaneous squamous cell carcinoma (SCC) has not been studied. We examined the expression of EZH2 in normal skin, actinic keratosis (AK), SCC in situ, well-differentiated (SCC-WD), moderately-differentiated (SCC-MD) and poorly-differentiated SCC (SCC-PD) to ascertain whether EZH2 expression differentiates these conditions. Immunohistochemical staining for EZH2 was performed on formalin-fixed paraffin-embedded biopsies and a tissue microarray containing normal skin, AK, SCC in situ, and SCC of different grades. In comparison to the normal skin, EZH2 expression in actinic keratosis was increased (p=0.03). Similarly, EZH2 expression in all of the neoplastic conditions studied (SCC in situ, SCC-WD, SCC-MD and SCC-PD) was greatly increased in comparison to both the normal skin and actinic keratosis (p≤0.001). EZH2 expression increases incrementally from normal skin to AK and further to SCC, suggesting a role for EZH2 in the progression and differentiation of SCC. EZH2 expression may be used as a diagnostic marker for differentiating SCC from AK or normal skin.
... forms of SMA still shorten life span, new approaches to ventilation and feeding have expanded what’s possible. ... 5-linked SMA in the last decade. Other approaches include less specific methods of helping motor neurons ...
The clinical landscape for SMA in a new therapeutic era
Talbot, K; Tizzano, E F
2017-01-01
Despite significant advances in basic research, the treatment of degenerative diseases of the nervous system remains one of the greatest challenges for translational medicine. The childhood onset motor neuron disorder spinal muscular atrophy (SMA) has been viewed as one of the more tractable targets for molecular therapy due to a detailed understanding of the molecular genetic basis of the disease. In SMA, inactivating mutations in the SMN1 gene can be partially compensated for by limited expression of SMN protein from a variable number of copies of the SMN2 gene, which provides both a molecular explanation for phenotypic severity and a target for therapy. The advent of the first tailored molecular therapy for SMA, based on modulating the splicing behaviour of the SMN2 gene provides, for the first time, a treatment which alters the natural history of motor neuron degeneration. Here we consider how this will change the landscape for diagnosis, clinical management and future therapeutic trials in SMA, as well as the implications for the molecular therapy of other neurological diseases. PMID:28644430
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. Copyright © 2011 Elsevier Inc. All rights reserved.
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. Copyright © 2016 Elsevier Inc. All rights reserved.
Liu, Yanfei; Wang, Zhenqing; Li, Hao; Sun, Min; Wang, Fangxin; Chen, Bingjie
2018-01-01
In this paper, a new shape memory alloy (SMA) hybrid basalt fibre reinforced polymer (BFRP) composite laminate was fabricated and a new surface modification method with both silane coupling agent KH550 and Al2O3 nanoparticles was conducted to enhance the interface performance. The mechanical performance of BFRP composite laminates with and without SMA fibres and the influence of SMA surface modification were studied in this paper. Different SMA fibre surface treatment methods, including etching with both H2SO4 and NaOH, modification with the silane coupling agent KH550 and new modification method with both KH550 and Al2O3 nanoparticles, were conducted to enhance the bonding between the SMA fibres and polymer matrix. Scanning electron microscopy (SEM) was used to observe the micromorphology of the SMA fibre surfaces exposed to different treatments and the damage morphology of composite laminates. The mechanical performance of the composites was investigated with tensile, three-point bending and low-velocity impact tests to study the influence of embedded SMA fibres and the different surface modifications of the SMA fibres. The results demonstrated that the embedded Ni-Ti SMA fibres can significantly enhance the mechanical performance of BFRP composite laminates. SMA fibres modified with both the silane coupling agent KH550 and Al2O3 nanoparticles illustrate the best mechanical performance among all samples. PMID:29300321
Zheng, Yu; Yun, Chenxia; Wang, Qihui; Smith, Wanli W; Leng, Jing
2015-02-01
The tree shrew (Tupaia belangeri) diverges from the primate order (Primates) and is classified as a separate taxonomic group of mammals - Scandentia. It has been suggested that the tree shrew can be used as an animal model for studying human diseases; however, the genomic sequence of the tree shrew is largely unidentified. In the present study, we reported the full-length cDNA sequence of the housekeeping gene, β-actin, in the tree shrew. The amino acid sequence of β-actin in the tree shrew was compared to that of humans and other species; a simple phylogenetic relationship was discovered. Quantitative polymerase chain reaction (qPCR) and western blot analysis further demonstrated that the expression profiles of β-actin, as a general conservative housekeeping gene, in the tree shrew were similar to those in humans, although the expression levels varied among different types of tissue in the tree shrew. Our data provide evidence that the tree shrew has a close phylogenetic association with humans. These findings further enhance the potential that the tree shrew, as a species, may be used as an animal model for studying human disorders.
Loss of cytokeratin 10 indicates malignant transformation in actinic cheilitis.
Garcia, Natália Galvão; Oliveira, Denise Tostes; Lauris, José Roberto Pereira; Domingues, Maria Aparecida Custódio; Minicucci, Eliana Maria; Soares, Cléverson Teixeira
2016-05-01
The aim of this study was to investigate the relationship the expression of cytokeratins (CK10 and CK13) and the cell proliferation index determined by Ki-67 of lip squamous cell carcinoma and actinic cheilitis with different degrees of dysplasia. Forty-five paraffin-embedded actinic cheilitis with and without dysplasia and 20 lip squamous cell carcinoma were analyzed by immunohistochemistry using anti-human anti-CK10, anti-CK13, and anti-Ki-67 antibodies. The majority of actinic cheilitis showed immunopositivity for CK10 and CK13 with decrease or loss of expression in dysplastic areas. In lip squamous cell carcinoma of the lip, heterogeneous expression of CK13 and immunonegativity for CK10 were observed. There was a statistically significant difference between CK10 expression in lip squamous cell carcinoma and in actinic cheilitis with or without dysplasia (p < 0.001). The cell proliferation index was higher in actinic cheilitis with dysplasia and lip squamous cell carcinoma than in actinic cheilitis without epithelial dysplasia. A significant correlation was found between the intensity of the epithelial dysplasia and the cell proliferation index (p < 0.001). These results provide evidence that there is a downregulation of CK10 expression in dysplastic areas of patients with actinic cheilitis and in those with lip squamous cell carcinoma (LSCC) and that the index of cell proliferation, determined by Ki-67, is directly correlated with the intensity of the epithelial dysplasia. Altogether, these results suggest that CK10 expression and the epithelial cell proliferation index can help to identify malignant transformation in the lip region.
Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R
2017-10-01
Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Fascin regulates nuclear actin during Drosophila oogenesis
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
Actinous enigma or enigmatic actin
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. PMID:28232879
Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*
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
Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.
Matthews, Paul R; Eastwood, Sharon L; Harrison, Paul J
2012-01-01
Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.
Actin-induced dimerization of palladin promotes actin-bundling
Vattepu, Ravi; Yadav, Rahul; Beck, Moriah R
2015-01-01
A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics. PMID:25307943
Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes
Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani
2010-01-01
The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during
Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.
Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani
2010-07-12
The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac
Rojas, Isolde G; Boza, Yadira V; Spencer, Maria Loreto; Flores, Maritza; Martínez, Alejandra
2012-01-01
Actinic cheilitis (AC) is characterized by epithelial and connective tissue alterations caused by ultraviolet sunlight overexposure known as photodamage. Fibroblasts have been linked to photodamage and tumor progression during skin carcinogenesis; however, their role in early lip carcinogenesis remains unknown. The aim of this study was to assess the density of fibroblasts in AC and normal lip (NL) samples and determine their association with markers of lip photodamage. Fibroblasts, mast cells, p53, COX-2, and elastin were detected in NL (n = 20) and AC (n = 28) biopsies using immunohistochemistry/histochemistry. Mast cell and fibroblast density and epithelial p53 and COX-2 expression scores were then obtained. Elastosis was scored 1-4 according to elastin fiber density and tortuosity. Fibroblasts, mast cells, p53, COX-2, and elastosis were increased in AC as compared to NL (P < 0.001). Multivariate analysis showed an association between fibroblast and mast cell density at the papillary and reticular areas of AC and NL (P < 0.05). Papillary fibroblast density was also associated with epithelial p53 and COX-2 expression (P < 0.05). Increased fibroblast density, both papillary and reticular, was found in the high elastosis group (scores 3-4) as compared to the low elastosis group (scores 1-2) (P < 0.01). Increased reticular mast cell density was detected only in the high elastosis group (P < 0.01). Fibroblasts are increased in AC, and they are associated with mast cell density, epithelial p53 and COX-2 expression, and actinic elastosis. Therefore, fibroblasts may contribute to lip photodamage and could be considered useful markers of early lip carcinogenesis. © 2011 John Wiley & Sons A/S.
Rab8 Regulates the Actin-based Movement of MelanosomesV⃞
Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne
2005-01-01
Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612
NASA Astrophysics Data System (ADS)
Hübler, M.; Gurka, M.; Schmeer, S.; Breuer, U. P.
2013-09-01
In this contribution we present a comprehensive theoretical and experimental description of an active shape memory alloy (SMA) fiber reinforced composite (FRP) hybrid structure. The major influences on actuation performance arising from variations in the design and manufacturing process are discussed, utilizing a new phenomenological model to describe the actuating SMA material. The different material properties for the activated, respective the unactivated, SMA as well as the influence of different loading conditions or pre-treatment of the material are taken into account in this model. To validate our material model we performed new actuation experiments with an exemplary SMA-FRP structure, which we compared to finite element (FE) simulation results. Our FE-model is based on a material model for the actuating SMA elements derived from experiments and data on the actual microscopic geometry of the hybrid composite. Therefore it is able to predict very precisely the actuation behavior of a typical FRP structure for industrial use cases: a thin walled CFRP sheet with SMA wires attached to the top for performing a bending motion with a maximum deflection of approx. 25% of its length.
Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi
2016-01-01
Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.
Are non-muscle actin isoforms functionally equivalent?
Simiczyjew, Aleksandra; Pietraszek-Gremplewicz, Katarzyna; Mazur, Antonina Joanna; Nowak, Dorota
2017-11-01
Actin is highly conserved and it is the most widespread protein in eukaryotic cells. One of the most important features of actin, which allows it to have many different functions, is its ability to polymerize and interact with many other proteins. Actins are the major constituent of the actin cytoskeleton, which is an important system that is involved in various aspects of cell function, including cell motility, structure, integrity, regulation of signal transduction and transcription. Six mammal actin isoforms are highly conserved and share common functions. Two of them, β and γ non-muscle actin isoforms, which differ only by four amino acids located at the N-terminus of the polypeptide chain, are required for survival and proper cell functioning. We also summarized data about actbl2, which is suggested to be a newly discovered isoactin. Here, we review the current knowledge about tissue-specific expression of the non-muscle actin isoforms and possible functional differences between them. We also discuss molecular tools, which in recent years have allowed for a better understanding of the role of these proteins in cell functioning.
Tobacco Arp3 is localized to actin-nucleating sites in vivo
Maisch, Jan; Fišerová, Jindřiška; Fischer, Lukáš; Nick, Peter
2009-01-01
The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP–ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)–FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP–ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP–ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization. PMID:19129161
SMA-MAP: a plasma protein panel for spinal muscular atrophy.
Kobayashi, Dione T; Shi, Jing; Stephen, Laurie; Ballard, Karri L; Dewey, Ruth; Mapes, James; Chung, Brett; McCarthy, Kathleen; Swoboda, Kathryn J; Crawford, Thomas O; Li, Rebecca; Plasterer, Thomas; Joyce, Cynthia; Chung, Wendy K; Kaufmann, Petra; Darras, Basil T; Finkel, Richard S; Sproule, Douglas M; Martens, William B; McDermott, Michael P; De Vivo, Darryl C; Walker, Michael G; Chen, Karen S
2013-01-01
Spinal Muscular Atrophy (SMA) presents challenges in (i) monitoring disease activity and predicting progression, (ii) designing trials that allow rapid assessment of candidate therapies, and (iii) understanding molecular causes and consequences of the disease. Validated biomarkers of SMA motor and non-motor function would offer utility in addressing these challenges. Our objectives were (i) to discover additional markers from the Biomarkers for SMA (BforSMA) study using an immunoassay platform, and (ii) to validate the putative biomarkers in an independent cohort of SMA patients collected from a multi-site natural history study (NHS). BforSMA study plasma samples (N = 129) were analyzed by immunoassay to identify new analytes correlating to SMA motor function. These immunoassays included the strongest candidate biomarkers identified previously by chromatography. We selected 35 biomarkers to validate in an independent cohort SMA type 1, 2, and 3 samples (N = 158) from an SMA NHS. The putative biomarkers were tested for association to multiple motor scales and to pulmonary function, neurophysiology, strength, and quality of life measures. We implemented a Tobit model to predict SMA motor function scores. 12 of the 35 putative SMA biomarkers were significantly associated (p<0.05) with motor function, with a 13(th) analyte being nearly significant. Several other analytes associated with non-motor SMA outcome measures. From these 35 biomarkers, 27 analytes were selected for inclusion in a commercial panel (SMA-MAP) for association with motor and other functional measures. Discovery and validation using independent cohorts yielded a set of SMA biomarkers significantly associated with motor function and other measures of SMA disease activity. A commercial SMA-MAP biomarker panel was generated for further testing in other SMA collections and interventional trials. Future work includes evaluating the panel in other neuromuscular diseases, for pharmacodynamic
Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.
Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun
2017-07-18
As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.
Tumour endothelial marker-1 is expressed in canine Haemangiopericytomas.
Fujii, Y; Tsuchiya, T; Morita, R; Kimura, M; Suzuki, K; Machida, N; Mitsumori, K; Shibutani, M
2013-01-01
The aim of this study was to characterize immunohistochemically 18 cases of canine haemangiopericytoma (CHP) using two new candidate markers for pericytes, tumour endothelial marker (TEM)-1 and new glue (NG)-2, as well as the conventional mesenchymal cellular markers, vimentin, α-smooth muscle actin (α-SMA), desmin and von Willebrand factor (vWF). Because pericytes may have the same origin as endothelial or smooth muscle cells or the same differentiation potential as myofibroblasts, 17 cases of leiomyosarcoma (LMS), 20 cases of haemangiosarcoma (HS) and three cases of myofibroblastic sarcoma (MFS) were also examined. Expression of TEM-1 by >10% of the neoplastic population was observed in 94.4% (17/18) of haemangiopericytomas, 23.5% (4/17) of LMSs, 30.0% (6/20) of HSs and 66.7% (2/3) of MFSs. NG-2 expression by >10% of the neoplastic population was observed in 16.7% (3/18) of haemangiopericytomas, 52.9% (9/17) of LMSs, 0% (0/20) of HSs and 33.3% (1/3) of MFSs. Vimentin was expressed by all of tumours. In haemangiopericytoma, the incidence of positive immunoreactivity in >10% of the neoplastic population was 5.6% (1/18) for both α-SMA and desmin and 0% (0/18) for vWF. Considering the phenotypic features of cells expressing TEM-1, CHPs are thought to originate from immature vascular mural cells sharing their phenotype with myofibroblasts. NG-2 expression may be a phenotype of smooth muscle cells rather than pericytes in dogs. Copyright © 2012 Elsevier Ltd. All rights reserved.
Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin
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
Liu, Li-Bo; Xue, Yi-Xue; Liu, Yun-Hui; Wang, Yi-Bao
2008-04-01
Bradykinin (BK) has been shown to open blood-tumor barrier (BTB) selectively and to increase permeability of the BTB transiently, but the mechanism is unclear. This study was performed to determine whether BK opens the BTB by affecting the tight junction (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin, and caludin-5 and cytoskeleton protein filamentous actin (F-actin). In rat brain glioma model and BTB model in vitro, we find that the protein expression levels of ZO-1, occludin, and claudin-5 are attenuated by BK induction. Immunohistochemistry and immunofluorescence assays show that the attenuated expression of ZO-1, occludin, and claudin-5 and F-actin is most obvious in the smaller tumor capillaries (<20 microm) after BK infusion, and there is no change in the larger tumor capillaries (>20 microm). The redistribution of ZO-1, occludin, and claudin-5 and rearrangement of F-actin in brain microvascular endothelial cells are observed at the same time. Meanwhile, Evans blue assay shows that the permeability of BTB increases after BK infusion. Transmission electron microscopy indicates that TJ is opened and that pinocytotic vesicular density is increased. Transendothelial electrical resistance (TEER) and horseradish peroxidase flux assays also reveal that TJ is opened by BK induction. In addition, radioimmunity and Western blot assay reveal a significant decrease in expression levels of cAMP and catalytic subunit of protien kinase A (PKAcs) of tumor tissue. This study demonstrates that the increase of BK-mediated BTB permeability is associated with the down-regulation of ZO-1, occludin, and claudin-5 and the rearrangement of F-actin and that cAMP/PKA signal transduction system might be involved in the modulating process.
Study of MDM2 and SUMO-1 expression in actinic cheilitis and lip cancer.
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.
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Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena
2011-01-01
The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077
Lundberg, M S; Sadhu, D N; Grumman, V E; Chilian, W M; Ramos, K S
1995-09-01
The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.
Control of actin-based motility through localized actin binding
Banigan, Edward J.; Lee, Kun-Chun; Liu, Andrea J.
2014-01-01
A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disk. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. PMID:24225232
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
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.
Souza Freitas, Valéria; de Andrade Santos, Pedro Paulo; de Almeida Freitas, Roseana; Pereira Pinto, Leão; de Souza, Lélia Batista
2011-09-01
The aim of this study was to evaluate mast cell (MC) density and migration and their association with matrix metalloproteinase (MMP) 9 expression in squamous cell carcinoma (SCC) and actinic cheilitis (AC). Tryptase, c-Kit, and MMP-9 expression was evaluated in 20 cases of SCC, 20 cases of AC, and 7 cases of normal lip (control samples) by immunohistochemistry techniques. Tryptase(+) and c-Kit(+) MC densities were significantly higher in SCCs than in ACs and control samples (P < .001). However, no significant difference was found when comparing tryptase(+) and c-Kit(+) MC densities between ACs and control samples (P values .185 and .516, respectively). MMP-9 was strongly expressed in SCCs and moderately expressed in ACs and control samples. A highly significant association was found between tryptase(+) MC density and the expression of MMP-9 (P < .001). The increase in MC density associated with the strong expression of MMP-9 may favor SCC progression. Copyright © 2011 Mosby, Inc. All rights reserved.
Cofilin-2 controls actin filament length in muscle sarcomeres
Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka
2014-01-01
SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779
A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin
Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...
2017-01-27
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 H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » 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. Furthermore, 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.« less
Hagstrom, J N; Couto, L B; Scallan, C; Burton, M; McCleland, M L; Fields, P A; Arruda, V R; Herzog, R W; High, K A
2000-04-15
Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)
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.
Model and Simulation of an SMA Enhanced Lip Seal
NASA Astrophysics Data System (ADS)
Qiao, Rui; Gao, Xiujie; Brinson, L. Catherine
2011-07-01
The feasibility of using SMA wires to improve the seal effectiveness has been studied experimentally and numerically. In this article, we present only the numerical study of simulating the thermo-mechanical behavior for an SMA enhanced lip seal, leaving the test setup and results in the experimental counterpart. A pseudo 3D SMA model, considering 1D SMA behavior in the major loading direction and elastic response in other directions, was used to capture the thermo-mechanical behavior of SMA wires. The model was then implemented into ABAQUS using the user-defined material subroutine to inherit most features of the commercial finite element package. Two-way shape memory effect was also considered since the SMA material exhibits strong two-way effects. An axisymmetric finite element model was constructed to simulate a seal mounting on a shaft and the sealing pressure was calculated for both the regular seal and the SMA enhanced seal. Finally, the result was qualitatively compared with the experimental observation.
de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W.
2016-01-01
Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function is still poorly understood. In this study we show that L. amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin and phosphorylated FAK when compared to non-infected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. PMID:27641840
Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?
Pinson, Benoît; Salin, Bénédicte; Daignan-Fornier, Bertrand
2006-01-01
Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle. PMID:16914523
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. © The Author(s) 2014.
Wang, Yuping; Cheng, Mingliang; Zhang, Baofang; Nie, Fei; Jiang, Hongmei
2013-01-01
Aim To investigate the effect of blueberry juice intake on rat liver fibrosis and its influence on hepatic antioxidant defense. Methods Rabbiteye blueberry was used to prepare fresh juice to feed rats by daily gastric gavage. Dan-shao-hua-xian capsule (DSHX) was used as a positive control for liver fibrosis protection. Liver fibrosis was induced in male Sprague-Dawley rats by subcutaneous injection of CCl4 and feeding a high-lipid/low-protein diet for 8 weeks. Hepatic fibrosis was evaluated by Masson staining. The expression of α-smooth muscle actin (α-SMA) and collagen III (Col III) were determined by immunohistochemical techniques. The activities of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenates were determined. Metallothionein (MT) expression was detected by real-time RT-PCR and immunohistochemical techniques. Results Blueberry juice consumption significantly attenuates CCl4-induced rat hepatic fibrosis, which was associated with elevated expression of metallothionein (MT), increased SOD activity, reduced oxidative stress, and decreased levels of α-SMA and Col III in the liver. Conclusion Our study suggests that dietary supplementation of blueberry juice can augment antioxidative capability of the liver presumably via stimulating MT expression and SOD activity, which in turn promotes HSC inactivation and thus decreases extracellular matrix collagen accumulation in the liver, and thereby alleviating hepatic fibrosis. PMID:23554912
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.
Kuznetsova, E V; Snarskaya, E S; Zavalishina, L E; Tkachenko, S B
Matrix metalloproteinases (MMPs) mediate the degradation of all types of collagens and other extracellular matrix components (elastin, proteoglycans, and laminin), their synthesis and accumulation play a key role in the hydrolysis of basement membrane. MMPs are involved in a wide range of proteolytic processes in the presence of different physiological and pathological changes, including inflammation, wound healing, angiogenesis, and carcinogenesis. to study the specific features of MMP-1 and MMP-9 expression in different stages of skin photoaging, in the foci of actinic keratosis and basal cell carcinoma by immunohistochemical examination. 12 samples of the healthy skin (6 samples of the eyelid skin with Glogau grade II photoaging; 6 ones of eyelid skin with Glogau grades III-IV photoaging) and biopsies from 8 foci of actinic keratosis and from 8 ones of basal cell carcinoma were examined. A positive reaction to MMPs was shown as different brown staining intensity in the cytoplasm of keratinocytes/tumor cells. MMP-1 and MMP-9 expression was recorded in 67% of the histological specimens of the Glogau grade III photoaged skin and in 100% of those of Glogau grade IV. In the foci of actinic keratosis, the expression of MMP-1 was observed in 62.5% of cases and that of MMP-9 was seen in 87.5%. In basal cell carcinoma, the expression of MMP-1 and MMP-9 was detected in all investigated samples. The immunomorphological findings are indicative of the important role of the level of MMP-1 and MMP-9 expression that is associated with the degree of progression of skin photoaging processes. Minimal MMP-1 and MMP-9 expression was recorded even in grades III-IV photoaging and in the foci of actinic keratosis. Intense MMP-1 and MMP-9 expression was detected in malignant skin epithelial neoplasms as different clinicomorphological types of basal cell carcinoma.
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.
Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K
2000-10-01
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.
Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.
Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J
2014-02-01
The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.
de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W
2017-03-01
Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. © 2016 John Wiley & Sons Ltd.
Steady-state nuclear actin levels are determined by export competent actin pool.
Skarp, Kari-Pekka; Huet, Guillaume; Vartiainen, Maria K
2013-10-01
A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. Copyright © 2013 Wiley Periodicals, Inc.
Rubenstein, Peter A; Wen, Kuo-Kuang
2005-10-01
Actin participates in many important biological processes. Currently, intensive investigation is being carried out in a number of laboratories concerning the function of actin in these processes and the molecular basis of its functions. We present a glimpse into four of these areas: actin-like proteins in bacterial cells, actin in the eukaryotic nucleus, the conformational plasticity of the actin filament, and finally, Arp2/3-dependent regulation of actin filament branching and creation of new filament barbed ends. IUBMB Life, 57: 683-687, 2005.
Actin-binding proteins sensitively mediate F-actin bundle stiffness
NASA Astrophysics Data System (ADS)
Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.
2006-09-01
Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.
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.
NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.
Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi
2016-02-01
The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.
Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L
2012-09-01
The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.
IFT88 influences chondrocyte actin organization and biomechanics.
Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M
2016-03-01
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. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). 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. IFT88(orpk) 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 IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) 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. 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. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen
2015-07-01
The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.
Variable stiffness mechanisms with SMA actuators
NASA Astrophysics Data System (ADS)
Siler, Damin J.; Demoret, Kimberly B. J.
1996-05-01
Variable stiffness is a new branch of smart structures development with several applications related to aircraft. Previous research indicates that temporarily reducing the stiffness of an airplane wing can decrease control actuator sizing and improve aeroelastic roll performance. Some smart materials like shape memory alloys (SMA) can change their material stiffness properties, but they tend to gain stiffness in their `power on' state. An alternative is to integrate mechanisms into a structure and change stiffness by altering boundary conditions and structural load paths. An innovative concept for an axial strut mechanism was discovered as part of research into variable stiffness. It employs SMA springs (specifically Ni-Ti) in a way that reduces overall stiffness when the SMA springs gain stiffness. A simplified mathematical model for static analysis was developed, and a 70% reduction in stiffness was obtained for a particular selection of springs. The small force capacity of commercially available SMA springs limits the practicality of this concept for large load applications. However, smart material technology is still immature, and future advances may permit development of a heavy-duty, variable stiffness strut that is small and light enough for use in aircraft structures.
Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B
2012-05-01
Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.
Kim, Sokho; Kwon, Jungkee
2015-01-01
The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs. PMID:25640761
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp
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-143more » 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
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.
PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility
Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.
2014-01-01
Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693
Garagounis, Constantine; Kostaki, Kalliopi-Ioanna; Hawkins, Tim J; Cummins, Ian; Fricker, Mark D; Hussey, Patrick J; Hetherington, Alistair M; Sweetlove, Lee J
2017-02-01
Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Kim, Jae-Hun; Lee, Jong-Min; Jo, Hang Joon; Kim, Sook Hui; Lee, Jung Hee; Kim, Sung Tae; Seo, Sang Won; Cox, Robert W; Na, Duk L; Kim, Sun I; Saad, Ziad S
2010-02-01
Noninvasive parcellation of the human cerebral cortex is an important goal for understanding and examining brain functions. Recently, the patterns of anatomical connections using diffusion tensor imaging (DTI) have been used to parcellate brain regions. Here, we present a noninvasive parcellation approach that uses "functional fingerprints" obtained by correlation measures on resting state functional magnetic resonance imaging (fMRI) data to parcellate brain regions. In other terms, brain regions are parcellated based on the similarity of their connection--as reflected by correlation during resting state--to the whole brain. The proposed method was used to parcellate the medial frontal cortex (MFC) into supplementary motor areas (SMA) and pre-SMA subregions. In agreement with anatomical landmark-based parcellation, we find that functional fingerprint clustering of the MFC results in anterior and posterior clusters. The probabilistic maps from 12 subjects showed that the anterior cluster is mainly located rostral to the vertical commissure anterior (VCA) line, whereas the posterior cluster is mainly located caudal to VCA line, suggesting the homologues of pre-SMA and SMA. The functional connections from the putative pre-SMA cluster were connected to brain regions which are responsible for complex/cognitive motor control, whereas those from the putative SMA cluster were connected to brain regions which are related to the simple motor control. These findings demonstrate the feasibility of the functional connectivity-based parcellation of the human cerebral cortex using resting state fMRI. Copyright (c) 2009 Elsevier Inc. All rights reserved.
Calcium-mediated actin reset (CaAR) mediates acute cell adaptations.
Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland
2016-12-06
Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.
Visa, Neus; Percipalle, Piergiorgio
2010-01-01
Actin participates in several essential processes in the cell nucleus. Even though the presence of actin in the nucleus was proposed more than 30 years ago, nuclear processes that require actin have been only recently identified. Actin is part of chromatin remodeling complexes; it is associated with the transcription machineries; it becomes incorporated into newly synthesized ribonucleoproteins; and it influences long-range chromatin organization. As in the cytoplasm, nuclear actin works in conjunction with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and other nuclear components. PMID:20452941
Nazari, Fatemeh; Parham, Abbas; Maleki, Adham Fani
2015-01-01
Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.
Yoshiba, Nagako; Yoshiba, Kunihiko; Ohkura, Naoto; Takei, Erika; Edanami, Naoki; Oda, Youhei; Hosoya, Akihiro; Nakamura, Hiroaki; Okiji, Takashi
2015-01-01
Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing. PMID:25805839
Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization
Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio
2009-01-01
We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923
Induction of HoxB transcription by retinoic acid requires actin polymerization.
Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco
2009-08-01
We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.
How actin network dynamics control the onset of actin-based motility
Kawska, Agnieszka; Carvalho, Kévin; Manzi, John; Boujemaa-Paterski, Rajaa; Blanchoin, Laurent; Martiel, Jean-Louis; Sykes, Cécile
2012-01-01
Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or “gels” are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including “primer” contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility. PMID:22908255
Effects of increased Kindlin-2 expression in bladder cancer stromal fibroblasts.
Wu, Jitao; Yu, Cuicui; Cai, Li; Lu, Youyi; Jiang, Lei; Liu, Chu; Li, Yongwei; Feng, Fan; Gao, Zhenli; Zhu, Zhe; Yu, Shengqiang; Yuan, Hejia; Cui, Yuanshan
2017-08-01
Kindlin-2 is a focal adhesion protein highly expressed in bladder cancer stromal fibroblasts. We investigated the prognostic significance of Kindlin-2 in bladder cancer stromal fibroblasts and evaluated the effects of Kindlin-2 on the malignant behaviors of tumor cells. Immunohistochemical staining of 203 paraffin-embedded bladder cancer tissues showed that Kindlin-2 expression correlated with advanced stage, high grade, and relapse of bladder cancer. Kaplan-Meier survival analysis demonstrated that patients exhibiting high Kindlin-2 expression had shorter survival times than those with low Kindlin-2 expression ( p < 0.01). Multivariate analysis revealed that high Kindlin-2 expression leads to poor prognosis in bladder cancer. Using cancer-associated fibroblasts (CAFs) isolated from human bladder cancer tissue, we observed that Kindlin-2 knockdown decreased CAFs activation, resulting in decreased expression of α-smooth muscle actin (α-SMA) and the extracellular matrix protein fibronectin. Kindlin-2 suppression also reduced CAF-induced bladder cancer cell migration and invasion. Moreover, we found that Kindlin-2 activates CAFs and promotes the invasiveness of bladder cancer cells by stimulating TGF-β-induced epithelial-mesenchymal transition. These results support targeting Kindlin-2 and the corresponding activated CAFs in bladder cancer therapy.
Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein
Roof, Dorothy J.; Hayes, Annmarie; Adamian, Michael; Chishti, Athar H.; Li, Tiansen
1997-01-01
Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments. PMID:9245787
Resemblance of actin-binding protein/actin gels to covalently crosslinked networks
NASA Astrophysics Data System (ADS)
Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.
1990-05-01
THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro
2013-08-02
Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: 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-β4more » (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.« less
Analysis of MUC4 expression in human pancreatic cancer xenografts in immunodeficient mice.
Ansari, Daniel; Bauden, Monika P; Sasor, Agata; Gundewar, Chinmay; Andersson, Roland
2014-08-01
Mucin 4 (MUC4) is a cell surface glycoprotein that is overexpressed in most pancreatic tumors. The aim of the present study was to characterize MUC4 expression in experimental pancreatic cancer in order to clarify the correlation between MUC4 and pancreatic cancer histology in vivo. Pancreatic xenograft tumors were generated in immunodeficient mice (n=15) by subcutaneous injection of MUC4(+) human pancreatic cancer cell lines Capan-1, HPAF-II or CD18/HPAF. MUC4 immunoreactivity was compared between the cancer models. Alpha-smooth muscle actin (α-SMA) was used to identify cancer-associated fibroblasts and the amount of collagen fibers was quantified with sirius red. Tumor incidence was 100%. Tumor size showed no difference across groups (p=0.796). The median MUC4 count was highest in Capan-1 tumors (p=0.002). α-SMA and collagen extent were also highest in Capan-1 tumors (p=0.018). The Capan-1 xenograft model could serve as a valuable resource to test new therapeutic strategies targeting MUC4 in pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Tensile and fatigue behavior of polymer composites reinforced with superelastic SMA strands
NASA Astrophysics Data System (ADS)
Daghash, Sherif M.; Ozbulut, Osman E.
2018-06-01
This study explores the use of superelastic shape memory alloy (SMA) strands, which consist of seven individual small-diameter wires, in an epoxy matrix and characterizes the tensile and fatigue responses of the developed SMA/epoxy composites. Using a vacuum assisted hand lay-up technique, twelve SMA fiber reinforced polymer (FRP) specimens were fabricated. The developed SMA-FRP composites had a fiber volume ratio of 50%. Tensile response of SMA-FRP specimens were characterized under both monotonic loading and increasing amplitude loading and unloading cycles. The degradation in superelastic properties of the developed SMA-FRP composites during fatigue loading at different strain amplitudes was investigated. The effect of loading rate on the fatigue response of SMA-FRP composites was also explored. In addition, fractured specimens were examined using the scanning electron microscopy (SEM) technique to study the failure mechanisms of the tested specimens. A good interfacial bonding between the SMA strands and epoxy matrix was observed. The developed SMA-FRP composites exhibited good superelastic behavior at different strain amplitudes up to at least 800 cycle after which significant degradation occurred.
Jiang, Chang-Jie; Weeds, Alan G.; Khan, Safina; Hussey, Patrick J.
1997-01-01
Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond. PMID:9275236
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.
Calcium-mediated actin reset (CaAR) mediates acute cell adaptations
Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland
2016-01-01
Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress. DOI: http://dx.doi.org/10.7554/eLife.19850.001 PMID:27919320
Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction
Palani, Saravanan; Sommese, Ruth; Kamnev, Anton; Hatano, Tomoyuki; Sivaramakrishnan, Sivaraj
2017-01-01
Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially. PMID:28655757
Changes in actin dynamics are involved in salicylic acid signaling pathway.
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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M
2016-01-25
The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.
... kids of the same age or have trouble lifting things. Kids with SMA can develop scoliosis (a ... Nervous System Your Muscles Wheelchairs Scoliosis Steven's Story: Power Player Kyphosis Muscular Dystrophy Spinal Muscular Atrophy: Steven's ...
Synthetic peptides that cause F-actin bundling and block actin depolymerization
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.
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.
Overview of engineering activities at the SMA
NASA Astrophysics Data System (ADS)
Christensen, R. D.; Kubo, D. Y.; Rao, Ramprasad
2008-07-01
The Submillmeter Array (SMA) consists of 8 6-meter telescopes on the summit of Mauna Kea. The array has been designed to operate from the summit of Mauna Kea and from 3 remote facilities: Hilo, Hawaii, Cambridge, Massachusetts and Taipei, Taiwan. The SMA provides high-resolution scientific observations in most of the major atmospheric windows from 180 to 700 GHz. Each telescope can house up to 8 receivers in a single cryostat and can operate with one or two receiver bands simultaneously. The array being a fully operational observatory, the demand for science time is extremely high. As a result specific time frames have been set-aside during both the day and night for engineering activities. This ensures that the proper amount of time can be spent on maintaining existing equipment or upgrading the system to provide high quality scientific output during nighttime observations. This paper describes the methods employed at the SMA to optimize engineering development of the telescopes and systems such that the time available for scientific observations is not compromised. It will also examine some of the tools used to monitor the SMA during engineering and science observations both at the site and remote facilities.
Ion-dependent Polymerization Differences between Mammalian β- and γ-Nonmuscle Actin Isoforms*
Bergeron, Sarah E.; Zhu, Mei; Thiem, Suzanne M.; Friderici, Karen H.; Rubenstein, Peter A.
2010-01-01
β- and γ-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. β-Actin contains an Asp1-Asp2-Asp3 and Val10 whereas γ-actin has a Glu1-Glu2-Glu3 and Ile10. Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20–25% insect actin. Surprisingly, Ca-γ-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than β-actin. In the Mg-form, this difference between the two is much smaller. Ca-γ-actin depolymerizes half as fast as does β-actin. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing β-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with γ-actin. Phosphate release during treadmilling is twice as fast with β- as with γ-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of γ-actin may cause a selective energy barrier relative to β-actin that retards the equilibration between G- and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the γ-actin-rich cochlear hair cell stereocilium where local mm calcium concentrations may exist. PMID:20308063
Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk
2017-05-01
Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.
Microscopy basics and the study of actin-actin-binding protein interactions.
Thomasson, Maggie S; Macnaughtan, Megan A
2013-12-15
Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.
RAI14 (retinoic acid induced protein 14) is an F-actin regulator
Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-ho; Cheng, C. Yan
2013-01-01
RAI14 (retinoic acid induced protein 14) is an actin-binding protein first identified in the liver. In the testis, RAI14 is expressed by both Sertoli and germ cells in the seminiferous epithelium. Besides binding to actin in the testis, RAI14 is also a binding protein for palladin, an actin cross-linking and bundling protein. A recent report has shown that RAI14 displays stage-specific and spatiotemporal expression at the ES [ectoplasmic specialization, a testis-specific filamentous (F)-actin-rich adherens junction] in the seminiferous epithelium of adult rat testes during the epithelial cycle of spermatogenesis, illustrating its likely involvement in F-actin organization at the ES. Functional studies in which RAI14 was knocked down by RNAi in Sertoli cells in vitro and also in testicular cells in vivo have illustrated its role in conferring the integrity of actin filament bundles at the ES, perturbing the Sertoli cell tight junction (TJ)-pemeability barrier function in vitro, and also spermatid polarity and adhesion in vivo, thereby regulating spermatid transport at spermiation. Herein, we critically evaluate these earlier findings and also provide a likely hypothetic model based on the functional role of RAI14 at the ES, and how RAI14 is working with palladin and other actin regulatory proteins in the testis to regulate the transport of (1) spermatids and (2) preleptotene spermatocytes across the seminiferous epithelium and the blood-testis barrier (BTB), respectively, during spermatogenesis. This model should serve as a framework upon which functional experiments can be designed to better understand the biology of RAI14 and other actin-binding and regulatory proteins in the testis. PMID:23885305
Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast
NASA Astrophysics Data System (ADS)
Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios
2010-03-01
The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09
Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells
Hepler, Peter K.; Bezanilla, Magdalena
2009-01-01
Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells. PMID:19478943
Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.
Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M; Ikonen, Teemu P; Wohlhoefler, Michael; Schmoller, Kurt M; Bausch, Andreas R; Joel, Peteranne; Trybus, Kathleen M; Noegel, Angelika A; Schleicher, Michael; Huber, Robert; Holak, Tad A
2011-12-06
The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.
Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly
Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M.; Ikonen, Teemu P.; Wohlhoefler, Michael; Schmoller, Kurt M.; Bausch, Andreas R.; Joel, Peteranne; Trybus, Kathleen M.; Noegel, Angelika A.; Schleicher, Michael; Huber, Robert; Holak, Tad A.
2011-01-01
The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire–actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire–actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott–Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire–actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire–actin module. In addition, we find that preformed, isolated Spire–actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs—even a single WH2 repeat—sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem. PMID:22106272
DOE Office of Scientific and Technical Information (OSTI.GOV)
Choi, Hoo-Kyun; Pokharel, Yuba Raj; Lim, Sung Chul
2009-11-01
Coenzyme Q10 (CoQ10), an endogenous antioxidant, is important in oxidative phosphorylation in mitochondria. It has anti-diabetic and anti-cardiovascular disease effects, but its ability to protect against liver fibrosis has not been studied. Here, we assessed the ability of solubilized CoQ10 to improve dimethylnitrosamine (DMN)-induced liver fibrogenesis in mice. DMN treatments for 3 weeks produced a marked liver fibrosis as assessed by histopathological examination and tissue 4-hydroxyproline content. Solubilized CoQ10 (10 and 30 mg/kg) significantly inhibited both the increases in fibrosis score and 4-hydroxyproline content induced by DMN. Reverse transcription-polymerase chain reaction and Western blot analyses revealed that solubilized CoQ10 inhibitedmore » increases in the transforming growth factor-beta1 (TGF-beta1) mRNA and alpha-smooth muscle actin (alpha-SMA) protein by DMN. Interestingly, hepatic glutamate-cysteine ligase (GCL) and glutathione S-transferase A2 (GSTA2) were up-regulated in mice treated with CoQ10. Solubilized CoQ10 also up-regulated antioxidant enzymes such as catalytic subunits of GCL and GSTA2 via activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) in H4IIE hepatoma cells. Moreover, CoQ10's inhibition of alpha-SMA and TGF-beta1 expressions disappeared in Nrf2-null MEF cells. In contrast, Nrf2 overexpression significantly decreased the basal expression levels of alpha-SMA and TGF-beta1 in Nrf2-null MEF cells. These results demonstrated that solubilized CoQ10 inhibited DMN-induced liver fibrosis through suppression of TGF-beta1 expression via Nrf2/ARE activation.« less
Fabrication and testing of SMA composite beam with shape control
NASA Astrophysics Data System (ADS)
Noolvi, Basavaraj; S, Raja; Nagaraj, Shanmukha; Mudradi, Varada Raj
2017-07-01
Smart materials are the advanced materials that have characteristics of sensing and actuation in response to the external stimuli like pressure, heat or electric charge etc. These materials can be integrated in to any structure to make it smart. From the different types of smart materials available, Shape Memory Alloy (SMA) is found to be more useful in designing new applications, which can offer more actuating speed, reduce the overall weight of the structure. The unique property of SMA is the ability to remember and recover from large strains of upto 8% without permanent deformation. Embedding the SMA wire/sheet in fiber-epoxy/flexible resin systems has many potential applications in Aerospace, Automobile, Medical, Robotics and various other fields. In this work the design, fabrication, and testing of smart SMA composite beam has been carried out. Two types of epoxy based resin systems namely LY 5210 resin system and EPOLAM 2063 resin system are used in fabricating the SMA composite specimens. An appropriate mould is designed and fabricated to retain the pre-strain of SMA wire during high temperature post curing of composite specimens. The specimens are fabricated using vacuum bag technique.
Energy-dissipating and self-repairing SMA-ECC composite material system
NASA Astrophysics Data System (ADS)
Li, Xiaopeng; Li, Mo; Song, Gangbing
2015-02-01
Structural component ductility and energy dissipation capacity are crucial factors for achieving reinforced concrete structures more resistant to dynamic loading such as earthquakes. Furthermore, limiting post-event residual damage and deformation allows for immediate re-operation or minimal repairs. These desirable characteristics for structural ‘resilience’, however, present significant challenges due to the brittle nature of concrete, its deformation incompatibility with ductile steel, and the plastic yielding of steel reinforcement. Here, we developed a new composite material system that integrates the unique ductile feature of engineered cementitious composites (ECC) with superelastic shape memory alloy (SMA). In contrast to steel reinforced concrete (RC) and SMA reinforced concrete (SMA-RC), the SMA-ECC beams studied in this research exhibited extraordinary energy dissipation capacity, minimal residual deformation, and full self-recovery of damage under cyclic flexural loading. We found that the tensile strain capacity of ECC, tailored up to 5.5% in this study, allows it to work compatibly with superelastic SMA. Furthermore, the distributed microcracking damage mechanism in ECC is critical for sufficient and reliable recovery of damage upon unloading. This research demonstrates the potential of SMA-ECC for improving resilience of concrete structures under extreme hazard events.
Numerical tool for SMA material simulation: application to composite structure design
NASA Astrophysics Data System (ADS)
Chemisky, Yves; Duval, Arnaud; Piotrowski, Boris; Ben Zineb, Tarak; Tahiri, Vanessa; Patoor, Etienne
2009-10-01
Composite materials based on shape memory alloys (SMA) have received growing attention over these last few years. In this paper, two particular morphologies of composites are studied. The first one is an SMA/elastomer composite in which a snake-like wire NiTi SMA is embedded into an elastomer ribbon. The second one is a commercial Ni47Ti44Nb9 which presents elastic-plastic inclusions in an NiTi SMA matrix. In both cases, the design of such composites required the development of an SMA design tool, based on a macroscopic 3D constitutive law for NiTi alloys. Two different strategies are then applied to compute these composite behaviors. For the SMA/elastomer composite, the macroscopic behavior law is implemented in commercial FEM software, and for the Ni47Ti44Nb9 a scale transition approach based on the Mori-Tanaka scheme is developed. In both cases, simulations are compared to experimental data.
Chandel, Nirupama; Sharma, Bipin; Husain, Mohammad; Salhan, Divya; Singh, Tejinder; Rai, Partab; Mathieson, Peter W.; Saleem, Moin A.; Malhotra, Ashwani
2013-01-01
Alterations in the podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on the podocyte actin cytoskeleton and the mechanism involved. We hypothesized that HIV may be compromising the actin cytoskeleton via downregulation of the vitamin D receptor (VDR) of conditionally immortalized differentiated human podocytes (CIDHPs). HIV-transduced podocytes (HIV/CIDHPs) not only displayed downregulation of VDR but also showed activation of the renin-angiotensin system (RAS) in the form of enhanced expression of renin and increased production of ANG II. Moreover, CIDHPs lacking VDR displayed enhanced ANG II production, and treatment of HIV/CIDHPs with EB1089 (vitamin D3; VD) attenuated ANG II production. HIV/CIDHPs as well as ANG II-treated CIDHPs exhibited enhanced expression of cathepsin (CTS) L. Additionally, losartan (an ANG II type I receptor blocker) inhibited both HIV- and ANG II-induced podocyte cathepsin L expression. Furthermore, VD downregulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV- and ANG II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization; on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphological evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression, and HIV/CIDHPs expressing CTSL exhibited downregulation of dynamin. These findings indicate that HIV-induced downregulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised the actin cytoskeleton. PMID:23467424
Expression of matrix metalloproteinase-1, -2 and -3 in squamous cell carcinoma and actinic keratosis
Tsukifuji, R; Tagawa, K; Hatamochi, A; Shinkai, H
1999-01-01
Matrix metalloproteinase (MMP) plays an important role in extracellular matrix degradation associated with cancer invasion. An expression of MMP-1 (interstitial collagenase), MMP-2 (72-kDa type IV collagenase) and MMP-3 (stromelysin-1) was investigated in squamous cell carcinoma (SCC) and its precancerous condition, actinic keratosis (AK), using in situ hybridization techniques. MMP-1 mRNA was detected in tumour cells and/or in stromal cells in all cases of SCC, four of six AKs adjacent to SCC and four of 16 AKs. MMP-2 and MMP-3 mRNAs were detected in SCC but not in AK. The expression of MMP-3 correlated to that of MMP-1 (P = 0.03) localized at the tumour mass and stroma of the invasive area, while MMP-2 mRNA was detected widely throughout the stroma independent of MMP-1 expression. Our results indicated that the expression of MMP-1, -2 and -3 showed different localization patterns, suggesting a unique role of each MMP in tumour progression. Moreover, MMP-1 expression could be an early event in the development of SCC, and AK demonstrating MMP-1 mRNA, might be in a more advanced dysplastic state, progressing to SCC. © 1999 Cancer Research Campaign PMID:10362121
Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal
2014-01-01
TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Blom, Magdalena; Reis, Katarina; Heldin, Johan
RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less
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
Gates, Michael A; Kannan, Ramakrishnan; Giniger, Edward
2011-11-30
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. 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. 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.
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.
Daniel, Filipe I; Alves, Soraia R; Vieira, Daniella S C; Biz, Michelle T; Daniel, Inah W B S; Modolo, Filipe
2016-11-01
Epigenetic modifications, including DNA methylation of tumor suppressor genes carried out by DNA methyltransferases (DNMTs), are important events in carcinogenesis. Although there are studies concerning to its expression in several cancer types, DNMTs expression pattern is not known in photoinduced lip carcinogenesis. The aim of this study was to investigate the immunoexpression of DNMTs 1, 3a, and 3b in lip precancerous lesion (actinic cheilitis) and cancer. Thirty cases of actinic cheilitis (AC), thirty cases of lip squamous cell carcinoma (LSCC), and twenty cases of non-neoplastic tissue (NNT) were selected for immunohistochemical investigation of DNMTs 1, 3a, and 3b. Nuclear DNMT 1 immunoreactivity was significantly higher in the LSCC group (68.6%) compared with NNT (47%), and nuclear DNMT 3b was higher in LSCC (70.9%) than in NNT (37.9%) and in AC (44%). Only DNMT 3a showed both higher nuclear and cytoplasmic expression in AC (35.9% and 35.5%, respectively) than in NNT (4.4% and 16.1%, respectively) and LSCC (8.8% and 13.2%, respectively) (P < 0.05). The results suggested that DNMT 3a could play a key role in the methylation process of initial steps of UV carcinogenesis present in AC while DNMT 3b could be responsible for de novo methylation in already established lip cancer. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function
McGough, Amy; Pope, Brian; Chiu, Wah; Weeds, Alan
1997-01-01
Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled. PMID:9265645
Actin isoform specificity is required for the maintenance of lactation
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
Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro
2016-03-04
Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami
2014-01-01
The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811
Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.
Urbancíková, M; Vozárová, G; Lesko, J; Golais, F
1999-10-01
Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.
NASA Astrophysics Data System (ADS)
Tsuda, Yuri; Yasutake, Hironori; Ishijima, Akihiko; Yanagida, Toshio
1996-11-01
Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10-26 Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.
Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen; Meagher, Richard B.
2012-01-01
Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals. PMID:22589468
Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton
Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent
2007-01-01
Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that
Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng
2012-08-15
Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.
Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew; Xu, Yan; Perl, Anne Karina
2017-08-01
The following data are derived from key stages of acinar lung development and define the developmental role of lung interstitial fibroblasts expressing platelet-derived growth factor alpha (PDGFRα). This dataset is related to the research article entitled "Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development" (Endale et al., 2017) [1]. At E16.5 (canalicular), E18.5 (saccular), P7 (early alveolar) and P28 (late alveolar), PDGFRα GFP mice, in conjunction with immunohistochemical markers, were utilized to define the spatiotemporal relationship of PDGFRα + fibroblasts to endothelial, stromal and epithelial cells in both the proximal and distal acinar lung. Complimentary analysis with flow cytometry was employed to determine changes in cellular proliferation, define lipofibroblast and myofibroblast populations via the presence of intracellular lipid or alpha smooth muscle actin (αSMA), and evaluate the expression of CD34, CD29, and Sca-1. Finally, PDGFRα + cells isolated at each stage of acinar lung development were subjected to RNA-Seq analysis, data was subjected to Bayesian timeline analysis and transcriptional factor promoter enrichment analysis.
Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pécot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John J; Thomas, Jessica; Cole, Sara L; Moldovan, Leni; Moldovan, Nicanor I
2015-06-01
Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-ε-capro-lactone (PCL) fibers with diameters in 5-20 μm range ('scaffold microfibers', SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them 'actin grips'. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.
Zhang, Lei; Han, Changsong; Sun, Chuanhui; Meng, Hongxue; Ye, Fei; Na, Shiping; Chen, Fulai; Zhang, Duo; Jin, Xiaoming
2013-01-01
Approximately 40% of patients with Henoch-Schonlein purpura (HSP) develop Henoch-Schonlein purpura nephritis (HSPN) after 4 to 6 weeks of subcutaneous hemorrhaging. Immunoglobulin-A nephropathy (IgAN) and HSPN have numerous similarities, which can cause difficulty in correctly diagnosing the disorder during a differential diagnosis. The pathogenesis of the 2 diseases is not clear. We enrolled 137 patients with HSPN, 107 patients with IgAN, and 28 healthy (control) patients in our study. The levels of alpha-smooth muscle actin (α-SMA), c-Met, and Gal-deficient IgA1 (Gd-IgA1) in the 3 patient groups were determined and compared. The α-SMA, c-Met, and Gd-IgA1 levels and the clinical data from the patients with HSPN were analyzed for any correlations. The α-SMA and c-Met levels of the HSPN group were significantly higher than those of the IgAN and healthy control groups (P < 0.01). The Gd-IgA1 levels of the HSPN and IgAN groups were significantly different from the Gd-IgA1 level of the healthy control group (P < 0.01). The α-SMA levels of the HSPN group were positively correlated with blood urea nitrogen levels, serum creatinine levels, hematuria index, and proteinuria levels (P < 0.01). The c-Met levels of the HSPN group were positively correlated with the blood urea nitrogen and serum creatinine levels (P < 0.01). There were no significant differences among the α-SMA, c-Met, and Gd-IgA1 levels or the clinical data for the child and adult patients with HSPN. The serum levels of α-SMA and c-Met in patients with HSPN may be associated with the degree of disease severity. Gd-IgA1 is involved in the common immunologic pathogenesis of HSPN and IgAN. Copyright © 2013 Mosby, Inc. All rights reserved.
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.
Feasibility study of an active soft catheter actuated by SMA wires
NASA Astrophysics Data System (ADS)
Konh, Bardia; Karimi, Saeed; Miller, Scott
2018-03-01
This study aims to assess the feasibility of using a combination of thin elastomer tubes and SMA wires to develop an active catheter. Cardiac catheters have been widely used in investigational and interventional procedures such as angiography, angioplasty, electro- physiology, and endocardial ablation. The commercial models manually steer inside the patient's body via internally installed pull wires. Active catheters, on the other hand, have the potential to revolutionize surgical procedures because of their computer-controlled and enhanced motion. Shape memory alloys have been used for almost a decade as a trustworthy actuator for biomedical applications. In this work, SMA wires were attached to a small pressurized elastomer tube to realize deflection. The tube was pressurized to maintain a constant stress on the SMA wires. The tip motion via actuation of SMA wires was then measured and reported. The results of this study showed that by adopting an appropriate training process for the SMA wires prior to performing the experiments and adopting an appropriate internal pressure for the elastomer tube, less external loads on SMA wires would be needed for a consistent actuation.
Jensen, Helene H; Holst, Mikkel R; Login, Frédéric H; Morgen, Jeanette J; Nejsum, Lene N
2018-06-01
Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.
Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.
2012-01-01
Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942
Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier
2015-01-01
The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337
Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission
Rehklau, Katharina; Hoffmann, Lena; Gurniak, Christine B; Ott, Martin; Witke, Walter; Scorrano, Luca; Culmsee, Carsten; Rust, Marco B
2017-01-01
Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles’ morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization. PMID:28981113
Ling, Li; Chen, Libo; Zhang, Changning; Gui, Shuyan; Zhao, Haiyan; Li, Zhengzhang
2018-01-01
Abnormal expression of matrix metalloproteinase 9 (MMP9) is correlated with podocyte epithelial-to-mesenchymal transition (EMT) in diabetic nephropathy (DN). However, the mechanisms underlying this process are not well defined. Site-specific demethylation may sustain high expression levels of target genes. In the present study, in order to investigate the association between DNA demethylation of MMP9 promoter and podocyte EMT in DN, human podocytes were cultured in high-glucose (HG) medium and a rat model of DN was established by intraperitoneal injection of streptozotocin (STZ) to determine whether site-specific demethylation of the MMP9 promoter was involved in regulating podocyte EMT in DN. The MTT assay was used to assess the effects of HG culture on the growth of podocytes, and the demethylation status of the MMP9 promoter was assessed by bisulfite sequencing polymerase chain reaction. mRNA and protein expression levels of MMP9, α-smooth muscle actin (α-SMA), podocalyxin and fibronectin-1 in podocytes were assessed by reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses. The results demonstrated that HG treatment up regulated the expression of MMP9, α-SMA and fibronectin-1, but down regulated the expression of podocalyxin in podocytes. The MMP9 promoter region was revealed to contain a variety of demethylated CpG sites, and HG treatment reduced the rate of MMP9 promotermethylation, which, in turn, enhanced its promoter activity. In summary, these data suggested that demethylation of the MMP9 promoter may serve an important role in podocyte EMT in DN. The demethylation status of the MMP9 promoter maybe used as an important prognostic marker of DN in clinic. PMID:29436620
Nucleation of actin polymerization by gelsolin.
Ditsch, A; Wegner, A
1994-08-15
The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.
Tanaka, Hideki; Tanabe, Natsuko; Kawato, Takayuki; Nakai, Kumiko; Kariya, Taro; Matsumoto, Sakurako; Zhao, Ning; Motohashi, Masafumi; Maeno, Masao
2013-01-01
Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization. PMID
Performance of SMA-reinforced composites in an aerodynamic profile
NASA Astrophysics Data System (ADS)
Simpson, John; Boller, Christian
2002-07-01
Within the European collaborative applied fundamental research project ADAPT, fundamentals of SMA-reinforced composites were evaluated and the specific manufacturing techniques for these composites developed and realised. The involved partners are listed at the end. To demonstrate applicability of these composites a realistically scaled aerodynamic profile of around 0.5m span by 0.5m root chord was designed, manufactured and assembled. The curved skins were manufactured as SMA composites with two layers of SMA-wires integrated into the layup of aramid fibre prepregs. All SMA wires were connected such that they can be operated as individual sets of wires and at low voltages, similar to the conditions for electrical energy generation in a real aircraft. The profile was then mounted on a vibration test rig and activated and excited by a shaker at its tip which allowed to test the dynamic performance of the profile under different external loading conditions with various internal actuation conditions through the SMA wires. The paper includes some background of the design and manufacturing of the aerodynamic profile and will discuss some of the results determined recently on the test rig. A view with regard to future wind tunnel testing will be given as well.
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.
Cervical Spinal Cord Atrophy Profile in Adult SMN1-Linked SMA
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
PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions
Hong, Nan Hyung; Qi, Aidong
2015-01-01
Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691
Walther, Christa G; Whitfield, Robert; James, David C
2016-04-01
The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.
2017-01-01
Mature skin is prone to bruising, resulting in a condition known as actinic purpura, characterized by unsightly ecchymosis and purple patches. Similar to other skin conditions, the incidence of actinic purpura increases with advancing age and occurs with equal frequency among men and women. The unsightly appearance of actinic purpura may be a source of emotional distress among the elderly. A new product has been formulated specifically for the treatment of actinic purpura. This product contains retinol, α-hydroxy acids, arnica oil, ceramides, niacinamide, and phytonadione, which effectively treat actinic purpura by improving local circulation, thickening the skin, and repairing the skin barrier. The objective of this paper is to review the beneficial properties of these ingredients and their respective roles in the treatment of actinic purpura. PMID:28979656
2011-01-01
Introduction Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS. Methods Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry. Results Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1. Conclusions Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and
Fazal, Fabeha; Minhajuddin, Mohd; Bijli, Kaiser M; McGrath, James L; Rahman, Arshad
2007-02-09
Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.
Identification of Actin-Binding Proteins from Maize Pollen
DOE Office of Scientific and Technical Information (OSTI.GOV)
Staiger, C.J.
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 ABPsmore » (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.« less
Actin assembly factors regulate the gelation kinetics and architecture of F-actin networks.
Falzone, Tobias T; Oakes, Patrick W; Sees, Jennifer; Kovar, David R; Gardel, Margaret L
2013-04-16
Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Superelastic SMA U-shaped dampers with self-centering functions
NASA Astrophysics Data System (ADS)
Wang, Bin; Zhu, Songye
2018-05-01
As high-performance metallic materials, shape memory alloys (SMAs) have been investigated increasingly by the earthquake engineering community in recent years, because of their remarkable self-centering (SC) and energy-dissipating capabilities. This paper systematically presents an experimental study on a novel superelastic SMA U-shaped damper (SMA-UD) with SC function under cyclic loading. The mechanical properties, including strength, SC ability, and energy-dissipating capability with varying loading amplitudes and strain rates are evaluated. Test results show that excellent and stable flag-shaped hysteresis loops are exhibited in multiple loading cycles. Strain rate has a negligible effect on the cyclic behavior of the SMA-UD within the dynamic frequency range of typical interest in earthquake engineering. Furthermore, a numerical investigation is performed to understand the mechanical behavior of the SMA-UD. The numerical model is calibrated against the experimental results with reasonable accuracy. Then, the stress–strain states with different phase transformations are also discussed.
Hassan, Sobia; Syed, Serajuddaula; Kehar, Shahnaz Imdad
2014-01-01
Objective: This study aims to determine expression of Glial Fibrillary Acidic Protein and of Alpha Smooth Muscle Actin (α-SMA) in hepatic stellate cells of CHC cases and their association with stage of fibrosis. Methods: The study was conducted at Ziauddin University, Clifton Campus during the year 2010-2012. Sixty Chronic Hepatitis C cases were immmunostained using anti α-SMA antibody and anti-GFAP antibody. Semi quantitative scoring in pericentral, periportal and perisinusoidal area of each case was done to assess immunoexpression of each marker. Results : Immunoexpression of GFAP showed significant association with α-SMA. GFAP expression was inversely correlated with progression of fibrosis. Conclusion : GFAP could represent a useful marker for early hepatic stellate cells activation. Follow up biopsies showing decline in GFAP levels may help identify the target group requiring aggressive therapy. PMID:25225520
NASA Astrophysics Data System (ADS)
Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.
2002-08-01
The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.
Confinement of NORMAL- AND HIGH-STRENGTH CONCRETE by Shape Memory Alloy (SMA) Spirals
NASA Astrophysics Data System (ADS)
Gholampour, A.; Ozbakkaloglu, T.
2018-01-01
This paper presents the results of an experimental study on the axial compressive behaviour of normal- and high-strength concrete (NSC and HSC) confined by shape memory alloy (SMA) spirals. A spiral pitch space of 36 and 20 mm was used for SMA confinement of NSC and HSC columns, respectively. The confining pressure was applied on the concrete cylinders by SMA spirals that were prestrained at 0, 5.5, and 9.5%. The compression test results on the SMA-confined specimens indicate that the prestrain level of SMA significantly affects the axial compressive behaviour of both NSC and HSC. An increase in the level of prestrain leads to an increase in the peak axial stress and corresponding strain of SMA-confined concrete.
Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations
Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten
2013-01-01
The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176
Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K
2014-03-01
The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.
Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu
2016-04-01
The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM
DOE Office of Scientific and Technical Information (OSTI.GOV)
Imamura, Ryoichi; Isaka, Yoshitaka; Ichimaru, Naotsugu
Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys from tubular apoptosis and inhibit subsequent tubulointerstitial injury without erythropoiesis. The therapeutic effect of CEPO was evaluated using a rat ischemia-reperfusion injury model. Saline-treated kidneys exhibited increased tubular apoptosis with interstitial expression of {alpha}-smooth muscle actin ({alpha}-SMA), while EPO treatment inhibited tubular apoptosismore » and {alpha}-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis with limited expression of {alpha}-SMA. Moreover, CEPO significantly promoted tubular epithelial cell proliferation without erythropoiesis. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury.« less
Nakamura, Hideaki; Fang, Jun; Gahininath, Bharate; Tsukigawa, Kenji; Maeda, Hiroshi
2011-11-07
SMA-ZnPP and PEG-ZnPP are micellar drugs, encapsulating zinc protoporphyrin IX (ZnPP) with styrene maleic acid copolymer (SMA) and covalent conjugate of ZnPP with polyethylene glycol (PEG) respectively. Their intracellular uptake rate and subcellular localization were investigated. We found SMA-ZnPP showed higher and more efficient (about 2.5 times) intracellular uptake rate than PEG-ZnPP, although both SMA-ZnPP and PEG-ZnPP micelles were localized at endoplasmic reticulum (ER) and inhibited the target enzyme heme oxygenase 1 (HO-1) similarly. Both micellar ZnPP were taken up into the tumor cells by endocytosis. Furthermore SMA-ZnPP and PEG-ZnPP were examined for their drug releasing mechanisms. Liberation of ZnPP from the SMA micelle appears to depend on cellular amphiphilic components such as lecithin, while that for PEG-ZnPP depends on hydrolytic cleavage. These results indicate that these micelle formulations make water insoluble ZnPP to water soluble practical anticancer agents. Copyright © 2011 Elsevier B.V. All rights reserved.
Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin
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
Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells
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. PMID:28357356
Co-transcriptional nuclear actin dynamics
Percipalle, Piergiorgio
2013-01-01
Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849
Case, Lindsay B.; Waterman, Clare M.
2011-01-01
At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459
Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H.
2013-01-01
Summary 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. PMID:24091012
Hydroxyurea enhances SMN2 gene expression through nitric oxide release.
Xu, Cheng; Chen, Xin; Grzeschik, Susanna M; Ganta, Madhuri; Wang, Ching H
2011-02-01
Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.
Nucleus-associated actin in Amoeba proteus.
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. Copyright © 2016 Elsevier GmbH. All rights reserved.
DNA vaccine encoding Haemonchus contortus actin induces partial protection in goats.
Yan, Ruofeng; Wang, Jingjing; Xu, Lixin; Song, Xiaokai; Li, Xiangrui
2014-10-01
Actin is a globular multi-functional protein that forms microfilaments, and participates in many important cellular processes. Previous study found that Haemonchus contortus actin could be recognized by the serum of goats infected with the homology parasite. This indicated that H. contortus actin could be a potential candidate for vaccine. In this study, DNA vaccine encoding H. contortus actin was tested for protection against experimental H. contortus infections in goats. Fifteen goats were allocated into three trial groups. The animals of Actin group were vaccinated with the DNA vaccine on day 0 and 14, and challenged with 5000 infective H. contortus third stage larval (L3) on day 28. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results showed that DNA vaccine were transcribed at local injection sites and expressed in vivo post immunizations respectively. For goats in Actin vaccinated group, higher levels of serum IgG, serum IgA and mucosal IgA were produced, the percentages of CD4(+) T lymphocytes, CD8(+) T lymphocytes and B lymphocytes and the concentrations of TGF-β were increased significantly (P<0.05). Following L3 challenge, the mean eggs per gram feces (EPG) and worm burdens of Actin group were reduced by 34.4% and 33.1%, respectively. This study suggest that recombinant H. contortus Actin DNA vaccine induced partial immune response and has protective potential against goat haemonchosis.
FE analysis of SMA-based bio-inspired bone-joint system
NASA Astrophysics Data System (ADS)
Yang, S.; Seelecke, S.
2009-10-01
This paper presents the finite element (FE) analysis of a bio-inspired bone-joint system. Motivated by the BATMAV project, which aims at the development of a micro-air-vehicle platform that implements bat-like flapping flight capabilities, we study the actuation of a typical elbow joint, using shape memory alloy (SMA) in a dual manner. Micro-scale martensitic SMA wires are used as 'metal muscles' to actuate a system of humerus, elbow joint and radius, in concert with austenitic wires, which operate as flexible joints due to their superelastic character. For the FE analysis, the humerus and radius are modeled as standard elastic beams, while the elbow joint and muscle wires use the Achenbach-Muller-Seelecke SMA model as beams and cable elements, respectively. The particular focus of the paper is on the implementation of the above SMA model in COMSOL.
Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel
2015-10-01
It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. © 2015 Wiley Periodicals, Inc.
SMA Foils for MEMS: From Material Properties to the Engineering of Microdevices
NASA Astrophysics Data System (ADS)
Kohl, Manfred; Ossmer, Hinnerk; Gueltig, Marcel; Megnin, Christof
2018-03-01
In the early nineties, microelectromechanical systems (MEMS) technology has been still in its infancy. As silicon (Si) is not a transducer material, it was clear at the very beginning that mechanically active materials had to be introduced to MEMS in order to enable functional microdevices with actuation capability beyond electrostatics. At that time, shape memory alloys (SMAs) have been available in bulk form, mainly as SMA wires and SMA plates. On the macro scale, these materials show highest work densities compared to other actuation principles in the order of 107 J/m3, which stimulated research on the integration of SMA to MEMS. Subsequently, two approaches for producing planar materials have been initiated (1) magnetron sputtering of SMA thin films and (2) the integration of rolled SMA foils, which both turned out to be very successful creating a paradigm change in microactuation technology. The following review covers important milestones of the research and development of SMA foil-based microactuators including materials characterization, design engineering, technology, and demonstrator development as well as first commercial products.
SMA Foils for MEMS: From Material Properties to the Engineering of Microdevices
NASA Astrophysics Data System (ADS)
Kohl, Manfred; Ossmer, Hinnerk; Gueltig, Marcel; Megnin, Christof
2017-12-01
In the early nineties, microelectromechanical systems (MEMS) technology has been still in its infancy. As silicon (Si) is not a transducer material, it was clear at the very beginning that mechanically active materials had to be introduced to MEMS in order to enable functional microdevices with actuation capability beyond electrostatics. At that time, shape memory alloys (SMAs) have been available in bulk form, mainly as SMA wires and SMA plates. On the macro scale, these materials show highest work densities compared to other actuation principles in the order of 107 J/m3, which stimulated research on the integration of SMA to MEMS. Subsequently, two approaches for producing planar materials have been initiated (1) magnetron sputtering of SMA thin films and (2) the integration of rolled SMA foils, which both turned out to be very successful creating a paradigm change in microactuation technology. The following review covers important milestones of the research and development of SMA foil-based microactuators including materials characterization, design engineering, technology, and demonstrator development as well as first commercial products.
Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M
2018-05-01
Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema
2007-09-07
Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.
Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks
Falzone, Tobias T.; Oakes, Patrick W.; Sees, Jennifer; Kovar, David R.; Gardel, Margaret L.
2013-01-01
Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. PMID:23601318
Investigations on Vibration Characteristics of Sma Embedded Horizontal Axis Wind Turbine Blade
NASA Astrophysics Data System (ADS)
Jagadeesh, V.; Yuvaraja, M.; Chandhru, A.; Viswanathan, P.; Senthil kumar, M.
2018-02-01
Vibration induced in wind turbine blade is a solemn problem as it reduces the life of the blade and also it can create critical vibration onto the tower, which may cause serious damage to the tower. The aim of this paper is to investigate the vibration characteristics of the prototype horizontal axis wind turbine blade. Shape memory alloys (SMA), with its variable physical properties, provides an alternative actuating mechanism. Heating an SMA causes a change in the elastic modulus of the material and hence SMAs are used as a damping material. A prototype blade with S1223 profile has been manufactured and the natural frequency is found. The natural frequency is found by incorporating the single SMA wire of 0.5mm diameter over the surface of the blade for a length of 240 mm. Similarly, number of SMA wires over the blade is increased up to 3 and the natural frequency is found. Frequency responses showed that the embedment of SMA over the blade’s surface will increase the natural frequency and reduce the amplitude of vibration. This is because of super elastic nature of SMA. In this paper, when SMA wire of 0.5 mm diameter and of length of 720 mm is embedded on the blade, an increase in the natural frequency by 6.3% and reducing the amplitude by 64.8%. Results of the experimental modal and harmonic indicates the effectiveness of SMA as a passive vibration absorber and that it has potential as a modest and high-performance method for controlling vibration of the blade.
Investigation of residual stresses in shape memory alloy (SMA) composites
NASA Astrophysics Data System (ADS)
Berman, Justin Bradley
Shape memory alloy (SMA) composites are a class of smart materials in which SMA actuators are embedded in a host matrix. The shape memory effect allows for stress induced phase transformations and large recoverable strains that make SMA composites promising candidates for structural shape/vibration control, impact absorption, aircraft deicing or in-flight airfoil shape control systems. However, the difference in thermal expansion between the SMA and the host material leads to residual stresses during processing. In addition, the SMA transformation from martensite to austenite, or the reverse, also generate stresses. These stresses acting in combination can lead to SMA/polymer interfacial debonding or microcracking of the host matrix. The present work was undertaken to study the behavior of nitinol shape memory alloys embedded in epoxy and glass/epoxy matrices and to investigate the development of residual stresses during their manufacture and actuation. A three-phase concentric cylinder micromechanics model and an SMA composite thermoelastic beam theory were developed to analyze the micromechanical and structural-level thermal and transformational stresses for nitinol composites induced by nitinol wires embedded in a host matrix. A series of warpage experiments were conducted on nitinol composite beams during heating cycles to provide experimental validation of model predictions and to assess their thermoelastic structural behavior under non-mechanical loading. Micromechanical model results indicate that excessive residual hoop stresses in nitino/graphite/epoxy composites leads to radial cracking around the embedded nitinol wires. Based on modeling results, the most important factor in reducing residual stresses (and thereby preventing radial cracking) is increasing the level of recovery strain for the nitinol wire. The SMA composite beam model agrees well with experimental data captured for the nitinol/epoxy beam series. Warpage experiments on nitinol
Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun
2016-01-01
Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.
Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun
2016-01-01
Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648
Yusof, Alia Md; Abd Ghafar, Norzana; Kamarudin, Taty Anna; Hui, Chua Kien; Yusof, Yasmin Anum Mohd
2016-02-24
This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry. Corneal keratocytes from New Zealand white rabbits were cultured in basal medium (BM) and serum enriched medium (BMS). Serial dilutions of Gelam honey (GH) were added to both media and cells were cultured until passage 1. MTT assay was performed on corneal keratocytes in both media to ascertain the optimal dose of GH that produced maximum proliferation. Gelam honey at the concentration of 0.0015% in both media showed the highest proliferative capacity with no morphological changes compared to their respective controls. The gene expression of aldehyde dehydrogenase (ALDH), a marker for quiescent keratocytes and vimentin, a marker for fibroblast, were higher in the GH enriched groups. The alpha smooth muscle actin (α-SMA) expression, marker for myofibroblast, was lower in GH treated groups compared to the controls. Immunocytochemistry results were in accordance to the gene expression analyses. Gelam honey at a concentration of 0.0015% promotes ex vivo corneal keratocytes proliferation while retaining desirable phenotype expression. The results serve as a basis for the development of Gelam honey as a potential natural product in promoting corneal wound healing.
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
Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi
2013-04-01
The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying
2013-03-01
For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.
Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.
Uda, Munehiro; Kawasaki, Hiroaki; Iizumi, Kyoichi; Shigenaga, Ayako; Baba, Takeshi; Naito, Hisashi; Yoshioka, Toshitada; Yamakura, Fumiyuki
2015-11-01
Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.
Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A
2006-01-01
Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.
Carlier, Marie-France; Laurent, Valérie; Santolini, Jérôme; Melki, Ronald; Didry, Dominique; Xia, Gui-Xian; Hong, Yan; Chua, Nam-Hai; Pantaloni, Dominique
1997-01-01
Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics. PMID:9087445
Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton
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
The confining effectiveness of NiTiNb and NiTi SMA wire jackets for concrete
NASA Astrophysics Data System (ADS)
Choi, Eunsoo; Chung, Young-Soo; Choi, Jun-Hyeok; Kim, Hong-Taek; Lee, Hacksoo
2010-03-01
The purpose of this study is to assess the confining effectiveness of shape memory alloy (SMA) wire jackets for concrete. The performance of SMA wire jackets was compared to that of steel jackets. A prestrained martensitic SMA wire was wrapped around a concrete cylinder and then heated by a heating jacket. In the process, a confining stress around the cylinder was developed in the SMA wire due to the shape memory effect; this jacketing method can increase the strength and ductility of the cylinder under an axial compressive load. In this study, NiTi and NiTiNb SMA wires of 1.0 mm in diameter were used for the confinement. Recovery tests were conducted on the wires to assess their recovery and residual stress. The confinement by SMA wire jackets increased the strength slightly and greatly increased the ductility compared to the strength and ductility of plain concrete cylinders. The NiTiNb SMA wire jacket showed better performance than that of the NiTi SMA wire jacket. The confining effectiveness of the SMA wire jackets of this study was estimated to be similar to that of the steel jackets. This study showed the potential of the SMA wire jacketing method to retrofit reinforced concrete columns and protect them from seismic risks.
Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.
Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle
2018-04-26
Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.
Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.; Colin-York, Huw; Clausen, Mathias P.; Felce, James H.; Galiani, Silvia; Erlenkämper, Christoph; Santos, Ana M.; Heddleston, John M.; Pedroza-Pacheco, Isabela; Waithe, Dominic; de la Serna, Jorge Bernardino; Lagerholm, B. Christoffer; Liu, Tsung-li; Chew, Teng-Leong; Betzig, Eric; Davis, Simon J.; Eggeling, Christian
2017-01-01
T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions. PMID:28691087
Potential of using stone matrix asphalt (SMA) for thin overlays
DOT National Transportation Integrated Search
2003-04-01
Stone matrix asphalt (SMA) has been used within the U.S. since 1991. To date almost all of the SMA mixes have had either a 12.5 or 19.0 mm nominal maximum aggregate size (NMAS). These two NMASs have been predominant because they conform to informatio...
Development of damage suppression system using embedded SMA foil sensor and actuator
NASA Astrophysics Data System (ADS)
Ogisu, Toshimichi; Nomura, Masato; Ando, Norio; Takaki, Junji; Song, Dong Y.; Takeda, Nobuo
2000-06-01
The recent studies suggest 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 sensors and actuators in CFRP laminates. The goal of this research is suppression of damage growth in CFRP laminates. At first, the authors proposed a concept of damage suppression in CFRP laminates. Then, the development studies are conducted in three phases. The first phase is the improvement of interlaminar shear strength between SMA and CFRP laminates. Some surface treatments were investigated for the improvement of bonding property by peel resistance test and single lap shear strength test. The second phase is the investigation of fabrication technique for producing a CFRP panel with embedded SMA foils. Fixture jigs were devised to introduce tensile loads during the fabrication process. The third phase is the strength demonstration of CFRP laminates with embedded SMA foils. Some strength test were conducted to obtain the design data for aircraft structures. It is confirmed that the shrinking force of pre-strained SMA influences to the strength and the crack density of CFRP panel.
Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D
2018-06-01
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which Ac
NASA Astrophysics Data System (ADS)
Dingal, P. C. Dave P.; Bradshaw, Andrew M.; Cho, Sangkyun; Raab, Matthew; Buxboim, Amnon; Swift, Joe; Discher, Dennis E.
2015-09-01
Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that copolymerization of collagen I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fibre bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally--as in scars--while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the `scar marker' smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, whereas cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a `mechanical memory' of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.
Satish, Latha; O'Gorman, David B; Johnson, Sandra; Raykha, Christina; Gan, Bing Siang; Wang, James H-C; Kathju, Sandeep
2013-07-01
Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.
Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia
Goldman, Robert D.; Louvard, Daniel
2010-01-01
Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration. PMID:20421424
Zhang, Li; Chen, Yan; Li, Guixia; Chen, Minggang; Huang, Wei; Liu, Yanrui; Li, Yumei
2016-01-05
Pulmonary adventitial fibroblasts (PAFs) are activated under stress stimuli leading to their differentiation into myofibroblasts, which is involved in vessel remodeling. 15-HETE is known as an important factor in vessel remodeling under hypoxia; however, the role of 15-HETE in PAF phenotypic alteration is not clear. The effect of 15-HETE on PAF phenotypic alterations was investigated in the present study. PAFs were treated with 15-HETE (0.5 μM) for 24 h, and the myofibroblast marker α-smooth muscle actin (α-SMA) was analyzed. The 15-HETE induced α-SMA expression and cell morphology. 15-HETE upregulated FGF-2 levels in PAFs, and knockdown FGF-2 by siRNAs blocked the enhanced α-SMA expression induced by 15-HETE. p38 kinase was activated, and blocked depressed 15-HETE-induced FGF-2 expression. The downstream of p38 pathway, Egr-1 activation, was also raised by 15-HETE treatment, and silenced Egr-1 suppressed the 15-HETE-induced upregulation of FGF-2. TGF-β1 was upregulated with FGF-2 treatment, and α-SMA expression induced by FGF-2 was inhibited after the cell was transferred with TGF-β1 siRNA. Meanwhile, FGF-2 increased α-SMA expression and improved proliferation, which was associated with p27(kip1) and cyclin E variation. The above results suggest that p38/Egr-1 pathway-mediated FGF-2 is involved in 15-HETE-induced differentiation of PAFs into myofibroblasts and cell proliferation.
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.
Tropomodulin Capping of Actin Filaments in Striated Muscle Development and Physiology
Gokhin, David S.; Fowler, Velia M.
2011-01-01
Efficient striated muscle contraction requires precise assembly and regulation of diverse actin filament systems, most notably the sarcomeric thin filaments of the contractile apparatus. By capping the pointed ends of actin filaments, tropomodulins (Tmods) regulate actin filament assembly, lengths, and stability. Here, we explore the current understanding of the expression patterns, localizations, and functions of Tmods in both cardiac and skeletal muscle. We first describe the mechanisms by which Tmods regulate myofibril assembly and thin filament lengths, as well as the roles of closely related Tmod family variants, the leiomodins (Lmods), in these processes. We also discuss emerging functions for Tmods in the sarcoplasmic reticulum. This paper provides abundant evidence that Tmods are key structural regulators of striated muscle cytoarchitecture and physiology. PMID:22013379
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.
Praveen, Kavita; Wen, Ying; Gray, Kelsey M.; Noto, John J.; Patlolla, Akash R.; Van Duyne, Gregory D.; Matera, A. Gregory
2014-01-01
Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN. PMID:25144193
Praveen, Kavita; Wen, Ying; Gray, Kelsey M; Noto, John J; Patlolla, Akash R; Van Duyne, Gregory D; Matera, A Gregory
2014-08-01
Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.
Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.
Azevedo, Maria M; Domingues, Helena S; Cordelières, Fabrice P; Sampaio, Paula; Seixas, Ana I; Relvas, João B
2018-05-06
During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development. © 2018 Wiley Periodicals, Inc.
Actin filaments as tension sensors.
Galkin, Vitold E; Orlova, Albina; Egelman, Edward H
2012-02-07
The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.
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
Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells
Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G.; Kuemmerle, John F.; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I.
2014-01-01
Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA–depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. PMID:25143399
DasGupta, G; White, J; Cheung, P; Reisler, E
1990-09-11
The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.
Aldolase sequesters WASP and affects WASP/Arp2/3-stimulated actin dynamics.
Ritterson Lew, Carolyn; Tolan, Dean R
2013-08-01
In addition to its roles in sugar metabolism, fructose-1,6-bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed "moonlighting functions." These moonlighting functions likely involve the known aldolase-actin interaction, as many proteins with which aldolase interacts are involved in actin-dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott-Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP-dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3-dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP-dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP-dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin-binding activity. While the actin-binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP-mediated processes. Copyright © 2013 Wiley Periodicals, Inc.
An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.
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.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp
Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less
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
Theoretical modelling of residual and transformational stresses in SMA composites
NASA Astrophysics Data System (ADS)
Berman, J. B.; White, S. R.
1996-12-01
SMA composites are a class of smart materials in which shape memory alloy (SMA) actuators are embedded in a polymer matrix composite. The difference in thermal expansion between the SMA and the host material leads to residual stresses during processing. Similarly, the SMA transformations from martensite to austenite, or the reverse, also generate stresses. These stresses acting in combination can lead to SMA/epoxy interfacial debonding or microcracking of the composite phase. In this study the residual and transformational stresses are investigated for a nitinol wire embedded in a graphite/epoxy composite. A three-phase micromechanical model is developed. The nitinol wire is assumed to behave as a thermoelastic material. Nitinol austenitic and martensitic transformations are modelled using linear piecewise interpolation of experimental data. The interphase is modelled as a thermoelastic polymer. A transversely isotropic thermoelastic composite is used for the outer phase. Stress-free conditions are assumed immediately before cool down from the cure temperature. The effect of nitinol, coating and composite properties on residual and transformational stresses are evaluated. Fiber architectures favoring the axial direction decrease the magnitude of all residual stresses. A decrease in stresses at the composite/coating interface is also predicted through the use of thick, compliant coatings. Reducing the recovery strain and moving the transformation to higher temperatures were found to be most effective in reducing residual stresses.
Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast
Tang, Haosu; Bidone, Tamara C.
2015-01-01
The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307
Finite Element Analysis of Adaptive-Stiffening and Shape-Control SMA Hybrid Composites
NASA Technical Reports Server (NTRS)
Gao, Xiujie; Burton, Deborah; Turner, Travis L.; Brinson, Catherine
2005-01-01
Shape memory alloy hybrid composites with adaptive-stiffening or morphing functions are simulated using finite element analysis. The composite structure is a laminated fiber-polymer composite beam with embedded SMA ribbons at various positions with respect to the neutral axis of the beam. Adaptive stiffening or morphing is activated via selective resistance heating of the SMA ribbons or uniform thermal loads on the beam. The thermomechanical behavior of these composites was simulated in ABAQUS using user-defined SMA elements. The examples demonstrate the usefulness of the methods for the design and simulation of SMA hybrid composites. Keywords: shape memory alloys, Nitinol, ABAQUS, finite element analysis, post-buckling control, shape control, deflection control, adaptive stiffening, morphing, constitutive modeling, user element
Optimal design of damping layers in SMA/GFRP laminated hybrid composites
NASA Astrophysics Data System (ADS)
Haghdoust, P.; Cinquemani, S.; Lo Conte, A.; Lecis, N.
2017-10-01
This work describes the optimization of the shape profiles for shape memory alloys (SMA) sheets in hybrid layered composite structures, i.e. slender beams or thinner plates, designed for the passive attenuation of flexural vibrations. The paper starts with the description of the material and architecture of the investigated hybrid layered composite. An analytical method, for evaluating the energy dissipation inside a vibrating cantilever beam is developed. The analytical solution is then followed by a shape profile optimization of the inserts, using a genetic algorithm to minimize the SMA material layer usage, while maintaining target level of structural damping. Delamination problem at SMA/glass fiber reinforced polymer interface is discussed. At the end, the proposed methodology has been applied to study the hybridization of a wind turbine layered structure blade with SMA material, in order to increase its passive damping.
Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R
2015-07-06
Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.
The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag
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
Membrane wrinkling patterns and control with SMA and SMPC actuators
NASA Astrophysics Data System (ADS)
Lu, Mingyu; Li, Yunliang; Tan, Huifeng; Zhou, Limin
2009-07-01
Wrinkling is a main factor affecting the performance of the membrane structures and is always considered to be a failure as it can cause dramatic decrease of shape accuracy. The study of membrane wrinkling control has the analytical and experimental meanings. In this paper, a feasible membrane shape control method is presented. An expression of wrinkle wavelength using stress extremum principle is established based on the tension field theory and the Von Karman large deflection formula which verifies the generation and evolution reason of membrane wrinkles. The control mechanism for membrane wrinkles is developed using shape memory alloy (SMA) and shape memory polymer composite (SMPC) actuators which are attached to the boundaries of the membrane for producing contraction/expansion forces to adjust the shape of the membrane. The whole control process is monitored by photogrammetric technique. Numerical simulations are also conducted using ANSYS finite element software with the nonlinear post-buckling analytical method. Both the experimental and numerical results show that the amplitudes of wrinkles are effectively controlled by SMA and SMPC actuators. The method introduced in this paper provides the foundation for shape control of the membrane wrinkling and is important to the future work on vibration control of space membrane structures.
Deaton, J D; Guerrero, T; Howard, T H
1992-01-01
In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin
Deaton, J D; Guerrero, T; Howard, T H
1992-12-01
In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin
Behavior of NiTiNb SMA wires under recovery stress or prestressing.
Choi, Eunsoo; Nam, Tae-Hyun; Chung, Young-Soo; Kim, Yeon-Wook; Lee, Seung-Yong
2012-01-05
The recovery stress of martensitic shape-memory alloy [SMA] wires can be used to confine concrete, and the confining effectiveness of the SMA wires was previously proved through experimental tests. However, the behavior of SMA wires under recovery stress has not been seriously investigated. Thus, this study conducted a series of tests of NiTiNb martensitic SMA wires under recovery stress with varying degrees of prestrain on the wires and compared the behavior under recovery stress with that under prestressing of the wires. The remaining stress was reduced by the procedure of additional strain loading and unloading. More additional strains reduced more remaining stresses. When the SMA wires were heated up to the transformation temperature under prestress, the stress on the wires increased due to the state transformation. Furthermore, the stress decreased with a decreasing temperature of the wires down to room temperature. The stress of the NiTiNb wires was higher than the prestress, and the developed stress seemed to depend on the composition of the SMAs. When an additional strain was subsequently loaded and unloaded on the prestressed SMA wires, the remaining stress decreased. Finally, the remaining stress becomes zero when loading and unloading a specific large strain.
TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics
Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland
2016-01-01
Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424
Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.
Fiore, Ana Paula Zen Petisco; Spencer, Virginia A; Mori, Hidetoshi; Carvalho, Hernandes F; Bissell, Mina J; Bruni-Cardoso, Alexandre
2017-06-06
Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here that it is the LN1/N-actin pathway that is aberrant in human breast cancer cells, leading to continuous growth. Photobleaching assays revealed that N-actin exit in nonmalignant cells begins as early as 30 min after LN1 treatment. LN1 attenuates the PI3K pathway leading to upregulation of exportin-6 (XPO6) activity and shuttles actin out of the nucleus. Silencing XPO6 prevents quiescence. Malignant cells are impervious to LN1 signaling. These results shed light on the crucial role of LN1 in quiescence and differentiation and how defects in the LN1/PI3K/XPO6/N-actin axis explain the loss of tissue homeostasis and growth control that contributes to malignant progression. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.
2012-01-01
The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility. PMID:22711700
Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.
2018-01-01
During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019
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.
WHAMM links actin assembly via the Arp2/3 complex to autophagy.
Kast, David J; Dominguez, Roberto
2015-01-01
Macroautophagy (hereafter autophagy) is the process by which cytosolic material destined for degradation is enclosed inside a double-membrane cisterna known as the autophagosome and processed for secretion and/or recycling. This process requires a large collection of proteins that converge on certain sites of the ER membrane to generate the autophagosome membrane. Recently, it was shown that actin accumulates around autophagosome precursors and could play a role in this process, but the mechanism and role of actin polymerization in autophagy were unknown. Here, we discuss our recent finding that the nucleation-promoting factor (NPF) WHAMM recruits and activates the Arp2/3 complex for actin assembly at sites of autophagosome formation on the ER. Using high-resolution, live-cell imaging, we showed that WHAMM forms dynamic puncta on the ER that comigrate with several autophagy markers, and propels the spiral movement of these puncta by an Arp2/3 complex-dependent actin comet tail mechanism. In starved cells, WHAMM accumulates at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Conversely, knocking down WHAMM, inhibiting the Arp2/3 complex or interfering with actin polymerization reduces the size and number of autophagosomes. These findings establish a link between Arp2/3 complex-mediated actin assembly and autophagy.
Skau, Colleen T; Courson, David S; Bestul, Andrew J; Winkelman, Jonathan D; Rock, Ronald S; Sirotkin, Vladimir; Kovar, David R
2011-07-29
Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.
Wu, Yidi; Gunst, Susan J.
2015-01-01
Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM. PMID:25759389
Tang, Elizabeth I.; Lee, Will M.
2016-01-01
Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662
Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan
2014-01-01
STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by
The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation
Deng, Su; Bothe, Ingo; Baylies, Mary K.
2015-01-01
The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease. PMID:26295716
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.
Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.
2013-01-01
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621
Apoptosis and apoptotic pathway in actinic prurigo by immunohistochemistry
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
Prestressing effect of cold-drawn short NiTi SMA fibres in steel reinforced mortar beams
NASA Astrophysics Data System (ADS)
Choi, Eunsoo; Kim, Dong Joo; Hwang, Jin-Ha; Kim, Woo Jin
2016-08-01
This study investigated the prestressing effect of cold-drawn short NiTi shape memory alloy (SMA) fibres in steel reinforced mortar beams. The SMA fibres were mixed with 1.5% volume content in a mortar matrix with the compressive strength of 50 MPa. The SMA fibres had an average length of 34 mm, and they were manufactured with a dog-bone shape: the diameters of the end- and middle-parts were 1.024 and 1.0 mm, respectively. Twenty mortar beams with the dimensions of 40 mm × 40 mm × 160 mm (B × H × L) were prepared. Two types of tests were conducted. One was to investigate the prestressing effect of the SMA fibres, and the beams with the SMA fibres were heated at the bottom. The other was to assess the bending behaviour of the beams prestressed by the SMA fibres. The SMA fibres induced upward deflection and cracking at the top surface by heating at the bottom; thus, they achieved an obvious prestressing effect. The beams that were prestressed by the SMA fibres did not show a significant difference in bending behaviour from that of the SMA fibre reinforced beams that were not subjected to heating. Stress analysis of the beams indicated that the prestressing effect decreased in relation to the cooling temperature.
Geometrical Determinants of Neuronal Actin Waves.
Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M; Gov, Nir S; Villard, Catherine
2017-01-01
Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments.
Geometrical Determinants of Neuronal Actin Waves
Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M.; Gov, Nir S.; Villard, Catherine
2017-01-01
Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments. PMID:28424590
Actinic keratosis among seafarers.
Oldenburg, M; Kuechmeister, B; Ohnemus, U; Baur, X; Moll, I
2013-11-01
The aim of this study was to assess the prevalence of UV-induced actinic keratosis and further skin lesions. A newly developed questionnaire about lifetime UV radiation exposure was completed by 514 seafarers. An experienced dermatologist inspected the whole-body skin status of all participants. The questionnaire revealed a pre-employment UV radiation exposure in 104 seafarers, sunbed use in 26 subjects and a median work-related UV radiation exposure at sea of 20 years. The diagnosis of actinic keratoses was made in 94 seafarers and the clinical diagnosis of skin cancers in 48 seafarers (28 basal cell carcinoma, 11 squamous cell carcinoma, 9 malignant melanoma). After age standardisation according to a European reference population, the male European seafarers in this study had a 1.80-fold increased risk of actinic keratosis. Actinic keratoses [OR 1.03 (1.01-1.05)] and squamous cell carcinoma [OR 1.07 (1.01-1.13)] were related to the duration of seafaring time in years. A significant association was also found between actinic keratosis/squamous cell carcinoma and sunlight exposure during home leave [OR 1.67 (1.03-2.81) and OR 6.19 (1.18-32.40)]. Furthermore, the engine room personnel-especially the technical officers-were at higher risk of developing actinic keratosis. Due to the high prevalence of actinic keratosis especially among older seafarers with fair skin, with longer duration of seafaring employment at sea and with higher UV exposure during home leave, more intensive advice should be given on sun protection both at sea and ashore.
Towards the development of a triple SMA actuated vertical tube
NASA Astrophysics Data System (ADS)
Karimi, Saeed; Konh, Bardia; Seidi, Ebrahim
2018-03-01
In this work an active vertically hung tube has been designed, fabricated and tested. The active tube was made of three separate 3D printed parts assembled and glued together. Shape Memory Alloy (SMA) wires were embedded as actuators in the body of the tube to privilege from their robust actuation and high energy density. Three SMA wires were trained and installed evenly on the exterior peripheral side of the tubes to realize motion in multiple directions. A deadweight was hung to one end of the tube to exert a certain amount of pre-stress on actuators. This design offers a restricted actuation because the two wires on the opposite side always resist the intended deflection. Hence, for a proper actuation, each wire was stressed to a certain level to exhibit either expansion or contraction upon demand. This amount of stress was selected based on rigorous experimental data. Power supply units were integrated and linked to a python program to control the amount of power passed through each SMA wire. The active tube was tested, and its movement was captured via a camera and analyzed by ImageJ software for the two cases free of stress and with an applied external load. The electrical resistance of the each SMA wire was measured and used for controlling the tube's deflection in each direction. This work demonstrated the feasibility of using three evenly distributed SMA wires on a tube to create motion in 3D direction.
Izoré, Thierry; van den Ent, Fusinita
2017-01-01
A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.
Types of SMA (Spinal Muscular Atrophy)
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Crack-closing of cement mortar beams using NiTi cold-drawn SMA short fibers
NASA Astrophysics Data System (ADS)
Choi, Eunsoo; Kim, Dong Joo; Chung, Young-Soo; Kim, Hee Sun; Jung, Chungsung
2015-01-01
In this study, crack-closing tests of mortar beams reinforced by shape memory alloy (SMA) short fibers were performed. For this purpose, NiTi SMA fibers with a diameter of 0.965 mm and a length of 30 mm were made from SMA wires of 1.0 mm diameter by cold drawing. Four types of SMA fibers were prepared, namely, straight and dog-bone-shaped fiber and the two types of fibers with paper wrapping in the middle of the fibers. The paper provides an unbonded length of 15 mm. For bending tests, six types of mortar beams with the dimensions of 40 mm × 40 mm × 160 mm (B×H×L) were prepared. The SMA fibers were placed at the bottom center of the beams along with an artificial crack of 10 mm depth and 1 mm thickness. This study investigated the influence of SMA fibers on the flexural strength of the beams from the measured force- deflection curves. After cracking, the beams were heated at the bottom by fire to activate the SMA fibers. Then, the beams recovered the deflection, and the cracks were closed. This study evaluated crack-closing capacity using the degree of crack recovery and deflection-recovery factor. The first factor is estimated from the crack-width before and after crack-closing, and the second one is obtained from the downward deflection due to loading and the upward deflection due to the closing force of the SMA fibers.
Baum, Jake; Tonkin, Christopher J; Paul, Aditya S; Rug, Melanie; Smith, Brian J; Gould, Sven B; Richard, Dave; Pollard, Thomas D; Cowman, Alan F
2008-03-13
Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.
Surface-induced polymerization of actin.
Renault, A; Lenne, P F; Zakri, C; Aradian, A; Vénien-Bryan, C; Amblard, F
1999-01-01
Living cells contain a very large amount of membrane surface area, which potentially influences the direction, the kinetics, and the localization of biochemical reactions. This paper quantitatively evaluates the possibility that a lipid monolayer can adsorb actin from a nonpolymerizing solution, induce its polymerization, and form a 2D network of individual actin filaments, in conditions that forbid bulk polymerization. G- and F-actin solutions were studied beneath saturated Langmuir monolayers containing phosphatidylcholine (PC, neutral) and stearylamine (SA, a positively charged surfactant) at PC:SA = 3:1 molar ratio. Ellipsometry, tensiometry, shear elastic measurements, electron microscopy, and dark-field light microscopy were used to characterize the adsorption kinetics and the interfacial polymerization of actin. In all cases studied, actin follows a monoexponential reaction-limited adsorption with similar time constants (approximately 10(3) s). At a longer time scale the shear elasticity of the monomeric actin adsorbate increases only in the presence of lipids, to a 2D shear elastic modulus of mu approximately 30 mN/m, indicating the formation of a structure coupled to the monolayer. Electron microscopy shows the formation of a 2D network of actin filaments at the PC:SA surface, and several arguments strongly suggest that this network is indeed causing the observed elasticity. Adsorption of F-actin to PC:SA leads more quickly to a slightly more rigid interface with a modulus of mu approximately 50 mN/m. PMID:10049338
Contreras, Osvaldo; Rebolledo, Daniela L; Oyarzún, Juan Esteban; Olguín, Hugo C; Brandan, Enrique
2016-06-01
Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.
Chen, Bo; Tang, Kanglai; Zhang, Jiqiang; Guo, Yupeng; Liu, Xiangzhou; Shi, Youxin
2015-02-01
To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/ soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor y (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein (aP2). The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P < 0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary
Mesoscopic model of actin-based propulsion.
Zhu, Jie; Mogilner, Alex
2012-01-01
Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.
Polycation induced actin bundles.
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. Copyright © 2011 Elsevier B.V. All rights reserved.
Eskin, Julian A.; Jaiswal, Richa
2017-01-01
Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT–actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration. PMID:28663347
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.
Xu, Feifei; Wang, You; Tao, Tianqi; Song, Dandan; Liu, Xiuhua
2017-01-01
Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm 2 , for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.
França, Maria Eduarda Rocha de; Rocha, Sura Wanessa Santos; Oliveira, Wilma Helena; Santos, Laise Aline; de Oliveira, Anne Gabrielle Vasconcelos; Barbosa, Karla Patrícia Sousa; Nunes, Ana Karolina Santana; Rodrigues, Gabriel Barros; Lós, Deniele Bezerra; Peixoto, Christina Alves
2018-04-01
While diethylcarbamazine citrate (DEC) displays important anti-inflammatory effects in experimental models of liver injury, the mechanisms of its action remain poorly understood. The aim of the present study was to investigate the fibrolytic potential of DEC. Mice receive two injections of carbon tetrachloride (CCl 4 ) per week for 8 weeks. DEC 50 mg/kg body weight was administered through drinking water during the last 12 days of liver injury. The expression of hepatic stellate cells (HSCs) activation markers, including smooth muscle α-actin (α-SMA), collagen I, transforming growth factor-β 1 (TGF-β1), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) was assessed. The influence of DEC on the intracellular MAPK pathways of the HSCs (JNK and p38 MAPK) was also estimated. DEC inhibited HSCs activation measured as the production of α-SMA and collagen I. In addition, it down regulated the production of TGF-β1 and TIMP-1, and concomitantly increased MMP-2 activity. Furthermore, DEC significantly inhibited the activation of the JNK and p38 MAPK signaling pathways. In conclusion, DEC significantly attenuated the severity of CCl 4 -induced liver injury and the progression of liver fibrosis, exerting a potential fibrolytic effect in the CCl 4 -induced fibrosis model.
Zucca, A; Brizzi, S; Riccioni, R; Azzarà, A; Ghimenti, M; Carulli, G
2006-01-01
Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG-CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation.
Feghali-Bostwick, Carol A.; Amrani, Yassine; Bradding, Peter
2015-01-01
Idiopathic pulmonary fibrosis (IPF) is a common, progressive, and invariably lethal interstitial lung disease with no effective therapy. The key cell driving the development of fibrosis is the myofibroblast. Lipoxin A4 (LXA4) is an anti-inflammatory lipid, important in the resolution of inflammation, and it has potential antifibrotic activity. However, the effects of LXA4 on primary human lung myofibroblasts (HLMFs) have not previously been investigated. Therefore, the aim of this study was to examine the effects of LXA4 on TGF-β1–dependent responses in IPF- and nonfibrotic control (NFC)–derived HLMFs. HLMFs were isolated from IPF and NFC patients and grown in vitro. The effects of LXA4 on HLMF proliferation, collagen secretion, α-smooth muscle actin (αSMA) expression, and Smad2/3 activation were examined constitutively and following TGF-β1 stimulation. The LXA4 receptor (ALXR) was expressed in both NFC- and IPF-derived HLMFs. LXA4 (10−10 and 10−8 mol) reduced constitutive αSMA expression, actin stress fiber formation, contraction, and nuclear Smad2/3, indicating regression from a myofibroblast to fibroblast phenotype. LXA4 also significantly inhibited FBS-dependent proliferation and TGF-β1–dependent collagen secretion, αSMA expression, and Smad2/3 nuclear translocation in IPF-derived HLMFs. LXA4 did not inhibit Smad2/3 phosphorylation. In summary, LXA4 attenuated profibrotic HLMF activity and promoted HLMF regression to a quiescent fibroblast phenotype. LXA4 or its stable analogs delivered by aerosol may offer a novel approach to the treatment of IPF. PMID:26276873
NASA Astrophysics Data System (ADS)
Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.
2008-06-01
Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.
Novel Adult Stem Cells for Peripheral Nerve Regeneration
2013-09-01
conduit (cross sections). (a) Neurofilamen (NFM) staining shows that the implanted MVSC (GFP+) formed circular structure wrapping the newly...F-actin (g–i) (nuclei were stained with DAPI) or used for qPCR to measure the gene expression of smA and Cnn1 (j–k). 18s ribosomal RnA was used to...Publishers Limited. All rights reserved. To determine whether MVSCs could differentiate into mature SMCs and turn on EGFP expression, we activated Notch
Preliminary studies on SMA embedded wind turbine blades for passive control of vibration
NASA Astrophysics Data System (ADS)
Haghdoust, P.; Cinquemani, S.; Lo Conte, A.
2018-03-01
Wind turbine blades are being bigger and bigger, thus requiring lightweight structures that are more flexible and thus more sensitive to dynamic excitations and to vibration problems. This paper investigates a preliminary architecture of large wind turbine blades, embedding thin sheets of SMA to passively improve their total damping. A phenomenological material model is used for simulation of strain-dependent damping in SMA materials and an user defined material model was developed for this purpose. The response of different architectures of SMA embedded blades have been investigated in the time domain to find an optimal solution in which the less amount of SMA is used while the damping of the system is maximized
Design and quasi-static characterization of SMASH (SMA stabilizing handgrip)
NASA Astrophysics Data System (ADS)
Pathak, Anupam; Brei, Diann; Luntz, Jonathan; LaVigna, Chris; Kwatny, Harry
2007-04-01
Due to physiologically induced body tremors, there is a need for active stabilization in many hand-held devices such as surgical tools, optical equipment (cameras), manufacturing tools, and small arms weapons. While active stabilization has been achieved with electromagnetic and piezoceramics actuators for cameras and surgical equipment, the hostile environment along with larger loads introduced by manufacturing and battlefield environments make these approaches unsuitable. Shape Memory Alloy (SMA) actuators are capable of alleviating these limitations with their large force/stroke generation, smaller size, lower weight, and increased ruggedness. This paper presents the actuator design and quasi-static characterization of a SMA Stabilizing Handgrip (SMASH). SMASH is an antagonistically SMA actuated two degree-of-freedom stabilizer for disturbances in the elevation and azimuth directions. The design of the SMASH for a given application is challenging because of the difficulty in accurately modeling systems loads such as friction and unknown shakedown SMA material behavior (which is dependent upon the system loads). Thus, an iterative empirical design process is introduced that provides a method to estimate system loads, a SMA shakedown procedure using the system loads to reduce material creep, and a final selection and prediction for the full SMASH system performance. As means to demonstrate this process, a SMASH was designed, built and experimentally characterized for the extreme case study of small arms stabilization for a US Army M16 rifle. This study successfully demonstrated the new SMASH technology along with the unique design procedure that can be applied to small arms along with a variety of other hand-held devices.
Simulated and Experimental Damping Properties of a SMA/Fiber Glass Laminated Composite
NASA Astrophysics Data System (ADS)
Arnaboldi, S.; Bassani, P.; Biffi, C. A.; Tuissi, A.; Carnevale, M.; Lecis, N.; Loconte, A.; Previtali, B.
2011-07-01
In this article, an advanced laminated composite is developed, combining the high damping properties of shape memory alloy (SMA) with mechanical properties and light weight of a glass-fiber reinforced polymer. The composite is formed by stacking a glass-fiber reinforced epoxy core between two thin patterned strips of SMA alloy, and two further layers of fiber-glass reinforced epoxy. The bars of the laminated composite were assembled and cured in autoclave. The patterning was designed to enhance the interface adhesion between matrix and SMA inserts and optimally exploit the damping capacity of the SMA thin ribbons. The patterned ribbons of the SMA alloy were cut by means of a pulsed fiber laser source. Damping properties at different amplitudes on full scale samples were investigated at room temperature with a universal testing machine through dynamic tension tests, while temperature dependence was investigated by dynamic mechanical analyses (DMA) on smaller samples. Experimental results were used in conjunction with FEM analysis to optimize the geometry of the inserts. Experimental decay tests on the laminated composite have been carried out to identify the adimensional damping value related to their first flexural mode.
Zuo, Yan; Gao, Jing; Yeung, William S B; Lee, Kai-Fai
2010-10-15
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 β-actin proteins in vitro. Immunogold electron microscopic study localized VAD1.3 immunoreactivity to the acrosome membranes and matrix, and colocalized it with the β-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 β-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 β-actin for acrosome formation in spermatogenesis. Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.
Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen
2009-11-01
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.
Finite Element Analysis of Adaptive-Stiffening and Shape-Control SMA Hybrid Composites
NASA Technical Reports Server (NTRS)
Gao, Xiu-Jie; Turner, Travis L.; Burton, Deborah; Brinson, L. Catherine
2005-01-01
The usage of shape memory materials has extended rapidly to many fields, including medical devices, actuators, composites, structures and MEMS devices. For these various applications, shape memory alloys (SMAs) are available in various forms: bulk, wire, ribbon, thin film, and porous. In this work, the focus is on SMA hybrid composites with adaptive-stiffening or morphing functions. These composites are created by using SMA ribbons or wires embedded in a polymeric based composite panel/beam. Adaptive stiffening or morphing is activated via selective resistance heating or uniform thermal loads. To simulate the thermomechanical behavior of these composites, a SMA model was implemented using ABAQUS user element interface and finite element simulations of the systems were studied. Several examples are presented which show that the implemented model can be a very useful design and simulation tool for SMA hybrid composites.
Nuclear positioning by actin cables and perinuclear actin
Huelsmann, Sven; Brown, Nicholas H
2014-01-01
Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used. PMID:24905988
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. Copyright © 2010 Elsevier Inc. All rights reserved.
Non-Straub type actin from molluscan catch muscle
DOE Office of Scientific and Technical Information (OSTI.GOV)
Shelud'ko, Nikolay S., E-mail: sheludko@stl.ru; Girich, Ulyana V.; Lazarev, Stanislav S.
We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Crenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6 M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for themore » extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization–depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers. -- Highlights: •We developed method of repolymerizable invertebrate smooth muscle actin obtaining. •Our method does not involve use of denaturating agents, which could modify proteins. •Viscosity and polymerization rate of actin, gained that way, is similar to Straub one. •Electron microscopy showed that repolymerized mussel actin is similar to Straub one. •Repolymerized mussel actin has greater ATPase activating capacity, than Straub actin.« less
Circadian actin dynamics drive rhythmic fibroblast mobilisation during wound healing
Hoyle, Nathaniel P.; Seinkmane, Estere; Putker, Marrit; Feeney, Kevin A.; Krogager, Toke P.; Chesham, Johanna E.; Bray, Liam K.; Thomas, Justyn M.; Dunn, Ken; Blaikley, John; O’Neill, John S.
2017-01-01
Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping which imparts a ~24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately impact the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse’s active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than night-time wounds. We suggest that circadian regulation of the cytoskeleton influences wound healing efficacy from the cellular to the organismal scale. PMID:29118260
Analysis and Evaluation of the Dynamic Performance of SMA Actuators for Prosthetic Hand Design
NASA Astrophysics Data System (ADS)
O'Toole, Kevin T.; McGrath, Mark M.; Coyle, Eugene
2009-08-01
It is widely acknowledged within the biomedical engineering community that shape memory alloys (SMAs) exhibit great potential for application in the actuation of upper limb prosthesis designs. These lightweight actuators are particularly suitable for prosthetic hand solutions. A four-fingered, 12 degree-of-freedom prosthetic hand has been developed featuring SMA bundle actuators embedded within the palmar structure. Joule heating of the SMA bundle actuators generates sufficient torque at the fingers to allow a wide range of everyday tasks to be carried out. Transient characterization of SMA bundles has shown that performance/response during heating and cooling differs substantially. Natural convection is insufficient to provide for adequate cooling during elongation of the actuators. An experimental test-bed has been developed to facilitate analysis of the heat transfer characteristics of the appropriately sized SMA bundle actuators for use within the prosthetic hand design. Various modes of heat sinking are evaluated so that the most effective wire-cooling solution can be ascertained. SMA bundles of varying size will be used so that a generalized model of the SMA displacement performance under natural and forced cooling conditions can be obtained. The optimum cooling solution will be implemented onto the mechanical hand framework in future work. These results, coupled with phenomenological models of SMA behavior, will be used in the development of an effective control strategy for this application in future work.
Gordon, Jennifer L; Sibley, L David
2005-01-01
Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility. PMID:16343347
Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk
2013-01-01
Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888
Application of shape memory alloy (SMA) spars for aircraft maneuver enhancement
NASA Astrophysics Data System (ADS)
Nam, Changho; Chattopadhyay, Aditi; Kim, Youdan
2002-07-01
Modern combat aircraft are required to achieve aggressive maneuverability and high agility performance, while maintaining handling qualities over a wide range of flight conditions. Recently, a new adaptive-structural concept called variable stiffness spar is proposed in order to increase the maneuverability of the flexible aircraft. The variable stiffness spar controls wing torsional stiffness to enhance roll performance in the complete flight envelope. However, variable stiffness spar requires the mechanical actuation system in order to rotate the Variable stiffness spar during flight. The mechanical actuation system to rotate variable stiffness spar may cause an additional weight increase. In this paper, we will apply Shape Memory Alloy (SMA) spars for aeroelastic performance enhancement. In order to explore the potential of SMA spar design, roll performance of the composite smart wings will be investigated using ASTROS. Parametric study will be conducted to investigate the SMA spar effects by changing the spar locations and geometry. The results show that with activation of the SMA spar, the roll effectiveness can be increased up to 61% compared with the baseline model.
Application study on aircraft structures of CFRP laminates with embedded SMA foils
NASA Astrophysics Data System (ADS)
Ogisu, Toshimichi; Nomura, Masato; Ando, Norio; Takaki, Junji; Takeda, Nobuo
2002-07-01
This paper reports some research results for the application study of the smart materials an structural using Shape Memory Alloy (SMA) foils. First, the authors acquired the recovery strain of CFRP laminates generated by the recovery stress of the pre-strained SMA foils. Then, the quasi-static load-unload tests were conducted using several kinds of quasi-isotropic CFRP laminates with embedded SMA foils. Micro-mechanics of damage behavior due to the effects of the recovery strain and the first transverse crack strain were discussed. The improvement of maximum 40 percent for the onset strain of the transverse cracks and maximum 60 percent for the onset strain of delamination were achieved for CFRP laminates with embedded pre-strained SMA foils compared with standard CFRP laminates. Furthermore, the authors conducted the structural element test for application to actual structures. Testing technique and the manufacturing technique of the structural element specimen were established.
Apical and basal epitheliomuscular F-actin dynamics during Hydra bud evagination
Aufschnaiter, Roland; Wedlich-Söldner, Roland; Zhang, Xiaoming
2017-01-01
ABSTRACT Bending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analyzed actin-based processes during asexual bud evagination in the simple metazoan Hydra. We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells. We then combined live imaging with conventional phalloidin staining to directly follow actin reorganization. Bending of the Hydra epithelial double layer is initiated by a group of epitheliomuscular cells in the endodermal layer. These cells shorten their apical-basal axis and arrange their basal muscle processes in a circular configuration. We propose that this rearrangement generates the initial forces to bend the endoderm towards the ectoderm. Convergent tissue movement in both epithelial layers towards the centre of evagination then leads to elongation and extension of the bud along its new body axis. Tissue movement into the bud is associated with lateral intercalation of epithelial cells, remodelling of apical septate junctions, and rearrangement of basal muscle processes. The work presented here extends the analysis of morphogenetic mechanisms beyond embryonic tissues of model bilaterians. PMID:28630355
Xu, Bai-Hui; Liu, Yu; Wang, Ya-Long; Chen, Ming-Huang; Xu, Lin; Liao, Bao-Qiong; Lui, Rui; Li, Fei-Ping; Lin, Yan-Hong; Fu, Xian-Pei; Fu, Bin-Bin; Hong, Zi-Wei; Qi, Zhong-Quan
2017-01-01
ABSTRACT SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes. PMID:28933599
He, Shu-Wen; Xu, Bai-Hui; Liu, Yu; Wang, Ya-Long; Chen, Ming-Huang; Xu, Lin; Liao, Bao-Qiong; Lui, Rui; Li, Fei-Ping; Lin, Yan-Hong; Fu, Xian-Pei; Fu, Bin-Bin; Hong, Zi-Wei; Liu, Yu-Xin; Qi, Zhong-Quan; Wang, Hai-Long
2017-01-01
SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.
Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido
2016-05-15
The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles
Tong, Peter; Khayat, Zayna A.; Huang, Carol; Patel, Nish; Ueyama, Atsunori; Klip, Amira
2001-01-01
Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular compartment to the cell surface; this phenomenon is defective in type 2 diabetes. Here we examine the involvement of actin filaments in GLUT4 translocation and their possible defects in insulin resistance, using L6 myotubes expressing myc-tagged GLUT4. Insulin caused membrane ruffling, a dynamic distortion of the myotube dorsal surface. Fluorescence microscopy and immunogold staining of surface GLUT4myc coupled to backscatter electron microscopy revealed a high density of this protein in membrane ruffles. The t-SNAREs syntaxin4 and SNAP-23 were also abundant in these regions. Below the membrane, GLUT4 and the vesicular protein VAMP2, but not VAMP3, colocalized with the actin structures supporting the membrane ruffles. GLUT4myc externalization and membrane ruffles were reduced by jasplakinolide and by swinholide-A, drugs that affect actin filament stability and prevent actin branching, respectively. Insulin resistance generated by prolonged (24 hours) exposure of myotubes to high glucose and insulin diminished the acute insulin-dependent remodeling of cortical actin and GLUT4myc translocation, reminiscent of the effect of swinholide-A. We propose that GLUT4 vesicle incorporation into the plasma membrane involves insulin-dependent cortical actin remodeling and that defective actin remodeling contributes to insulin resistance. PMID:11489930
Baker, Joseph L.; Courtemanche, Naomi; Parton, Daniel L.; ...
2014-12-04
Formins catalyze nucleation and growth of actin filaments. In this paper, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and showed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L, or both) reduced the interactionmore » energies between the proteins, and in coarse-grained simulations, the formin lost more interprotein contacts with an actin dimer than with an actin 7-mer. Finally, biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins.« less
The design, hysteresis modeling and control of a novel SMA-fishing-line actuator
NASA Astrophysics Data System (ADS)
Xiang, Chaoqun; Yang, Hui; Sun, Zhiyong; Xue, Bangcan; Hao, Lina; Asadur Rahoman, M. D.; Davis, Steve
2017-03-01
Fishing line can be combined with shape memory alloy (SMA) to form novel artificial muscle actuators which have low cost, are lightweight and soft. They can be applied in bionic, wearable and rehabilitation robots, and can reduce system weight and cost, increase power-to-weight ratio and offer safer physical human-robot interaction. However, these actuators possess several disadvantages, for example fishing line based actuators possess low strength and are complex to drive, and SMA possesses a low percentage contraction and has high hysteresis. This paper presents a novel artificial actuator (known as an SMA-fishing-line) made of fishing line and SMA twisted then coiled together, which can be driven directly by an electrical voltage. Its output force can reach 2.65 N at 7.4 V drive voltage, and the percentage contraction at 4 V driven voltage with a 3 N load is 7.53%. An antagonistic bionic joint driven by the novel SMA-fishing-line actuators is presented, and based on an extended unparallel Prandtl-Ishlinskii (EUPI) model, its hysteresis behavior is established, and the error ratio of the EUPI model is determined to be 6.3%. A Joule heat model of the SMA-fishing-line is also presented, and the maximum error of the established model is 0.510 mm. Based on this accurate hysteresis model, a composite PID controller consisting of PID and an integral inverse (I-I) compensator is proposed and its performance is compared with a traditional PID controller through simulations and experimentation. These results show that the composite PID controller possesses higher control precision than basic PID, and is feasible for implementation in an SMA-fishing-line driven antagonistic bionic joint.
The actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations
NASA Astrophysics Data System (ADS)
Westendorf, Christian; Negrete, Jose, Jr.; Bae, Albert; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten
2013-03-01
We report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. The averaged F-actin response of many cells to a short-time pulse of cAMP is reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. We systematically exposed a large number of cells to periodic pulse trains. The results indicate a resonance peak at periodic inputs of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the actin regulatory network. To quantitatively test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and Aip1. These served as markers of the F-actin disassembly process and thus allow us to estimate the delay time. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed experimentally. Financial support by the Max-Planck Society and the DFG (SFB 937).
Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.
Sava, Parid; Ramanathan, Anand; Dobronyi, Amelia; Peng, Xueyan; Sun, Huanxing; Ledesma-Mendoza, Adrian; Herzog, Erica L; Gonzalez, Anjelica L
2017-12-21
Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-β1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.
Belsky, Justin B; Morris, Daniel C; Bouchebl, Ralph; Filbin, Michael R; Bobbitt, Kevin R; Jaehne, Anja K; Rivers, Emanuel P
2016-01-01
To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls. F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml). F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.
Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang
2017-03-01
In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.
Molecular and functional analyses of the contractile apparatus in lymphatic muscle
NASA Technical Reports Server (NTRS)
Muthuchamy, Mariappan; Gashev, Anatoliy; Boswell, Niven; Dawson, Nancy; Zawieja, David; Delp, Z. (Principal Investigator)
2003-01-01
Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM-MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM-MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow-twitch muscle-specific beta-MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac alpha-actin, vascular alpha-actin, enteric gamma-actin, and skeletal alpha-actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac alpha-actin and vascular alpha-actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular alpha-actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.
A systems-biology approach to yeast actin cables.
Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios
2012-01-01
We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin-cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin-monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions - among actin-monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins - and the emergence of cell-scale physical form as embodied by the actin cables themselves.
Human MSC gene expression under simulated microgravity (RPM)
NASA Astrophysics Data System (ADS)
Buravkova, Ludmila; Gershovich, Pavel; Grigoriev, Anatoly
It is generally supposed that microgravity cell response is mediated by some structures of actin cytoskeleton that can be implicated in cell mechanosensitivity. Cytoskeletal reorganization in the microgravity environment can affect gene expression, which results in alterations of cell function. However the direct impact of microgravity on expression of some cytoskeletal genes and encoded proteins remains unknown. Multipotential adult mesechymal stromal cells (MSCs) are the early precursors of bone marrow that can be induced to differentiate into bone-like cells as well as to the other mesenchymal tissues. In our previous experiments we revealed cytoskele-ton alterations and reduced human MSCs growth and osteogenesis in simulated microgravity by Random Positioning Machine. The purpose of this study was to determine the impact of low gravity on F-actin organization and gene expression level of α-, β-, γ-actin, vinculin, cofilin, small GTPase RhoA, Rho kinase (ROCK) and protein expression of some adhesion molecules in cultured hMSCs. Fluorescent microscopy have shown that even 30 min of SMG results in rearrangement of F-actin and the lack of stress fibers in cultured hMSCs. Cell number with abnormal F-actin organization was increased after 6 h, 24 h and 48 h of SMG. On the other hand, after 120 hours of SMG cells displayed partial restoration of F-actin fibers in comparison with 24 h and 48 h. Similarly, near the same restoration was seen in F-actin after readaptation for 24 h in 1g environment after 24 h of SMG. However, the observed alterations in F-actin dimensional organization were accompanied by changes in related proteins gene expression. Real-time PCR revealed slight up-regulation of α-actin expression that became more signifi-cant after 48 h of SMG. Down-regulation of γ-actin was observed after 48 hours of exposure in RPM. Moreover the up-regulation of β-tubulin, cofilin and small GTPase RhoA gene expres-sion was also detected after 48 h of SMG. On the
Kheir, Wassim Abou; Gevrey, Jean-Claude; Yamaguchi, Hideki; Isaac, Beth; Cox, Dianne
2005-11-15
Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.
Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis
2008-01-01
Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447
Geometrical and Mechanical Properties Control Actin Filament Organization
Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent
2015-01-01
The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model. PMID:26016478
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.
Actin filaments-A target for redox regulation.
Wilson, Carlos; Terman, Jonathan R; González-Billault, Christian; Ahmed, Giasuddin
2016-10-01
Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates-the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL-and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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. Copyright © 2012 Elsevier GmbH. All rights reserved.
Actin dynamics in Amoeba proteus motility.
Pomorski, P; Krzemiński, P; Wasik, A; Wierzbicka, K; Barańska, J; Kłopocka, W
2007-01-01
We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.
A Systems-Biology Approach to Yeast Actin Cables
Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios
2011-01-01
We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions—among actin monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins—and the emergence of cell-scale physical form as embodied by the actin cables themselves. PMID:22161338
Bryan, Keith E.; Rubenstein, Peter A.
2009-01-01
Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients. PMID:19419963
Primary motor cortex activity reduction under the regulation of SMA by real-time fMRI
NASA Astrophysics Data System (ADS)
Guo, Jia; Zhao, Xiaojie; Li, Yi; Yao, Li; Chen, Kewei
2012-03-01
Real-time fMRI (rtfMRI) is a new technology which allows human subjects to observe and control their own BOLD signal change from one or more localized brain regions during scanning. Current rtfMRI-neurofeedback studies mainly focused on the target region itself without considering other related regions influenced by the real-time feedback. However, there always exits important directional influence between many of cooperative regions. On the other hand, rtfMRI based on motor imagery mainly aimed at somatomotor cortex or primary motor area, whereas supplement motor area (SMA) was a relatively more integrated and pivotal region. In this study, we investigated whether the activities of SMA can be controlled utilizing different motor imagery strategies, and whether there exists any possible impact on an unregulated but related region, primary motor cortex (M1). SMA was first localized using overt finger tapping task, the activities of SMA were feedback to subjects visually on line during each of two subsequent imagery motor movement sessions. All thirteen healthy participants were found to be able to successfully control their SMA activities by self-fit imagery strategies which involved no actual motor movements. The activation of right M1 was also found to be significantly reduced in both intensity and extent with the neurofeedback process targeted at SMA, suggestive that not only the part of motor cortex activities were influenced under the regulation of a key region SMA, but also the increased difference between SMA and M1 might reflect the potential learning effect.
Yang, Cheng; Li, Li; Guo, Junhua; Zhang, Weiqiang; Zhu, Wenbiao; Rao, Xinhui; Huang, Wenjie
2017-04-01
Pim-3 belongs to the PIM kinase family and plays an important role in promoting inflammation, which is essential in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Immunohistochemistry (IHC), western blot, and RT-PCR analyses were performed to assess the expression of Pim-3 in both COPD and healthy lung tissue samples. SMA (Smooth Muscle Actin) and Cyclin D1 expression were detected by IHC. We also constructed animal models for the control, COPD, and Pim-3 inhibition groups, in order to analyze the effects of Pim-3 inhibition on COPD, and the role of Pim-3 in the p38 pathway. Compared with normal lung tissue, Pim-3 mRNA and protein were up-regulated in COPD tissue. Expression of Cyclin D1 and SMA were also up-regulated in the COPD group. In the animal model experiment, we found that suppression of Pim-3 decreased Pim-3, Cyclin D1, and SMA expression, as well as ameliorated lung damage in COPD patients. The inhibition of Pim-3 also resulted in the suppression of the p38 pathway. Our study suggests that up-regulation of Pim-3 successfully accelerated COPD development, and aggravated lung damage. The molecular mechanism of Pim-3 in COPD might be related to the p38 pathway, and is correlated with Cyclin D1 and SMA expression. Copyright © 2017 Elsevier GmbH. All rights reserved.
Yang, Lujun; Zhang, Dangui; Wu, Hongjuan; Xie, Sitian; Zhang, Mingjun; Zhang, Bingna; Tang, Shijie
2018-05-30
To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model. © 2018 S. Karger AG, Basel.
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.
Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.
Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha
2018-05-01
Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.
F-Actin Dynamics in Neurospora crassa ▿ †
Berepiki, Adokiye; Lichius, Alexander; Shoji, Jun-Ya; Tilsner, Jens; Read, Nick D.
2010-01-01
This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth. PMID:20139238
Xing, Fang-Zhou; Zhao, Yan-Gang; Zhang, Yuan-Yuan; He, Li; Zhao, Ji-Kai; Liu, Meng-Ying; Liu, Yan; Zhang, Ji-Qiang
2018-06-01
Estrogens play pivotal roles in hippocampal synaptic plasticity through nuclear receptors (nERs; including ERα and ERβ) and the membrane receptor (mER; also called GPR30), but the underlying mechanism and the contributions of nERs and mER remain unclear. Mammalian target of rapamycin complex 2 (mTORC2) is involved in actin cytoskeleton polymerization and long-term memory, but whether mTORC2 is involved in the regulation of hippocampal synaptic plasticity by ERs is unclear. We treated animals with nER antagonists (MPP/PHTPP) or the mER antagonist (G15) alone or in combination with A-443654, an activator of mTORC2. Then, we examined the changes in hippocampal SRC-1 expression, mTORC2 signaling (rictor and phospho-AKTSer473), actin polymerization (phospho-cofilin and profilin-1), synaptic protein expression (GluR1, PSD95, spinophilin, and synaptophysin), CA1 spine density, and synapse density. All of the examined parameters except synaptophysin expression were significantly decreased by MPP/PHTPP and G15 treatment. MPP/PHTPP and G15 induced a similar decrease in most parameters except p-cofilin, GluR1, and spinophilin expression. The ER antagonist-induced decreases in these parameters were significantly reversed by mTORC2 activation, except for the change in SRC-1, rictor, and synaptophysin expression. nERs and mER contribute similarly to the changes in proteins and structures associated with synaptic plasticity, and mTORC2 may be a novel target of hippocampal-dependent dementia such as Alzheimer's disease as proposed by previous studies. © 2018 John Wiley & Sons Ltd.
Eastern, J S; Martin, S
1980-12-01
Solitary plaques developed on the sun-exposed and damaged skin of five elderly, fair-skinned individuals. The lesions, erythematous to bluish confluent nodules and plaques with a cribriform appearance and comedone-like structures, presented a distinctive histologic picture of dilated, keratin-filled follicles within a matrix of amorphous, damaged collagen. We believe these cases demonstrate a distinct entity within the realm of actinic dermatoses, for which the name "actinic comedonal plaque" seems appropriate.
Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François
2006-11-01
APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.
STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.
Woroniuk, Anna; Porter, Andrew; White, Gavin; Newman, Daniel T; Diamantopoulou, Zoi; Waring, Thomas; Rooney, Claire; Strathdee, Douglas; Marston, Daniel J; Hahn, Klaus M; Sansom, Owen J; Zech, Tobias; Malliri, Angeliki
2018-05-29
The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.
Control of the actin cytoskeleton in root hair development.
Pei, Weike; Du, Fei; Zhang, Yi; He, Tian; Ren, Haiyun
2012-05-01
The development of root hair includes four stages: bulge site selection, bulge formation, tip growth, and maturation. The actin cytoskeleton is involved in all of these stages and is organized into distinct arrangements in the different stages. In addition to the actin configuration, actin isoforms also play distinct roles in the different stages. The actin cytoskeleton is regulated by actin-binding proteins, such as formin, Arp2/3 complex, profilin, actin depolymerizing factor, and villin. Some upstream signals, i.e. calcium, phospholipids, and small GTPase regulate the activity of these actin-binding proteins to produce the proper actin configuration. We constructed a working model on how the actin cytoskeleton is controlled by actin-binding proteins and upstream signaling in root hair development based on the current literature: at the tip of hairs, actin polymerization appears to be facilitated by Arp2/3 complex that is activated by small GTPase, and profilin that is regulated by phosphatidylinositol 4,5-bisphosphate. Meanwhile, actin depolymerization and turnover are likely mediated by villin and actin depolymerizing factor, which are stimulated by calcium. At the shank, actin cables are produced by formin and villin. Under the complicated interaction, the actin cytoskeleton is controlled spatially and temporally during root hair development. © 2012 Elsevier Ireland Ltd. All rights reserved.
Disrupting actin-myosin-actin connectivity in airway smooth muscle as a treatment for asthma?
Lavoie, Tera L; Dowell, Maria L; Lakser, Oren J; Gerthoffer, William T; Fredberg, Jeffrey J; Seow, Chun Y; Mitchell, Richard W; Solway, Julian
2009-05-01
Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.
Yu, Shan-Shan; Chen, Biao; Huang, Chen-Kai; Zhou, Juan-Juan; Huang, Xin; Wang, An-Jiang; Li, Bi-Min; He, Wen-Hua; Zhu, Xuan
2017-01-01
Activation of quiescent hepatic stellate cells (q-HSCs) and their transformation to myofibroblasts (MFBs) is a key event in liver fibrosis. Hedgehog (Hh) signaling stimulates q-HSCs to differentiate into MFBs, and NADPH oxidase (NOX) may be involved in regulating Hh signaling. The author's preliminary study demonstrated that ursolic acid (UA) selectively induces apoptosis in activated HSCs and inhibits their proliferation in vitro via negative regulation of NOX activity and expression. However, the effect of UA on q-HSCs remains to be elucidated. The present study aimed to investigate the effect of UA on q-HSC activation and HSC transformation and to observe alterations in the NOX and Hh signaling pathways during q-HSC activation. q-HSC were isolated from adult male Sprague-Dawley rats. Following culture for 3 days, the cells were treated with or without transforming growth factor-β1 (TGF-β1; 5 µg/l); intervention groups were pretreated with UA (40 µM) or diphenyleneiodonium chloride (DPI; 10 µM) for 30 min prior to addition of TGF-β1. mRNA and protein expression of NOX and Hh signaling components and markers of q-HSC activation were examined by western blotting and reverse transcription-polymerase chain reaction. TGF-β1 induced activation of q-HSCs, with increased expression of α-smooth muscle actin (α-SMA) and type I collagen. In addition, expression of NOX subunits (gp91phox, p67phox, p22phox, and Rac1) and Hh signaling components, including sonic Hh, sterol-4-alpha-methyl oxidase, and Gli family zinc finger 2, were upregulated in activated HSCs. Pretreatment of q-HSCs with UA or DPI prior to TGF-β1 significantly downregulated expression of NOX subunits and Hh signaling components and additionally inhibited expression of α-SMA and type I collagen, thereby preventing transformation to MFBs. UA inhibited TGF-β1-induced activation of q-HSCs and their transformation by inhibiting expression of NOX subunits and the downstream Hh pathway. PMID:29042951
Olshina, Maya A; Angrisano, Fiona; Marapana, Danushka S; Riglar, David T; Bane, Kartik; Wong, Wilson; Catimel, Bruno; Yin, Meng-Xin; Holmes, Andrew B; Frischknecht, Friedrich; Kovar, David R; Baum, Jake
2015-07-18
Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions
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. Copyright © 2014 Elsevier B.V. All rights reserved.
Structure of the Rigor Actin-Tropomyosin-Myosin Complex
Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan
2014-01-01
The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895
Full-scale flight tests of aircraft morphing structures using SMA actuators
NASA Astrophysics Data System (ADS)
Mabe, James H.; Calkins, Frederick T.; Ruggeri, Robert T.
2007-04-01
In August of 2005 The Boeing Company conducted a full-scale flight test utilizing Shape Memory Alloy (SMA) actuators to morph an engine's fan exhaust to correlate exhaust geometry with jet noise reduction. The test was conducted on a 777-300ER with GE-115B engines. The presence of chevrons, serrated aerodynamic surfaces mounted at the trailing edge of the thrust reverser, have been shown to greatly reduce jet noise by encouraging advantageous mixing of the free, and fan streams. The morphing, or Variable Geometry Chevrons (VGC), utilized compact, light weight, and robust SMA actuators to morph the chevron shape to optimize the noise reduction or meet acoustic test objectives. The VGC system was designed for two modes of operation. The entirely autonomous operation utilized changes in the ambient temperature from take-off to cruise to activate the chevron shape change. It required no internal heaters, wiring, control system, or sensing. By design this provided one tip immersion at the warmer take-off temperatures to reduce community noise and another during the cooler cruise state for more efficient engine operation, i.e. reduced specific fuel consumption. For the flight tests a powered mode was added where internal heaters were used to individually control the VGC temperatures. This enabled us to vary the immersions and test a variety of chevron configurations. The flight test demonstrated the value of SMA actuators to solve a real world aerospace problem, validated that the technology could be safely integrated into the airplane's structure and flight system, and represented a large step forward in the realization of SMA actuators for production applications. In this paper the authors describe the development of the actuator system, the steps required to integrate the morphing structure into the thrust reverser, and the analysis and testing that was required to gain approval for flight. Issues related to material strength, thermal environment, vibration
Jenabi, Mehrnaz; Peck, Kyung K; Young, Robert J; Brennan, Nicole; Holodny, Andrei I
2014-12-01
Accurate localization of anatomically and functionally separate SMA tracts is important to improve planning prior to neurosurgery. Using fMRI and probabilistic DTI techniques, we assessed the connectivity between the frontal language area (Broca's area) and the rostral pre-SMA (language SMA) and caudal SMA proper (motor SMA). Twenty brain tumor patients completed motor and language fMRI paradigms and DTI. Peaks of functional activity in the language SMA, motor SMA and Broca's area were used to define seed regions for probabilistic tractography. fMRI and probabilistic tractography identified separate and unique pathways connecting the SMA to Broca's area - the language SMA pathway and the motor SMA pathway. For all subjects, the language SMA pathway had a larger number of voxels (P<0.0001) and higher connectivity (P<0.0001) to Broca's area than did the motor SMA pathway. In each patient, the number of voxels was greater in the language and motor SMA pathways than in background pathways (P<0.0001). No differences were found between patients with ipsilateral and those with contralateral tumors for either the language SMA pathway (degree of connectivity: P<0.36; number of voxels: 0.35) or the motor SMA pathway (degree of connectivity, P<0.28; number of voxels, P<0.74). Probabilistic tractography can identify unique white matter tracts that connect language SMA and motor SMA to Broca's area. The language SMA is more significantly connected to Broca's area than is the motor subdivision of the SMA proper. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths
Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.
2015-01-01
Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073
Actin growth profile in clathrin-mediated endocytosis
NASA Astrophysics Data System (ADS)
Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.
2017-05-01
Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.
Giovanini, Allan Fernando; de Sousa Passoni, Giuliene Nunes; Göhringer, Isabella; Deliberador, Tatiana Miranda; Zielak, João Cesar; Storrer, Carmem Lucia Muller; Costa-Casagrande, Thais Andrade; Scariot, Rafaela
2018-06-01
TGF-β1 is a cytokine that may induce both osteoneogenesis through Runx-2 or fibrosis via the transcription of α-smooth muscle actin (α-SMA). Because it has been previously known that alendronate increases the level of TGF-β1 and that under the usual condition of bone metabolism the estrogen may prevent the fibrotic effect of TGF-β1, the aim of this study was to evaluate if alendronate alters the cellular differentiation process post calvarial surgery in estrogen-deficient specimens. A transosseous defect that was 5 mm in diameter was created on the calvarium of each of 32 female rats with previous ovarian-salpingo-oophorectomy. All defects were treated with autografts, and 16 rats received the administration of 1 mg/kg of alendronate three times a week until euthanasia on the 15th and 60th day post surgery. Histomorphometric and immunohistochemical analyses of the expression of TGF-β1, estrogen receptor alpha nuclear (α-ER), α-SMA, BMPR1B, and Runx-2 were performed, and ELISA was used to measure the level of estrogen. All animals demonstrated low levels of estrogen post ovarian-salpingo-oophorectomy. The histological results demonstrated larger bone matrix deposition in specimens treated with alendronate on the 15th day post surgery. The result was associated with a higher co-expression of TGF-β1, BMPR1B, and Runx-2 when compared with the control group. In addition, on the 60th day post surgery, the increase of bone matrix deposition from 15th to 60th day was discrete in specimens treated with alendronate compared with the control group. This result coincided with the intense simultaneous expression of TGF-β1, α-ER, and α-SMA, whereas the expression of BMPR1B and Runx-2 decreased. The prolonged administration of alendronate altered the cranial repair in ovarian-salpingo-oophorectomized specimens due to the simultaneous occurrence of low estrogen and the presence of TGF-β1+/α-ER+ inducing the presence of α-SMA + , whereas BMPR1B and Runx-2 were
Interactions of histatin-3 and histatin-5 with actin.
Blotnick, Edna; Sol, Asaf; Bachrach, Gilad; Muhlrad, Andras
2017-03-06
Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and -5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. Histatin-3 and -5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and -5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and -5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. Both histatin-3 and -5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8
Roozdar, Alale; Hayes, Malcolm M; Pourseyedei, Bahram; Zeinalinejad, Hamid; Shamsi Meymandi, Manzumeh; Dabiri, Bahram; Dabiri, Shahriar
2018-05-01
Gene expression profiling of breast cancer has demonstrated the importance of stromal response in determining the prognosis of invasive breast cancer. The host response to breast cancer is of increasing interest to pathologists and may be a future focus for novel pharmacological treatments. This study describes the pattern of distribution of stromal myofibroblasts using immunostains for CD10 and smooth muscle actin (SMA) in 50 primary breast cancers and their matched nodal metastases (68.6% nodes positive and 31.4% nodes negative). The stroma within the tumor (intratumoral) and at the advancing tumor edge (peri-tumoral) was studied in both primary and nodal sites. A simple quantitative scoring system was employed for both immunostains. The correlation between expression of these markers by stromal cells and standard pathological prognostic factors of stage, grade, hormone receptor and Her-2 status was analysed. SMA-positive stromal cells were more abundant in peri-tumoral stroma compared with intratumoral stroma in both primary and metastatic lesions. SMA expression in the lymph node metastases showed a significant correlation with tumor stage. SMA expression in peri-tumoral stroma correlated with Her-2 status. The results of this study suggest that myofibroblasts, particularly those expressing SMA, might potentiate the progression of the carcinomatous process especially in nodal metastases. Thus these cells may be a potential therapeutic target. © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Yamazaki, Daisuke; Oikawa, Tsukasa; Takenawa, Tadaomi
2007-01-01
During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.
SMA spring-based artificial muscle actuated by hot and cool water using faucet-like valve
NASA Astrophysics Data System (ADS)
Park, Cheol Hoon; Son, Young Su
2017-04-01
An artificial muscle for a human arm-like manipulator with high strain and high power density are under development, and an SMA(Shape memory alloy) spring is a good actuator for this application. In this study, an artificial muscle composed of a silicon tube and a bundle of SMA(Shape memory alloy) springs is evaluated. A bundle of SMA springs consists of five SMA springs which are fabricated by using SMA wires with a diameter of 0.5 mm, and hot and cool water actuates it by heating and cooling SMA springs. A faucet-like valve was also developed to mix hot water and cool water and control the water temperature. The mass of silicon tube and a bundle of SMA springs is only 3.3 g and 2.25 g, respectively, and the total mass of artificial muscle is 5.55 g. It showed good actuating performance for a load with a mass of 2.3 kg and the power density was more than 800 W/kg for continuous valve switching with a cycle of 0.6 s. The faucet-like valve can switch a water output from hot water to cold water within 0.3s, and the artificial muscle is actuated well in response to the valve position and speed. It is also presented that the temperature of the mixed water can be controlled depending on the valve position, and the displacement of the artificial muscle can be controlled well by the mixed water. Based on these results, SMA spring-based artificial muscle actuated by hot and cool water could be applicable to the human arm-like robot manipulators.
Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek
2008-01-01
Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100
Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael
2016-01-01
The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641
Wasik, Anita A; Koskelainen, Susanna; Hyvönen, Mervi E; Musante, Luca; Lehtonen, Eero; Koskenniemi, Kerttu; Tienari, Jukka; Vaheri, Antti; Kerjaschki, Dontscho; Szalay, Csaba; Révész, Csaba; Varmanen, Pekka; Nyman, Tuula A; Hamar, Peter; Holthöfer, Harry; Lehtonen, Sanna
2014-06-01
Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
An epidermal plakin that integrates actin and microtubule networks at cellular junctions.
Karakesisoglou, I; Yang, Y; Fuchs, E
2000-04-03
Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.
Lee, Sook-Jeong; Seo, Bo-Ra; Koh, Jae-Young
2015-12-04
Astrocytes may play important roles in the pathogenesis of Alzheimer's disease (AD) by clearing extracellular amyloid beta (Aβ) through endocytosis and degradation. We recently showed that metallothionein 3 (Mt3), a zinc-binding metallothionein that is enriched in the central nervous system, contributes to actin polymerization in astrocytes. Because actin is likely involved in the endocytosis of Aβ, we investigated the possible role of Mt3 in Aβ endocytosis by cortical astrocytes in this study. To assess the route of Aβ uptake, we exposed cultured astrocytes to fluorescently labeled Aβ1-40 or Aβ1-42 together with chloropromazine (CP) or methyl-beta-cyclodextrin (MβCD), inhibitors of clathrin- and caveolin-dependent endocytosis, respectively. CP treatment almost completely blocked Aβ1-40 and Aβ1-42 endocytosis, whereas exposure to MβCD had no significant effect. Actin disruption with cytochalasin D (CytD) or latrunculin B also completely blocked Aβ1-40 and Aβ1-42 endocytosis. Because the absence of Mt3 also results in actin disruption, we examined Aβ1-40 and Aβ1-42 uptake and expression in Mt3 (-/-) astrocytes. Compared with wild-type (WT) cells, Mt3 (-/-) cells exhibited markedly reduced Aβ1-40 and Aβ1-42 endocytosis and expression of Aβ1-42 monomers and oligomers. A similar reduction was observed in CytD-treated WT cells. Finally, actin disruption and Mt3 knockout each increased the overall levels of clathrin and the associated protein phosphatidylinositol-binding clathrin assembly protein (PICALM) in astrocytes. Our results suggest that the absence of Mt3 reduces Aβ uptake in astrocytes through an abnormality in actin polymerization. In light of evidence that Mt3 is downregulated in AD, our findings indicate that this mechanism may contribute to the extracellular accumulation of Aβ in this disease.
A dynamic formin-dependent deep F-actin network in axons
Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe
2015-01-01
Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902
Three’s company: The fission yeast actin cytoskeleton
Kovar, David R.; Sirotkin, Vladimir; Lord, Matthew
2010-01-01
How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly vexing in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review, we explore recent studies in fission yeast that help unravel how different actin structures operate in cells. PMID:21145239
Xiang, Yangfei; Zheng, Kai; Ju, Huaiqiang; Wang, Shaoxiang; Pei, Ying; Ding, Weichao; Chen, Zhenping; Wang, Qiaoli; Qiu, Xianxiu; Zhong, Meigong; Zeng, Fanli; Ren, Zhe; Qian, Chuiwen; Liu, Ge
2012-01-01
Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis. PMID:22623803
The nature of the globular- to fibrous-actin transition.
Oda, Toshiro; Iwasa, Mitsusada; Aihara, Tomoki; Maéda, Yuichiro; Narita, Akihiro
2009-01-22
Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.
Rojas, I Gina; Martínez, Alejandra; Brethauer, Ursula; Grez, Patricia; Yefi, Roger; Luza, Sandra; Marchesani, Francisco J
2009-03-01
Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies, including oral cancers. Recent studies have shown that mast cell-derived protease tryptase can induce COX-2 expression by the cleavage of proteinase-activated receptor-2 (PAR-2). Actinic cheilitis (AC) is a premalignant form of lip cancer characterized by an increased density of tryptase-positive mast cells. To investigate the possible contribution of tryptase to COX-2 overexpression during early lip carcinogenesis, normal lip (n=24) and AC (n=45) biopsies were processed for COX-2, PAR-2 and tryptase detection, using RT-PCR and immunohistochemistry. Expression scores were obtained for each marker and tested for statistical significance using Mann-Whitney and Spearmann's correlation tests as well as multivariate logistic regression analysis. Increased epithelial co-expression of COX-2 and PAR-2, as well as, elevated subepithelial density of tryptase-positive mast cells were found in AC as compared to normal lip (P<0.001). COX-2 overexpression was found to be a significant predictor of AC (P<0.034, forward stepwise, Wald), and to be correlated with both tryptase-positive mast cells and PAR-2 expression (P<0.01). The results suggest that epithelial COX-2 overexpression is a key event in AC, which is associated with increased tryptase-positive mast cells and PAR-2. Therefore, tryptase may contribute to COX-2 up-regulation by epithelial PAR-2 activation during early lip carcinogenesis.
Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C
2016-01-01
The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.
Liquid behavior of cross-linked actin bundles.
Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L
2017-02-28
The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.
Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu
2012-01-01
Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.
Actin cytoskeleton as a putative target of the neem limonoid Azadirachtin A.
Anuradha, Aritakula; Annadurai, Ramaswamy S; Shashidhara, L S
2007-06-01
Limonoids isolated from the Indian neem tree (Azadirachta indica) have been gaining global acceptance in agricultural applications and in contemporary medicine for their myriad but discrete properties. However, their mode of action is still not very well understood. We have studied the mode of action of Azadirachtin A, the major limonoid of neem seed extracts, using Drosophila melanogaster as the model system. Azadirachtin A induces moderate-to-severe phenotypes in different tissues in a dose-dependent manner. At the cellular level, Azadirachtin A induces depolymerization of Actin leading to arrest of cells and subsequently apoptosis in a caspase-independent manner. Azadirachtin A-induced phenotypes were rescued by the over-expression of Cyclin E in a tissue-dependent manner. Cyclin E, which caused global rescue of Azadirachtin A-induced phenotypes, also effected rearrangement of the actin filaments. These results suggest that probably actin is a target of Azadirachtin A activity.
Bricheux, G; Brugerolle, G
1997-08-01
The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Johnsson, Anna-Karin; Karlsson, Roger, E-mail: roger.karlsson@wgi.su.se
2012-01-15
Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a rangemore » of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.« less
Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics.
Ono, Shoichiro; Ono, Kanako
2002-03-18
Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.
Wang, Yuh-Shuh; Motes, Christy M; Mohamalawari, Deepti R; Blancaflor, Elison B
2004-10-01
The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.
Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2
NASA Astrophysics Data System (ADS)
Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui
2011-01-01
Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure
Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J
2007-11-01
Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.
Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J
2018-07-01
Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.
Managing actinic keratosis in primary care.
Salmon, Nicola; Tidman, Michael J
2016-10-01
Actinic, or solar, keratosis is caused by chronic ultraviolet-induced damage to the epidermis. In the UK, 15-23% of individuals have actinic keratosis lesions. Risk factors include: advanced age; male gender; cumulative sun exposure or phototherapy; Fitzpatrick skin phototypes I-II; long-term immuno-suppression and genetic syndromes e.g. xeroderma pigmentosum and albinism. Actinic keratoses are regarded by some authorities as premalignant lesions that may transform into invasive squamous cell carcinoma (SCC) and by others as in situ SCC that may progress to an invasive stage. The risk of malignant change appears low; up to 0.5% per lesion per year. Up to 20-30% of lesions may spontaneously regress but in the absence of any reliable prognostic clinical indicators regarding malignant potential active treatment is considered appropriate. Actinic keratosis lesions may present as discrete hyperkeratotic papules, cutaneous horns, or more subtle flat lesions on sun-exposed areas of skin. The single most helpful diagnostic sign is an irregularly roughened surface texture: a sandpaper-like feel almost always indicates actinic damage. Dermatoscopy can be helpful in excluding signs of basal cell carcinoma when actinic keratosis is non-keratotic. It is always important to consider the possibility of SCC. The principal indication for referral to secondary care is the possibility of cutaneous malignancy. However, widespread and severe actinic damage in patients who are immunosuppressed is also a reason for referral.
Sobierajska, Katarzyna; Skurzynski, Szymon; Stasiak, Marta; Kryczka, Jakub; Cierniewski, Czeslaw S.; Swiatkowska, Maria
2014-01-01
Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism. PMID:24415753
Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen
2014-02-01
During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.
Embedding of Superelastic SMA Wires into Composite Structures: Evaluation of Impact Properties
NASA Astrophysics Data System (ADS)
Pappadà, Silvio; Rametta, Rocco; Toia, Luca; Coda, Alberto; Fumagalli, Luca; Maffezzoli, Alfonso
2009-08-01
Shape memory alloy (SMA) represents the most versatile way to realize smart materials with sensing, controlling, and actuating functions. Due to their unique mechanical and thermodynamic properties and to the possibility to obtain SMA wires with very small diameters, they are used as smart components embedded into the conventional resins or composites, obtaining active abilities, tunable properties, self-healing properties, and damping capacity. Moreover, superelastic SMAs are used to increase the impact resistance properties of composite materials. In this study, the influence of the integration of thin superelastic wires to suppress propagating damage of composite structures has been investigated. Superelastic SMAs have very high strain to failure and recoverable elastic strain, due to a stress-induced martensitic phase transition creating a plateau region in the stress-strain curve. NiTi superelastic wires ( A f = -15 °C fully annealed) of 0.10 mm in diameter have been produced and characterized by SAES Getters. The straight annealed wire shows the typical flag stress-strain behavior. The measured loading plateau is about 450 MPa at ambient temperature with a recoverable elastic strain of more than 6%. For these reasons superelastic SMA fibers can absorb much more strain energy than other fibers before their failure, partly with a constant stress level. In this paper, the improvement of composite laminates impact properties by embedding SMA wires is evaluated and indications for design and manufacturing of SMA composites with high-impact properties are also given.
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.
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.
Kinetics of Binding of Caldesmon to Actin*
Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai
2005-01-01
The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374
[Photodynamic therapy for actinic cheilitis].
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.
Development of an artificial urethral valve using SMA actuators
NASA Astrophysics Data System (ADS)
Chonan, S.; Jiang, Z. W.; Tani, J.; Orikasa, S.; Tanahashi, Y.; Takagi, T.; Tanaka, M.; Tanikawa, J.
1997-08-01
The development of an artificial urethral valve for the treatment of urinary incontinence which occurs frequently in the aged is described. The prototype urethral valve is assembled in hand-drum form with four thin shape memory alloy (SMA) (nickel - titanium alloy) plates of 0.3 mm thickness. The shape memory effect in two directions is used to replace the urinary canal sphincter muscles and to control the canal opening and closing functions. The characteristic of the SMA is to assume the shape of a circular arc at normal temperatures and a flat shape at higher temperatures. Experiments have been conducted using a canine bladder and urinary canal.
Actin cable dynamics in budding yeast
Yang, Hyeong-Cheol; Pon, Liza A.
2002-01-01
Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity. We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast. We detected two populations of actin cables: (i) bud-associated cables, which extend from the bud along the mother-bud axis, and (ii) randomly oriented cables, which are relatively short. Time-lapse imaging of Abp140p–GFP revealed an apparent increase in the length of bud-associated actin cables. Analysis of movement of Abp140p–GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud. The rate of extension of the tip of an elongating actin cable is 0.29 ± 0.08 μm/s. Latrunculin A (Lat-A) treatment completely blocked this process. We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 ± 0.14 μm/s. Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables. However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell. Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation. PMID:11805329
Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation.
Branco, Rémi; Pearsall, Eliza-Jane; Rundle, Chelsea A; White, Rosemary G; Bradby, Jodie E; Hardham, Adrienne R
2017-03-01
Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 μN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.
Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai
2001-01-01
Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164
González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.
2017-01-01
Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963
Research notes : SMA, stone matrix resists ruts.
DOT National Transportation Integrated Search
2003-08-01
The first Oregon Department of Transportation SMA placement was part of a pilot project constructed on Interstate 5 (I-5) in 1996. : Four asphalt mixes were placed over a 3-mile section of concrete to evaluate the use of asphalt overlays for repair o...
Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin
Lu, Hailong; Fagnant, Patricia M.; Bookwalter, Carol S.; Joel, Peteranne; Trybus, Kathleen M.
2015-01-01
Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD. PMID:26153420
A Continuum Model of Actin Waves in Dictyostelium discoideum
Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.
2013-01-01
Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312
Actin filaments – a target for redox regulation
Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin
2016-01-01
Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342
Zhang, Yanqiu; Zhang, Daning; Zhang, Mianzhi
2012-12-01
To evaluate inhibition effect and mechanism of compound ethanol extracts from Wuweizi (Fructus Schisandrae Chinensis), Chuanxiong (Rhizoma Chuanxiong) and Muli (Cocha Ostreae) (FRC) on glomerular and tubular interstitial fibrosis in streptozocin (STZ)-induced diabetic nephropathy (ND) model mice. Twenty-seven male C57BL/6 mice were divided randomly into 3 groups: nondibetic (ND), STZ-induced diabetic (D), and STZ-induced diabetic that were treated with 5 g x kg(-1) x day(-1) of FRC by oral gavage (D(FRC)), with 9 in each group. The protein expressions of E-cadherin, alpha-smooth muscle actin (alpha-SMA), Plasminogen Activator Inhibitor-1 (PAL-1) in renal tissues were investigated by Western blotting. The expressions of fibronectin (FN) and alpha-SMA were detected by immunohistochemical method. The morphological changes of renal tissues were observed under a microscope. Renal tissues in the D(FRC) group showed a lessened degree of fibrosis. Meanwhile, the expressions of FN, alpha-SMA and PAI-1 were significantly lower in the D(FRC) group than those in the D group (all P < 0.05). FRC can ameliorate the DN in the C57BL/6 mice, and its mechanism may relate to inhibition on the epithelial to mesenchymal transdifferentiation, endothelial-myofibroblast transition and PAL-1 expression.
Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth; Halavatyi, Aliaksandr; Hoffmann, Céline; Moes, Michèle; Hadzic, Ermin; Catillon, Marie; Yatskou, Mikalai; Friederich, Evelyne
2010-01-01
Background Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. Methodology/Principal Findings To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. Conclusions/Significance Altogether these findings quantitatively
Erbium laser resurfacing for actinic cheilitis.
Cohen, Joel L
2013-11-01
Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA).
Tada, Hirobumi; Miyazaki, Tomoyuki; Takemoto, Kiwamu; Takase, Kenkichi; Jitsuki, Susumu; Nakajima, Waki; Koide, Mayu; Yamamoto, Naoko; Komiya, Kasane; Suyama, Kumiko; Sano, Akane; Taguchi, Akiko; Takahashi, Takuya
2016-10-25
Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.
Tada, Hirobumi; Miyazaki, Tomoyuki; Takemoto, Kiwamu; Takase, Kenkichi; Jitsuki, Susumu; Nakajima, Waki; Koide, Mayu; Yamamoto, Naoko; Komiya, Kasane; Suyama, Kumiko; Sano, Akane; Taguchi, Akiko; Takahashi, Takuya
2016-01-01
Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life. PMID:27791080
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.
Xenopus egg cytoplasm with intact actin.
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. © 2014 Elsevier Inc. All rights reserved.
NASA Astrophysics Data System (ADS)
Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae
2014-03-01
The semi-flexible biopolymer actin is a ubiquitous component of nearly all biological organisms, playing an important role in many biological processes such as cell structure and motility, cancer invasion and metastasis, muscle contraction, and cell signaling. Concentrated actin networks possess unique viscoelastic properties that have been the subject of much theoretical and experimental work. However, much is still unknown regarding the correlation of the applied stress on the network to the induced filament strain at the molecular level. Here, we use dual optical traps alongside fluorescence microscopy to carry out active microrheology measurements that link mechanical stress to structural response at the micron scale. Specifically, we actively drive microspheres through entangled actin networks while simultaneously measuring the force the surrounding filaments exert on the sphere and visualizing the deformation and subsequent relaxation of fluorescent labeled filaments within the network. These measurements, which provide much needed insight into the link between stress and strain in actin networks, are critical for clarifying our theoretical understanding of the complex viscoelastic behavior exhibited in actin networks.
Resistance of Actin to Cleavage during Apoptosis
NASA Astrophysics Data System (ADS)
Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.
1997-01-01
A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.
Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.
Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I
2014-10-15
Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Live-cell imaging of G-actin dynamics using sequential FDAP
Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku
2011-01-01
Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616
Development of a spined underwater biomimetic vehicle with SMA actuators
NASA Astrophysics Data System (ADS)
Rediniotis, Othon K.; Lagoudas, Dimitris C.; Garner, Luke J.; Wilson, Larry N.
1999-06-01
We present here our progress towards the development of a type of biomimetic active hydrofoil that utilizes Shape Memory Alloy (SMA) actuator technology. The actuation is presently applied to the control of hydrodynamic forces and moments, including thrust generation, on a 2D hydrofoil. The SMA actuation elements are two sets of thin wires (0.015' to 0.027') on either side of an elastomer element that provides the main structural support. Controlled heating and cooling of the two wire sets generates bi-directional bending of the elastomer, which in turn deflects (for quasi-static control) or oscillates (for thrust generation) the trailing edge of the hydrofoil. The aquatic environment of the hydrofoil lends itself to cooling schemes that utilize the excellent heat transfer properties of water. The SMA actuator was able to deflect the trailing edge by +/- 5 degree(s) at rates as high as 2 Hz. FEM modeling of hydrofoil response to thermoelectric heating has been carried out using a thermomechanical constitutive model for SMAs. FEM predictions are compared with experimental measurements.
Mechanics model for actin-based motility
NASA Astrophysics Data System (ADS)
Lin, Yuan
2009-02-01
We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.
Mechanics model for actin-based motility.
Lin, Yuan
2009-02-01
We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.
Diverse roles of actin in C. elegans early embryogenesis
Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio
2007-01-01
Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study
Gungor-Ordueri, N. Ece; Celik-Ozenci, Ciler
2014-01-01
In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326
Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*
Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland
2013-01-01
The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468
Myosin Vs organize actin cables in fission yeast
Lo Presti, Libera; Chang, Fred; Martin, Sophie G.
2012-01-01
Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734
Myosin Vs organize actin cables in fission yeast.
Lo Presti, Libera; Chang, Fred; Martin, Sophie G
2012-12-01
Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.
Actin filament curvature biases branching direction
NASA Astrophysics Data System (ADS)
Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel
2012-02-01
Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.
Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels
Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence
2014-01-01
The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460
Burke, Emily; Mahoney, Nicole M.; Almo, Steven C.; Barik, Sailen
2000-01-01
Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome. PMID:10623728
Phenotypic and genotypic studies of ALS cases in ALS-SMA families.
Corcia, Philippe; Vourc'h, Patrick; Blasco, Helene; Couratier, Philippe; Dangoumau, Audrey; Bellance, Remi; Desnuelle, Claude; Viader, Fausto; Pautot, Vivien; Millecamps, Stephanie; Bakkouche, Salah; Salachas, FranÇois; Andres, Christian R; Meininger, Vincent; Camu, William
2018-08-01
Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most frequent motor neuron disorders in adulthood and infancy, respectively. There is a growing literature supporting common pathophysiological patterns between those disorders. One important clinical issue for that is the co-occurrence of both diseases within a family. To collect families in which ALS and SMA patients co-exist and describe the phenotype and the genotype of ALS patients. Nine families with co-occurrence of SMA and ALS have been gathered over the last 15 years. Epidemiological, phenotype and genetic status were collected. Out of the nine families, six corresponded to the criteria of familial ALS (FALS). Clinical data were available for 11 patients out of the 15 ALS cases. Mean age of onset was 58.5 years, site of onset was lower limbs in nine cases (81.8%), median duration was 22 months. Four ALS patients carried a mutation: three mutations in SOD1 gene (G147N in two cases and one with E121G) and one repeat expansion in the C9ORF72 gene. Three patients had abnormal SMN1 copy numbers. While the high proportion of familial history of ALS cases in these ALS-SMA pedigrees could have suggested that these familial clusters of the two most frequent MND rely on a genetic background, we failed to exclude that this occurred by chance.
Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins
Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.
2013-01-01
Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011
Fatigue behavior of a thermally-activated NiTiNb SMA-FRP patch
NASA Astrophysics Data System (ADS)
El-Tahan, M.; Dawood, M.
2016-01-01
This paper presents the details of an experimental study that was conducted to characterize the fatigue behavior of a thermally-activated shape memory alloy (SMA)/carbon fiber reinforced polymer (CFRP) patch that can be used to repair cracked steel members. A total of 14 thermally-activated patches were fabricated and tested to evaluate the stability of the prestress under fatigue loading. The parameters considered in this study are the prestress level in the nickel-titanium-niobium SMA wires and the applied force range. An empirical model to predict the degradation of the prestress is also presented. The results indicate that patches for which the maximum applied loads in a fatigue cycle did not cause debonding of the SMA wires from the CFRP sustained two million loading cycles with less than 20% degradation of the prestress.
Medler, Scott; Mykles, Donald L
2003-10-01
Skeletal muscles are diverse in their contractile properties, with many of these differences being directly related to the assemblages of myofibrillar isoforms characteristic of different fibers. Crustacean muscles are similar to other muscles in this respect, although the majority of information about differences in muscle organization comes from vertebrate species. In the present study, we examined the correlation between myofibrillar protein isoforms and the patterns of myofibrillar gene expression in fast, slow-phasic (S(1)) and slow-tonic (S(2)) fibers of the American lobster Homarus americanus. SDS-PAGE and western blotting were used to identify isoform assemblages of myosin heavy chain (MHC), P75, troponin T (TnT) and troponin I (TnI). RT-PCR was used to monitor expression of fast and slow (S(1)) MHC, P75 and actin in different fiber types, and the MHC and actin levels were quantified by real-time PCR. Fast and slow fibers from the claw closers predominantly expressed fast and S(1) MHC, respectively, but also lower levels of the alternate MHC. By contrast, fast fibers from the deep abdominal muscle expressed fast MHC exclusively. In addition, slow muscles expressed significantly higher levels of actin than fast fibers. A distal bundle of fibers in the cutter claw closer muscle was found to be composed of a mixture of S(1) and S(2) fibers, many of which possessed a mixture of S(1) and S(2) MHC isoforms. This pattern supports the idea that S(1) and S(2) fibers represent extremes in a continuum of slow muscle phenotype. Overall, these patterns demonstrate that crustacean skeletal muscles cannot be strictly categorized into discrete fiber types, but a muscle's properties probably represent a point on a continuum of fiber types. This trend may result from differences in innervation pattern, as each muscle is controlled by a unique combination of phasic, tonic or both phasic and tonic motor nerves. In this respect, future studies examining how muscle phenotype
Specialized mouse embryonic stem cells for studying vascular development.
Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E
2014-01-01
Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.
Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue.
STUDY OF THE RHIC BPM SMA CONNECTOR FAILURE PROBLEM
DOE Office of Scientific and Technical Information (OSTI.GOV)
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 canmore » 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.« less
The Dynamic Actin Cytoskeleton in Smooth Muscle.
Tang, Dale D
2018-01-01
Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.
Cook, Mandy; Bolkan, Bonnie J; Kretzschmar, Doris
2014-01-01
loechrig (loe) mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK) complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG)-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH) phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.
Development of SMA Actuated Morphing Airfoil for Wind Turbine Load Alleviation
NASA Astrophysics Data System (ADS)
Karakalas, A.; Machairas, T.; Solomou, A.; Riziotis, V.; Saravanos, D.
Wind turbine rotor upscaling has entered a range of rotor diameters where the blade structure cannot sustain the increased aerodynamic loads without novel load alleviation concepts. Research on load alleviation using morphing blade sections is presented. Antagonistic shape memory alloy (SMA) actuators are implemented to deflect the section trailing edge (TE) to target shapes and target time-series relating TE movement with changes in lift coefficient. Challenges encountered by the complex thermomechanical response of morphing section and the enhancement of SMA transient response to achieve frequencies meaningful for aerodynamic load alleviation are addressed. Using a recently developed finite element for SMA actuators [1], actuator configurations are considered for fast cooling and heating cycles. Numerical results quantify the attained ranges of TE angle movement, the moving time period and the developed stresses. Estimations of the attained variations of lift coefficient vs. time are also presented to assess the performance of the morphing section.
Semantic memory retrieval circuit: role of pre-SMA, caudate, and thalamus.
Hart, John; Maguire, Mandy J; Motes, Michael; Mudar, Raksha Anand; Chiang, Hsueh-Sheng; Womack, Kyle B; Kraut, Michael A
2013-07-01
We propose that pre-supplementary motor area (pre-SMA)-thalamic interactions govern processes fundamental to semantic retrieval of an integrated object memory. At the onset of semantic retrieval, pre-SMA initiates electrical interactions between multiple cortical regions associated with semantic memory subsystems encodings as indexed by an increase in theta-band EEG power. This starts between 100-150 ms after stimulus presentation and is sustained throughout the task. We posit that this activity represents initiation of the object memory search, which continues in searching for an object memory. When the correct memory is retrieved, there is a high beta-band EEG power increase, which reflects communication between pre-SMA and thalamus, designates the end of the search process and resultant in object retrieval from multiple semantic memory subsystems. This high beta signal is also detected in cortical regions. This circuit is modulated by the caudate nuclei to facilitate correct and suppress incorrect target memories. Copyright © 2012 Elsevier Inc. All rights reserved.
A generalized analytical approach to the coupled effect of SMA actuation and elastica deflection
NASA Astrophysics Data System (ADS)
Sreekumar, M.; Singaperumal, M.
2009-11-01
A compliant miniature parallel manipulator made of superelastic nitinol pipe as its central pillar and actuated by three symmetrically attached shape memory alloy (SMA) wires is under development. The mobility for the platform is obtained by the selective actuation of one or two wires at a time. If one wire is actuated, the other two unactuated wires provide the counter effect. Similarly, if two wires are actuated simultaneously or in a differential manner, the third unactuated wire resists the movement of the platform. In an earlier work of the authors, the static displacement analysis was presented without considering the effect of unactuated wires. In this contribution, the force-displacement analysis is presented considering the effect of both actuated and unactuated wires. Subsequently, an attempt has been made to obtain a generalized approach from which six types of actuation methods are identified using a group of conditional parameters. Each method leads to a set of large deflection expressions suitable for a particular actuation method. As the large deflection expressions derived for the mechanism are nonlinear and involve interdependent parameters, their simplified form using a parametric approximation have also been obtained using Howell's algorithm. The generalized approach and the solution algorithm developed can be applied to any kind of compliant mechanism having large deflection capabilities, including planar and spatial MEMS devices and stability analysis of long slender columns supported by wires or cables. The procedure developed is also suitable for the static analysis of spatial compliant mechanisms actuated by multiple SMA actuators.
Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash
2013-01-01
It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522
Probing actin polymerization by intermolecular cross-linking.
Millonig, R; Salvo, H; Aebi, U
1988-03-01
We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant
Protective effects of long-term lithium administration in a slowly progressive SMA mouse model.
Biagioni, Francesca; Ferrucci, Michela; Ryskalin, Larisa; Fulceri, Federica; Lazzeri, Gloria; Calierno, Maria Teresa; Busceti, Carla L; Ruffoli, Riccardo; Fornai, Francesco
2017-12-01
In the present study we evaluated the long-term effects of lithium administration to a knock-out double transgenic mouse model (Smn-/-; SMN1A2G+/-; SMN2+/+) of Spinal Muscle Atrophy type III (SMA-III). This model is characterized by very low levels of the survival motor neuron protein, slow disease progression and motor neuron loss, which enables to detect disease-modifying effects at delayed time intervals. Lithium administration attenuates the decrease in motor activity and provides full protection from motor neuron loss occurring in SMA-III mice, throughout the disease course. In addition, lithium prevents motor neuron enlargement and motor neuron heterotopy and suppresses the occurrence of radial-like glial fibrillary acidic protein immunostaining in the ventral white matter of SMA-III mice. In SMA-III mice long-term lithium administration determines a dramatic increase of survival motor neuron protein levels in the spinal cord. These data demonstrate that long-term lithium administration during a long-lasting motor neuron disorder attenuates behavioural deficit and neuropathology. Since low level of survival motor neuron protein is bound to disease severity in SMA, the robust increase in protein level produced by lithium provides solid evidence which calls for further investigations considering lithium in the long-term treatment of spinal muscle atrophy.
Arima, Kengo; Tamaoki, Daisuke; Mineyuki, Yoshinobu; Yasuhara, Hiroki; Nakai, Tomonori; Shimmen, Teruo; Yoshihisa, Tohru; Sonobe, Seiji
2018-06-19
In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.
Grantham, Julie; Ruddock, Lloyd W.; Roobol, Anne; Carden, Martin J.
2002-01-01
We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers. PMID:12482199
Adipogenesis of the mesenchymal stromal cells and bone oedema in rheumatoid arthritis.
Okada, A; Yamasaki, S; Koga, T; Kawashiri, S Y; Tamai, M; Origuchi, T; Nakamura, H; Eguchi, K; Kawakami, A
2012-01-01
Bone oedema is a pathological change in rheumatoid arthritis (RA) that is detectable by magnetic resonance imaging (MRI). Recent histological analyses revealed that a prominent feature of bone oedema is the replacement of adipose tissue with inflammatory cells. Here, we demonstrate the possible roles of mesenchymal stromal cells (MSCs) in bone oedema formation and the pathogenic potential of the cells in RA. Adipogenesis of bone marrow-derived human MSCs was induced by a standard adipogenic induction medium in the presence or absence of cytokines. The cytokine productions from MSCs were screened by an antibody array system and confirmed by ELISA. The migration assay was performed to determine the locomotive abilities of undifferentiated MSCs or MSCs after adipogenesis. The expression of α smooth muscle actin (SMA) and F-actin was examined by immunostaining and phalloidin staining, respectively. TNF-α, interleukin (IL)-1β, IL-6, and TGF-β clearly inhibited the adipogenesis of MSCs. Production of IL-6 was markedly reduced, and IL-8 secretion was augmented in MSCs after adipogenesis. The mobility of MSCs after adipogenesis was clearly reduced in migration assays compared to that of undifferentiated MSCs. Consistent with these findings, SMA and F-actin expressions were clearly suppressed in MSCs committed to adipogenesis. Our data suggest that the inflammatory milieu promotes bone oedema by blocking adipogenesis of MSCs. In bone oedema, the enhanced IL-6 production and the increased mobility of MSCs may contribute to the progression of RA. Therefore, bone oedema may be an important target lesion in the treatment of RA.
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.
Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S
2017-11-01
Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2506-2512, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
Liquid droplets of cross-linked actin filaments
NASA Astrophysics Data System (ADS)
Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret
Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.
Sarcomeric Pattern Formation by Actin Cluster Coalescence
Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.
2012-01-01
Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394
Drosophila Spire is an actin nucleation factor.
Quinlan, Margot E; Heuser, John E; Kerkhoff, Eugen; Mullins, R Dyche
2005-01-27
The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.
Expression of JAKs/STATs pathway molecules in rat model of rapid focal segmental glomerulosclerosis.
Liang, Yaojun; Jin, Yu; Li, Yuning
2009-09-01
The objective of this study was to investigate the role of the Janus kinase-signal transducers and activators of transcription (JAKs/STATs) pathway in focal segmental glomerulosclerosis. Sixty specific pathogen-free male Wistar rats were randomly divided into two groups: a model group (MG) and a control group (CG). In the MG group, nephropathy was induced by unilateral nephrectomy and a single tail vein injection of adriamycin (5 mg/kg). Ten rats were sacrificed every 2 weeks in each group. The expressions of smooth muscle alpha actin (alpha-SMA), collagen (COL)-IV, STAT1, and STAT3 were examined using histochemical techniques, and Western blotting was used to examine the protein levels of STAT1, STAT3, phosphorylated (P)-STAT1, P-STAT3, and transforming growth factor beta1 (TGFbeta(1)). The expressions of JAK1, JAK2, STAT1, STAT3, suppressors of cytokine signaling (SOCS)1, SOCS3, protein inhibitors of activated STAT (PIAS)1, and PIAS3 were also measured by real-time quantitative reverse transcriptase-PCR. A steady and significant increase in the expressions of alpha-SMA, COL-IV and TGFbeta(1) were observed in MG rats over the whole experimental course. Increased STAT1 and P-STAT1 levels in MG rats were observed by week 6, whereas increased levels of STAT3 and P-STAT3 were noted by week 2. At the mRNA levels, JAK1, STAT1, and PIAS1 were significantly increased in MG rats in week 2, whereas JAK2 mRNA showed a significant decrease by weeks 2 and 4, followed by an significant increase in week 6. Significantly increased STAT3 levels were noted in week 2, followed by a steady and significant decrease in weeks 4 and 6. Significantly reduced levels of SOCS1, SOCS3, and PIAS3 mRNA were noted at all time points. We conclude that the JAKs/STATs signaling pathway may play an important role in the pathological process of rapid focal segmental glomerulosclerosis in the rat model.
Measurement and Analysis of in vitro Actin Polymerization
Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.
2014-01-01
Summary The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data. PMID:23868594
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. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
[Cytoskeletal actin and its associated proteins. Some examples in Protista].
Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J
1998-06-01
Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin
MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.
Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M
2006-10-19
Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.
Actin Turnover-Mediated Gravity Response in Maize Root Apices
Mancuso, Stefano; Barlow, Peter W; Volkmann, Dieter
2006-01-01
The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap. PMID:19521476
Nuclear positioning by actin cables and perinuclear actin: Special and general?
Huelsmann, Sven; Brown, Nicholas H
2014-01-01
Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used.
Metal muscles and nerves—a self-sensing SMA-actuated hand concept
NASA Astrophysics Data System (ADS)
Simone, F.; Rizzello, G.; Seelecke, S.
2017-09-01
Bio-inspired hand-like grippers actuated by Shape Memory Alloy (SMA) wires represent an emerging new technology with potential applications in many different fields, ranging from industrial assembly processes to biomedical systems. The inherently high energy density makes SMAs a natural choice for compact, lightweight, and silent actuator systems capable of producing a high amount of work, such as hand prostheses or robotic systems in industrial human/machine environments. In this work, a concept for a compact and versatile gripping system is developed, in which SMA wires are implemented as antagonistic muscles actuating an artificial hand with three fingers. In order to combine high gripping force with sufficient actuation speed, the muscle implementation pursues a multi-wire concept with several 0.1 mm diameter NiTi wires connected in parallel, in order to increase the surface-to-volume ratio for accelerated cooling. The paper starts with an illustration of the design concept of an individual 3-phalanx-finger, along with kinematic considerations for optimal placement of SMA wires. Three identical fingers are subsequently fabricated via 3D printing and assembled into a hand-like gripper. The maximum displacement of each finger phalanx is measured, and an average phalanxes dynamic responsiveness is evaluated. SMA self-sensing is documented by experiments relating the wires change in resistance to the finger motion. Several finger force measurements are also performed. The versatility of the gripper is finally documented by displaying a variety of achievable grasping configurations.
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.
miR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function.
Lim, Shok Ping; Ioannou, Nikolaos; Ramsay, Alan G; Darling, David; Gäken, Joop; Mufti, Ghulam J
2018-05-01
MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3 + T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions. ©2018 Society for Leukocyte Biology.
Biphasic interactions between a cationic dendrimer and actin.
Ruenraroengsak, Pakatip; Florence, Alexander T
2010-12-01
Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.
Mechanism of bandwidth improvement in passively cooled SMA position actuators
NASA Astrophysics Data System (ADS)
Gorbet, R. B.; Morris, K. A.; Chau, R. C. C.
2009-09-01
The heating of shape memory alloy (SMA) materials leads to a thermally driven phase change which can be used to do work. An SMA wire can be thermally cycled by controlling electric current through the wire, creating an electro-mechanical actuator. Such actuators are typically heated electrically and cooled through convection. The thermal time constants and lack of active cooling limit the operating frequencies. In this work, the bandwidth of a still-air-cooled SMA wire controlled with a PID controller is improved through optimization of the controller gains. Results confirm that optimization can improve the ability of the actuator to operate at a given frequency. Overshoot is observed in the optimal controllers at low frequencies. This is a result of hysteresis in the wire's contraction-temperature characteristic, since different input temperatures can achieve the same output value. The optimal controllers generate overshoot during heating, in order to cause the system to operate at a point on the hysteresis curve where faster cooling can be achieved. The optimization results in a controller which effectively takes advantage of the multi-valued nature of the hysteresis to improve performance.
Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding.
Frantz, Christian; Barreiro, Gabriela; Dominguez, Laura; Chen, Xiaoming; Eddy, Robert; Condeelis, John; Kelly, Mark J S; Jacobson, Matthew P; Barber, Diane L
2008-12-01
Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H(+) efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.
Das, Sujaan; Lemgruber, Leandro; Tay, Chwen L; Baum, Jake; Meissner, Markus
2017-08-15
The phylum Apicomplexa includes intracellular parasites causing immense global disease burden, the deadliest of them being the human malaria parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The cytoskeletal protein actin is well conserved within apicomplexans but divergent from mammalian actins, and was primarily reported to function during host cell invasion. However, novel invasion mechanisms have been described for several apicomplexans, and specific functions of the acto-myosin system are being reinvestigated. Of the two actin genes in P. falciparum, actin-1 (pfact1) is ubiquitously expressed in all life-cycle stages and is thought to be required for erythrocyte invasion, although its functions during parasite development are unknown, and definitive in vivo characterisation during invasion is lacking. Here we have used a conditional Cre-lox system to investigate the functions of PfACT1 during P. falciparum blood-stage development and host cell invasion. We demonstrate that PfACT1 is crucially required for segregation of the plastid-like organelle, the apicoplast, and for efficient daughter cell separation during the final stages of cytokinesis. Surprisingly, we observe that egress from the host cell is not an actin-dependent process. Finally, we show that parasites lacking PfACT1 are capable of microneme secretion, attachment and formation of a junction with the erythrocyte, but are incapable of host cell invasion. This study provides important mechanistic insights into the definitive essential functions of PfACT1 in P. falciparum, which are not only of biological interest, but owing to functional divergence from mammalian actins, could also form the basis for the development of novel therapeutics against apicomplexans.
GREM1 is expressed in the cancer-associated myofibroblasts of basal cell carcinomas.
Kim, Hye Sung; Shin, Myung Soo; Cheon, Min Seok; Kim, Jae Wang; Lee, Cheol; Kim, Woo Ho; Kim, Young Sill; Jang, Bo Gun
2017-01-01
Cancer-associated fibroblasts (CAFs) play important roles in cancer progression through their complex interactions with cancer cells. The secreted bone morphogenetic protein antagonist, gremlin1 (GREM1) is expressed by the CAFs of basal cell carcinomas (BCCs), and promotes the growth of cancer cells. In this study, we investigated the expression of GREM1 mRNAs in various benign and malignant skin tumors, including various BCC subtypes. Analysis by RNA in situ hybridization (ISH) revealed that fibroblasts in the scar tissue expressed GREM1 and α-smooth muscle actin (α-SMA), whereas resident fibroblasts in the dermis of the normal skin did not express GREM1. Real-time polymerase chain reaction analysis showed significantly higher GREM1 expression in skin cancers and pilomatricomas (PMCs) than in other benign skin tumors. Tissue microarrays analyzed by RNA ISH for GREM1 expression also demonstrated that 23% of BCCs, 42% of squamous cell carcinomas, 20% of melanomas, and 90% of PMCs were positive for GREM1 expression, whereas trichoepitheliomas, eccrine poromas, hidradenomas, and spiradenomas were negative for GREM1 expression. Most BCCs that were GREM1 expression positive were of desmoplastic or mixed subtypes, and GREM1 expression was localized to activated myofibroblasts at the tumoral-stromal interface. Interestingly, most PMCs harbored GREM1-expressing fibroblasts, probably because of the inflammatory responses caused by foreign body reactions to keratin. Additionally, in BCCs, stromal GREM1 expression had a strong correlation with CD10 expression. In conclusion, GREM1 is frequently expressed by myofibroblasts in scars or in the stroma of basal cell carcinomas, suggesting that GREM1 expression can be a marker for activated myofibroblasts in the cancer stroma or in scar tissue.
Soft Listeria: actin-based propulsion of liquid drops.
Boukellal, Hakim; Campás, Otger; Joanny, Jean-François; Prost, Jacques; Sykes, Cécile
2004-06-01
We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.
Actin Hydrophobic Loop (262-274) and Filament Nucleation and Elongation
Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Egelman, Edward H.; Reisler, Emil
2014-01-01
Summary The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently using a yeast actin mutant, L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked G-actin does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin - to assist with actin nucleation - and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not alone, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin the helical twist of F-actin changes by ~ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin, and a change of twist by ~ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or a competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics to both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors. PMID:18037437
Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts
DOE Office of Scientific and Technical Information (OSTI.GOV)
Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko
Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, butmore » F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.« less
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.
Development and Verification of Sputtered Thin-Film Nickel-Titanium (NiTi) Shape Memory Alloy (SMA)
2015-08-01
Shape Memory Alloy (SMA) by Cory R Knick and Christopher J Morris Approved for public release; distribution unlimited...Laboratory Development and Verification of Sputtered Thin-Film Nickel-Titanium (NiTi) Shape Memory Alloy (SMA) by Cory R Knick and Christopher
Correlative nanoscale imaging of actin filaments and their complexes
NASA Astrophysics Data System (ADS)
Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.
2013-06-01
Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.
Naresh, Sai
2016-02-01
Semen cryopreservation is broadly utilized as a part of the bovine reproducing industry, a large portion of the spermatozoa does not survive and the majority of those that do survive experience various molecular and physiological changes that influence their fertilizing capacity. The main aim of this study is to determine the effect of cooling (4 °C) and cryopreservation on cytoskeleton actin, tyrosine phosphorylation and quality of buffalo spermatozoa, and to determine the similarity between in vitro capacitation and cryopreservation induced capacitation like changes. To achieve this, Western blot was used to examine the changes in actin expression and protein tyrosine phosphorylation, whereas changes in actin polymerization, localization of actin and protein tyrosine phosphorylation during capacitation and cryopreservation were evaluated by indirect immunofluorescence technique. Localization studies revealed that the actin localized to flagella and acrosome membrane regions and following, capacitation it migrated towards the acrosome region of sperm. Time dependent increase in actin polymerization and protein tyrosine phosphorylation was observed during in vitro capacitation. The cooling phase (4 °C) and cryopreservation processes resulted in the loss/damage of cytoskeleton actin. In addition, we performed the actin polymerization and protein tyrosine phosphorylation in cooled and cryopreserved buffalo spermatozoa. Interestingly, cooling and cryopreservation induces actin polymerization and protein tyrosine phosphorylation, which were similar to in vitro capacitation (cryo-capacitation). These changes showed 1.3 folds reduction in the sperm quality parameters which includes motility, viability and plasma membrane integrity. Furthermore, our findings indicate that cooling and cryopreservation damages the cytoskeleton actin and also induces capacitation like changes such as protein tyrosine phosphorylation and actin polymerization. This could be one of the
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.
Porter, Katie; Day, Brad
2016-04-01
The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years, research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens, including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen perception. Based on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens. © 2015 Institute of Botany, Chinese Academy of Sciences.
NASA Astrophysics Data System (ADS)
Prahlad, Harsha
This dissertation presents the development of a torsional actuator to alter the twist distributions of a tiltrotor blade between hover and forward flight. The actuator uses a Shape Memory Alloy (SMA) tube as its active element. The historical development of the tiltrotor aircraft is discussed, with emphasis on advanced tiltrotor concepts. The central theme in these concepts is to reduce the compromises for tiltrotor blade design, thereby improving performance of the aircraft in both hover and forward flight modes. A survey of research conducted in the application of smart structures to performance enhancement of aircraft is conducted. A review of other torsional actuators that are based on SMAs is presented. An assessment of the state-of-the-art in SMA modeling and characterization, both in mechanical tensile and torsional loading, is also discussed. Shape Memory Alloys are "smart" actuation materials that are capable of providing high stroke and high force of actuation at relatively low bandwidth. However, their behavior is complex, and influenced by material non-linearities, thermo-mechanical conditions and history of loading. In addition, the behavior of torsional SMA actuators has not been investigated in detail. In order to address these issues, the current research carries out a comprehensive characterization of SMAs. Experimental characteristics of SMA wires under extensional loading, and SMA rods and tubes in both extensional and torsional loading under a variety of thermo-mechanical conditions are presented in this dissertation. It is demonstrated that the uniaxial quasistatic SMA models show good overall agreement with the experimental behavior of an SMA wire under extensional loading. In addition, an approach that incorporates these models with radial non-uniformity due to torsional deflections is shown to provide good predictions of torsional characteristics of SMA rods and tubes. Several differences of the material response under non
Kim, Eui-Hyun; Lee, Hyunbae; Kim, Jae-Hwan; Bae, Seung-Muk; Hwang, Heesu; Yang, Heesun; Choi, Eunsoo; Hwang, Jin-Ha
2018-02-22
Self-healing is an essential property of smart concrete structures. In contrast to other structural metals, shape memory alloys (SMAs) offer two unique effects: shape memory effects, and superelastic effects. Composites composed of SMA wires and conventional cements can overcome the mechanical weaknesses associated with tensile fractures in conventional concretes. Under specialized environments, the material interface between the cementitious component and the SMA materials plays an important role in achieving the enhanced mechanical performance and robustness of the SMA/cement interface. This material interface is traditionally evaluated in terms of mechanical aspects, i.e., strain-stress characteristics. However, the current work attempts to simultaneously characterize the mechanical load-displacement relationships synchronized with impedance spectroscopy as a function of displacement. Frequency-dependent impedance spectroscopy is tested as an in situ monitoring tool for structural variations in smart composites composed of non-conducting cementitious materials and conducting metals. The artificial geometry change in the SMA wires is associated with an improved anchoring action that is compatible with the smallest variation in resistance compared with prismatic SMA wires embedded into a cement matrix. The significant increase in resistance is interpreted to be associated with the slip of the SMA fibers following the elastic deformation and the debonding of the SMA fiber/matrix.
Low power consumption mini rotary actuator with SMA wires
NASA Astrophysics Data System (ADS)
Manfredi, Luigi; Huan, Yu; Cuschieri, Alfred
2017-11-01
Shape memory alloys (SMAs) are smart materials widely used as actuators for their high power to weight ratio despite their well-known low energy efficiency and limited mechanical bandwidth. For robotic applications, SMAs exhibit limitations due to high power consumption and limited stroke, varying from 4% to 7% of the total length. Hysteresis, during the contraction and extension cycle, requires a complex control algorithm. On the positive side, the small size and low weight are eminently suited for the design of mini actuators for robotic platforms. This paper describes the design and construction of a light weight and low power consuming mini rotary actuator with on-board contact-less position and force sensors. The design is specifically intended to reduce (i) energy consumption, (ii) dimensions of the sensory system, and (iii) provide a simple control without any need for SMA characterisation. The torque produced is controlled by on-board force sensors. Experiments were performed to investigate the energy consumption and performance (step and sinusoidal angle profiles with a frequency varying from 0.5 to 10 Hz and maximal amplitude of {15}\\circ ). We describe a transient capacitor effect related to the SMA wires during the sinusoidal profile when the active SMA wire is powered and the antagonist one switched-off, resulting in a transient current time varying from 300 to 400 ms.
Obeso, Ignacio; Robles, Noemí; Marrón, Elena M.; Redolar-Ripoll, Diego
2013-01-01
The pre-supplementary motor area (pre-SMA) is considered to be a key node in the cognitive control of actions that require rapid updating, inhibition, or switching, as well as working memory. It is now recognized that the pre-SMA is part of a “cognitive control” network involving the inferior frontal gyrus (IFG) and subcortical regions, such as the striatum and subthalamic nucleus. However, two important questions remain to be addressed. First, it is not clear if the main role of the pre-SMA in cognitive control lies in inhibition or switching of actions. From imaging evidence, the right pre-SMA is consistently recruited during inhibition and switching, but the extent to which it participates specifically in either of these processes is unknown. Secondly, the pre-SMA may perform inhibition and switching alone or as part of a larger brain network. The present study used online and offline transcranial magnetic stimulation (TMS) to dissociate the roles of pre-SMA in cognitive control, but also to investigate the potential contribution of connectivity between the pre-SMA and IFG. We applied continuous theta burst stimulation (cTBS) over the right IFG before participants performed a stop switching task while receiving single TMS pulses over the right pre-SMA. The results were compared to a sham cTBS session and pulses applied over the vertex region. Significant worsening of inhibition as well as response adaptation during inhibition was found when applying pulses over the pre-SMA. However, no such worsening was observed in switch trials. Additionally, after cTBS over the IFG, inhibition was also delayed, suggesting its critical necessity in stopping of actions. The results reveal a key contribution of the pre-SMA in inhibition and could suggest a dissociative role in the switching of actions. These findings indicate there is an essential union between IFG and pre-SMA during inhibition. PMID:23616761
Bernier, G; Mathieu, M; De Repentigny, Y; Vidal, S M; Kothary, R
1996-11-15
We have recently cloned the gene responsible for the mouse neurological disorder dystonia musculorum. The predicted product of this gene, dystonin (Dst), is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin binding domain. Here we report on the cloning and characterization of mouse ACF7. Sequence analysis revealed extended homology of mACF7 with both the actin binding domain (ABD) and the Bpag1 portions of dystonin. Moreover, mACF7 and Dst display similar isoform diversity and encode similar sized transcripts in the nervous system. Phylogenetic analysis of mACF7 and dystonin ABD sequences suggests a recent evolutionary origin and that these proteins form a separate novel subfamily within the beta-spectrin superfamily of actin binding proteins. Given the implication of several actin binding proteins in genetic disorders, it is important to know the pattern of mACF7 expression. mACF7 transcripts are detected principally in lung, brain, spinal cord, skeletal and cardiac muscle, and skin. Intriguingly, mACF7 expression in lung is strongly induced just before birth and is restricted to type II alveolar cells. To determine whether spontaneous mutants that may be defective in mACF7 exist, we have mapped the mACF7 gene to mouse chromosome 4.
Design and fabrication of a three-finger prosthetic hand using SMA muscle wires
NASA Astrophysics Data System (ADS)
Simone, Filomena; York, Alexander; Seelecke, Stefan
2015-03-01
Bio-inspired hand-like gripper systems based on shape memory alloy (SMA) wire actuation have the potential to enable a number of useful applications in, e.g., the biomedical field or industrial assembly systems. The inherent high energy density makes SMA solutions a natural choice for systems with lightweight, low noise and high force requirements, such as hand prostheses or robotic systems in a human/machine environment. The focus of this research is the development, design and realization of a SMA-actuated prosthetic hand prototype with three fingers. The use of thin wires (100 μm diameter) allows for high cooling rates and therefore fast movement of each finger. Grouping several small wires mechanically in parallel allows for high force actuation. To save space and to allow for a direct transmission of the motion to each finger, the SMA wires are attached directly within each finger, across each phalanx. In this way, the contraction of the wires will allow the movement of the fingers without the use of any additional gears. Within each finger, two different bundles of wires are mounted: protagonist ones that create bending movement and the antagonist ones that enable stretching of each phalanx. The resistance change in the SMA wires is measured during actuation, which allows for monitoring of the wire stroke and potentially the gripping force without the use of additional sensors. The hand is built with modern 3D-printing technologies and its performance while grasping objects of different size and shape is experimentally investigated illustrating the usefulness of the actuator concept.
Functional adaptation between yeast actin and its cognate myosin motors.
Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew
2011-09-02
We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.
Oligomerization of coronin: Implication on actin filament length in Leishmania.
Srivastava, Rashmi; Prasadareddy Kajuluri, Lova; Pathak, Neelam; Gupta, Chhitar M; Sahasrabuddhe, Amogh A
2015-12-01
Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network. © 2015 Wiley Periodicals, Inc.
Tian, Guilian; Zhou, Yun; Hajkova, Dagmar; Miyagi, Masaru; Dinculescu, Astra; Hauswirth, William W; Palczewski, Krzysztof; Geng, Ruishuang; Alagramam, Kumar N; Isosomppi, Juha; Sankila, Eeva-Marja; Flannery, John G; Imanishi, Yoshikazu
2009-07-10
Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1(-/-) mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.
Ferrer, María F; Pascuale, Carla A; Gomez, Ricardo M; Leguizamón, María S
2014-05-01
Chagas heart disease is a major public concern since 30% of infected patients develop cardiac alterations. The relationship between Trypanosoma cruzi discrete typing units (DTUs) and the biological properties exhibited by the parasite population has yet to be elucidated. In this study, we analysed the expression of α-smooth muscle actin (α-SMA) and galectin-3 (Gal-3) associated with cardiac extracellular matrix (ECM) remodelling a murine chronic cardiomyopathy induced by Tc I genotypes. We found the induction of myocarditis was associated with the upregulation of Col I, α-SMA, Gal-3, IFN-γ and IL-13, as analysed by q-PCR. In myocardial areas of fibrosis, the intensity of myocarditis and significant ECM remodelling correlated with the presence of Col I-, Gal-3- and α-SMA-positive cells. These results are promising for the further efforts to evaluate the relevance of Gal-3 in Chagas heart disease, since this galectin was proposed as a prognosis marker in heart failure patients.
Coactosin accelerates cell dynamism by promoting actin polymerization.
Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu
2013-07-01
During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.
Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Rubenstein, Peter A.
2011-01-01
In the Saccharomyces cerevisiae actin-profilin interface, Ala167 of the actin barbed end W-loop and His372 near the C terminus form a clamp around a profilin segment containing residue Arg81 and Tyr79. Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function. PMID:21956104
How actin binds and assembles onto plasma membranes from Dictyostelium discoideum
1988-01-01
We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099
Identification of sucrose synthase as an actin-binding protein
NASA Technical Reports Server (NTRS)
Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)
1998-01-01
Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.
Thymosin-beta(4) changes the conformation and dynamics of actin monomers.
De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D
2000-01-01
Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange. PMID:10777749
Treatment options for actinic keratoses.
McIntyre, William J; Downs, Michael R; Bedwell, Sondra A
2007-09-01
Actinic keratoses are rough, scaly lesions that commonly occur on sun-exposed areas of the skin. The prevalence of the condition increases with age. Actinic keratoses are thought to be carcinomas in situ, which can progress to squamous cell carcinomas. The decision to treat can be based on cosmetic reasons; symptom relief; or, most importantly, the prevention of malignancy and metastasis. Treatment options include ablative (destructive) therapies such as cryosurgery, curettage with electrosurgery, and photodynamic therapy. Topical therapies are used in patients with multiple lesions. Fluorouracil has been the traditional topical treatment for actinic keratoses, although imiquimod 5% cream and diclofenac 3% gel are effective alternative therapies. There are too few controlled trials comparing treatment modalities for physicians to make sound, evidence-based treatment decisions.
Side-binding proteins modulate actin filament dynamics
Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C
2015-01-01
Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001 PMID:25706231
Spatial control of actin polymerization during neutrophil chemotaxis
Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.
2010-01-01
Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877