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Sample records for cytoskeletal protein expression

  1. Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

    PubMed

    Reynoso-Ducoing, Olivia; Valverde-Islas, Laura; Paredes-Salomon, Cristina; Pérez-Reyes, América; Landa, Abraham; Robert, Lilia; Mendoza, Guillermo; Ambrosio, Javier R

    2014-05-01

    The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

  2. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    PubMed

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  3. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells.

  4. Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

    PubMed

    González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; González-Flores, Oscar; Galván-Rosas, Agustín; Porfirio Gómora-Arrati; Camacho-Arroyo, Ignacio

    2014-01-01

    Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

  5. Cytoskeletal Proteins of Actinobacteria

    PubMed Central

    Letek, Michal; Fiuza, María; Villadangos, Almudena F.; Mateos, Luís M.; Gil, José A.

    2012-01-01

    Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known about Actinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such as Mycobacterium, Corynebacterium, and Streptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals. PMID:22481946

  6. Purification of Tetrahymena cytoskeletal proteins.

    PubMed

    Honts, Jerry E

    2012-01-01

    Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

  7. Growth factors and steroid mediated regulation of cytoskeletal protein expression in serum-deprived primary astrocyte cultures.

    PubMed

    Bramanti, Vincenzo; Bronzi, Daniela; Tomassoni, Daniele; Costa, Antonino; Raciti, Giuseppina; Avitabile, Marcello; Amenta, Francesco; Avola, Roberto

    2008-12-01

    In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them together. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressful conditions.

  8. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chin-Mei Chang-Liu

    1995-06-01

    Experiments examined the effects of radiation dose-rate and protein synthesis inhibition expression of cytoskeletal and matrix elements in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for neutrons when comparing expression of {alpha}-tubulin and fibronectin genes. Cycloheximide repressed accumulation of {alpha}-tubulin-mRNA following exposure to high dose-rate neutrons or {gamma} rays. Cycloheximide did not affect accumulation of actin mRNA. Cycloheximide abrogated induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to radiation. 24 refs., 3 tabs.

  9. Kaempferol enhances intestinal barrier function through the cytoskeletal association and expression of tight junction proteins in Caco-2 cells.

    PubMed

    Suzuki, Takuya; Tanabe, Soichi; Hara, Hiroshi

    2011-01-01

    Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. We investigated the promotive effect of kaempferol on tight junction (TJ) barrier integrity in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER; a TJ integrity marker) across the monolayers rapidly and markedly increased during the first 6 h after kaempferol administration and remained elevated until 48 h without any changes in the lucifer yellow or dextran fluxes. Immunoblot analysis demonstrated that kaempferol promoted the actin cytoskeletal association of the TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, claudin-1, claudin-3, and claudin-4, which was associated with the increase in TER. Kaempferol-mediated ZO-2 and claudin-4 expression was relatively smaller or occurred later than the kaempferol-promoted cytoskeletal association. Confocal microscopy showed that kaempferol-induced assembly of occludin and claudin-3 occurred at the TJ at 6 h postadministration. Extraction of cholesterol with methyl-β-cyclodextrin suppressed the kaempferol-mediated increase in TER. Sucrose density gradient centrifugation showed that the kaempferol treatment increased the TJ protein distributions in the cholesterol-rich lipid microdomain fraction. Taken together, these results indicate that the membrane lipid microdomain is involved in the kaempferol-mediated promotion of TJ protein assembly and intestinal TJ integrity.

  10. Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites.

    PubMed

    Chen, Tao; Yu, Yan; Tang, Liu-Jiu; Kong, Li; Zhang, Cheng-Hong; Chu, Hai-Ying; Yin, Liang-Wei; Ma, Hai-Ying

    2017-03-01

    Cytoskeletal proteins are involved in neuronal survival. Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury. However, the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear. To examine this, we established a rat model of traumatic brain injury by controlled cortical impact. At 72 hours after injury, 2 × 10(7) cells/mL neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells (3 mL) were injected into the injured cortex. At 1-3 weeks after transplantation, expression of neurofilament 200, microtubule-associated protein 2, actin, calmodulin, and beta-catenin were remarkably increased in the injury sites. These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival, growth, and differentiation in the injury sites. The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.

  11. Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

    PubMed Central

    Chen, Tao; Yu, Yan; Tang, Liu-jiu; Kong, Li; Zhang, Cheng-hong; Chu, Hai-ying; Yin, Liang-wei; Ma, Hai-ying

    2017-01-01

    Cytoskeletal proteins are involved in neuronal survival. Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury. However, the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear. To examine this, we established a rat model of traumatic brain injury by controlled cortical impact. At 72 hours after injury, 2 × 107 cells/mL neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells (3 mL) were injected into the injured cortex. At 1–3 weeks after transplantation, expression of neurofilament 200, microtubule-associated protein 2, actin, calmodulin, and beta-catenin were remarkably increased in the injury sites. These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival, growth, and differentiation in the injury sites. The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway. PMID:28469658

  12. Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

    PubMed Central

    Higashibata, Akira; Hashizume, Toko; Nemoto, Kanako; Higashitani, Nahoko; Etheridge, Timothy; Mori, Chihiro; Harada, Shunsuke; Sugimoto, Tomoko; Szewczyk, Nathaniel J; Baba, Shoji A; Mogami, Yoshihiro; Fukui, Keiji; Higashitani, Atsushi

    2016-01-01

    Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts. PMID:28725720

  13. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1993-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for JANUS fission-spectrum neutrons when comparing expression of either a-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Cycloheximide, however, repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposures. Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation and that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  14. Neuroactive molecules and growth factors modulate cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture.

    PubMed

    Bramanti, Vincenzo; Grasso, Sonia; Tibullo, Daniele; Giallongo, Cesarina; Pappa, Rita; Brundo, Maria Violetta; Tomassoni, Daniele; Viola, Maria; Amenta, Francesco; Avola, Roberto

    2016-01-01

    Steroid hormones and neurotrophic factors regulate astroglial cell survival, proliferation, and differentiation in culture. The present study examines the interaction between glucocorticoids and growth factors (GFs) on cytoskeletal proteins and extracellular signal-regulated kinase 2 (ERK2) expression in stressed astroglial cultures at 25 days in vitro, according to the following experimental condition. Pretreatment with basic fibroblast growth factor alone or in combination with dexamethasone 10(-9) M for 48 hr induced an enhancement of glial fibrillary acidic protein, vimetin, and ERK2 expression. Treatment with "progression" GFs alone and in the last 12 hr significantly increased the above-mentioned markers' expression. The present study shows that glucocorticoids may cooperate with GFs or may abrogate their effects, depending on the experimental culture conditions used as well as the exposure time and the types of GFs added. Our findings provide evidence of interactive dialogue between GFs and neurosteroids in cultured astrocytes. This may have implications in the therapeutic approach to neurologic disorders associated with astrogliosis.

  15. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1994-05-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  16. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1992-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  17. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

    PubMed Central

    Boraas, Liana C.; Guidry, Julia B.; Pineda, Emma T.; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

  18. A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex.

    PubMed

    Cvrčková, Fatima; Oulehlová, Denisa

    2017-01-01

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

  19. Effects of transforming growth factor type beta on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

    SciTech Connect

    Lomri, A.; Marie, P.J. )

    1990-01-01

    Transforming growth factor beta (TGF beta) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGF beta on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGF beta (1.5-30 ng/mL) slightly (-23%) inhibited alkaline phosphatase activity. In parallel, TGF beta (0.5-30 ng/mL, 24 hours) greatly increased cell replication as evaluated by (3H)-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGF beta induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, alpha actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGF beta (1.5 ng/mL) increased the de novo biosynthesis of actin, alpha actinin, vimentin, and tubulins, as determined by {sup 35}S methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGF beta exposure. The results indicate that the stimulatory effect of TGF beta on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.

  20. Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression

    USDA-ARS?s Scientific Manuscript database

    Cellular prion protein (PrPC) is a multifunctional protein, whose exact physiological role remains elusive. Since previous studies indicated a neuroprotective function of PrPC, we investigated whether Prnp knockout mice(Prnp0/0)display age-dependent behavioral abnormalities. Matched sets of Prnp0/0 ...

  1. Differential expression of cytoskeletal proteins in the dendrites of parvalbumin-positive interneurons versus granule cells in the adult rat dentate gyrus.

    PubMed

    de Haas Ratzliff, A; Soltesz, I

    2000-01-01

    Parvalbumin-positive interneurons and granule cells of the dentate gyrus exhibit characteristic differences in morphological, cytochemical, physiological, and pathophysiological properties. Several of these defining features, including dendritic morphology, spine density, and sensitivity to insults, are likely to be influenced by the neuronal cytoskeleton. The data in this paper demonstrate striking differences in the expression levels of all three neurofilament triplet proteins, as well as alpha-internexin and beta-tubulin III, between the parvalbumin-positive interneurons and dentate granule cells. Therefore, the molecular composition of intermediate filaments and microtubules in the dendritic domain of parvalbumin-positive dentate interneurons is distinct from the cytoskeleton of neighboring granule cells, indicating the existence of highly cell type-specific cytoskeletal architecture within the dentate gyrus.

  2. The cytotoxic effect of memantine and its effect on cytoskeletal proteins expression in metastatic breast cancer cell line

    PubMed Central

    Seifabadi, Sima; Vaseghi, Golnaz; Javanmard, Shaghayegh Haghjooy; Omidi, Elham; Tajadini, Mohammadhasan; Zarrin, Bahareh

    2017-01-01

    Objective(s): Breast cancer is an important leading cause of death from cancer. Stathmin and tau proteins are regulators of cell motility, and their overexpression is associated with the progression and bad prognosis of breast cancer. Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is the potential inhibitor of tau protein in neurons. This study determines the effect of memantine on breast cancer cell migration and proliferation, tau and stathmin gene expression in cancer cells and its synergistic effect with paclitaxel. Materials and Methods: The cell proliferation was evaluated by MTT assay and for this purpose, MCF-7 breast cancer cells were treated with various concentration of memantine (2, 20 and 100 μg/ml). Tau and stathmin mRNA expression was evaluated through quantitative real time RT-PCR method. The migration of cancer cells treated with memantine for 24 hr was compared to non-treated cells using an in vitro transmembrane migration assay. Results: Incubation of breast cancer cells with memantine resulted in a dose dependent reduction in cell survival (P=0.0001). Paclitaxel (100 nM) showed synergistic effect with memantine (P=0.0001). Memantine significantly decreased tau and stathmin mRNA expression (by RT-PCR), so that 100 µmol/l of memantine decreased tau and stathmin expression by 46% (P=0.0341) and 33% (P=0.043), respectively. Migration of cells was also decreased by memantine (P=0.0001). Conclusion: The presented data shows that memantine reduced mRNA levels of tau and stathmin proteins and also reduced cellular migration. PMID:28133523

  3. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  4. Cytoskeletal integrity in interphase cells requires protein phosphatase activity.

    PubMed Central

    Eriksson, J E; Brautigan, D L; Vallee, R; Olmsted, J; Fujiki, H; Goldman, R D

    1992-01-01

    Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components. Images PMID:1332069

  5. Differential gene expression analysis by RNA-seq reveals the importance of actin cytoskeletal proteins in erythroleukemia cells

    PubMed Central

    Fernández-Calleja, Vanessa; Hernández, Pablo; Schvartzman, Jorge B.; García de Lacoba, Mario

    2017-01-01

    Development of drug resistance limits the effectiveness of anticancer treatments. Understanding the molecular mechanisms triggering this event in tumor cells may lead to improved therapeutic strategies. Here we used RNA-seq to compare the transcriptomes of a murine erythroleukemia cell line (MEL) and a derived cell line with induced resistance to differentiation (MEL-R). RNA-seq analysis identified a total of 596 genes (Benjamini–Hochberg adjusted p-value < 0.05) that were differentially expressed by more than two-fold, of which 81.5% (486/596) of genes were up-regulated in MEL cells and 110 up-regulated in MEL-R cells. These observations revealed that for some genes the relative expression of mRNA amount in the MEL cell line has decreased as the cells acquired the resistant phenotype. Clustering analysis of a group of genes showing the highest differential expression allowed identification of a sub-group among genes up-regulated in MEL cells. These genes are related to the organization of the actin cytoskeleton network. Moreover, the majority of these genes are preferentially expressed in the hematopoietic lineage and at least three of them, Was (Wiskott Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Rac2, when mutated in humans, give rise to severe hematopoietic deficiencies. Among the group of genes that were up-regulated in MEL-R cells, 16% of genes code for histone proteins, both canonical and variants. A potential implication of these results on the blockade of differentiation in resistant cells is discussed. PMID:28663935

  6. Expression of the cytoskeletal protein MAP5 and its regulation by neurotrophin 3 (NT3) in the inner ear sensory neurons.

    PubMed

    San José, I; Vázquez, E; García-Atarés, N; Rodriguez, S; Vega, J A; Represa, J

    1997-03-01

    Microtubule-associated proteins (MAPs) are essential cytoskeletal components during development for neurogenesis and neuronal plasticity. Inner ear innervation is accomplished by cochleovestibular ganglion (CVG) neurons in a highly specific, well-defined pattern, which is regulated by neurotrophic factors belonging to the neurotrophin family. The inner ear offers a suitable model for studying the expression of MAPs and assessing their role in neurotrophin-induced effects that are required for neuron-target innervation. The present study was undertaken to analyze the expression and localization of MAP5 isoforms during development of CVG neurons in vivo an in vitro; as well as the regulation of MAP5 by neurotrophin-3 (NT3) in cell culture. MAP5 expression in the inner ear of chick embryos and postnatal specimens was monitored using immunoblots and immunohistochemistry on frozen sections. MAP5 was highly expressed during the early stages of CVG development, at embryonic day (E)4, being located in both neuronal perikarya and neurites. Expression was maintained during the neurite outgrowth phase (E6-E12), when strong MAP5 immunostaining was observed at the same cellular locations. MAP5 expression decreased suddenly at E14, after the establishment of specific connections between the CVG neurons and their targets, the sensory epithelium of the inner ear. In cultured CVG neurons addition of NT3 led to increased MAP5 expression and produced neurite outgrowth. Both effects are differentially regulated in parallel by low (0.5 ng/ml) and high (5 ng/ml) NT3 concentrations. Present results suggest that MAP5 may be involved in neurotrophin-induced microtubule bundling during neurite outgrowth of auditory neurons.

  7. Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells*

    PubMed Central

    Radhakrishnan, Karthika; Bhagya, Kongattu P.; Kumar, Anil TR; Devi, Anandavalli N.; Sengottaiyan, Jeeva; Kumar, Pradeep G.

    2016-01-01

    Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)+ (spermatogonia), GFRα−/synaptonemal complex protein (SCP3)+ (meiotic), and GFRα−/Phosphoglycerate kinase 2 (PGK2)+ (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization

  8. Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

    PubMed

    Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

    2016-08-01

    Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein

  9. Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

    PubMed Central

    RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

    2007-01-01

    The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

  10. Inhibition of cytoskeletal protein carbonylation may protect against oxidative damage in traumatic brain injury

    PubMed Central

    Zhang, Qiusheng; Zhang, Meng; Huang, Xianjian; Liu, Xiaojia; Li, Weiping

    2016-01-01

    Oxidative stress is the principal factor in traumatic brain injury (TBI) that initiates protracted neuronal dysfunction and remodeling. Cytoskeletal proteins are known to be carbonylated under oxidative stress; however, the complex molecular and cellular mechanisms of cytoskeletal protein carbonylation remain poorly understood. In the present study, the expression levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were investigated in PC12 cells treated with H2O2. Western blot analysis was used to monitor the carbonylation levels of β-actin and β-tubulin. The results indicated that oxidative stress was increased in PC12 cells that were treated with H2O2 for 24 or 48 h. In addition, increased carbonylation levels of β-actin and β-tubulin were detected in H2O2-treated cells. However, these carbonylation levels were reduced by pretreatment with aminoguanidine, a type of reactive carbonyl species chelating agent, and a similar trend was observed following overexpression of proteasome β5 via transgenic technology. In conclusion, the present study results suggested that the development of TBI may cause carbonylation of cytoskeletal proteins, which would then undermine the stability of cytoskeletal proteins. Thus, the development of TBI may be improved via the inhibition of cytoskeletal protein carbonylation. PMID:28101189

  11. Viscum album agglutinin-I (VAA-I) increases cell surface expression of cytoskeletal proteins in apoptotic human neutrophils: moesin and ezrin are two novel targets of VAA-I.

    PubMed

    Simon, M M; Simard, J C; Girard, D

    2013-10-01

    Viscum album agglutinin-I (VAA-I) is a plant lectin, which possesses anti-inflammatory properties, including the ability to induce neutrophil apoptosis by a mechanism that is not completely understood. Among the three actin-binding membrane-anchoring proteins ezrin/radixin/moesin (ERM), neutrophils are known to express ezrin and moesin. The behavior of these proteins in apoptotic neutrophils is not well established. In the present study, the expression and localization of ezrin and moesin by Western blot and immunofluorescence revealed a clear degradation and relocalization of both the proteins during VAA-I-induced apoptosis. Also, flow cytometry analysis revealed that VAA-I markedly and significantly induced the cell surface expression of ezrin and moesin and this was reversed when cells were pretreated with the Syk inhibitor piceatannol. The expression of ezrin and moesin on the cell surface of apoptotic neutrophils may represent a mechanism responsible for the appearance of autoantibodies directed against ERM proteins, which have been found in the serum of patients suffering from autoimmune diseases. Therefore, the ability of VAA-I to increase cell surface expression of cytoskeletal proteins in apoptotic neutrophils provides important insight into a possible toxic mechanism of this plant lectin and this has to be considered for its potential utilization for in vivo treatment.

  12. Identification of paralogous life-cycle stage specific cytoskeletal proteins in the parasite Trypanosoma brucei.

    PubMed

    Portman, Neil; Gull, Keith

    2014-01-01

    The life cycle of the African trypanosome Trypanosoma brucei, is characterised by a transition between insect and mammalian hosts representing very different environments that present the parasite with very different challenges. These challenges are met by the expression of life-cycle stage-specific cohorts of proteins, which function in systems such as metabolism and immune evasion. These life-cycle transitions are also accompanied by morphological rearrangements orchestrated by microtubule dynamics and associated proteins of the subpellicular microtubule array. Here we employed a gel-based comparative proteomic technique, Difference Gel Electrophoresis, to identify cytoskeletal proteins that are expressed differentially in mammalian infective and insect form trypanosomes. From this analysis we identified a pair of novel, paralogous proteins, one of which is expressed in the procyclic form and the other in the bloodstream form. We show that these proteins, CAP51 and CAP51V, localise to the subpellicular corset of microtubules and are essential for correct organisation of the cytoskeleton and successful cytokinesis in their respective life cycle stages. We demonstrate for the first time redundancy of function between life-cycle stage specific paralogous sets in the cytoskeleton and reveal modification of cytoskeletal components in situ prior to their removal during differentiation from the bloodstream form to the insect form. These specific results emphasise a more generic concept that the trypanosome genome encodes a cohort of cytoskeletal components that are present in at least two forms with life-cycle stage-specific expression.

  13. Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

    PubMed Central

    Turkel, Nezaket; Portela, Marta; Poon, Carole; Li, Jason; Brumby, Anthony M.; Richardson, Helena E.

    2015-01-01

    ABSTRACT The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems. PMID:26187947

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  16. Topographic regulation of cytoskeletal protein phosphorylation by multimeric complexes in the squid giant fiber system.

    PubMed

    Grant, P; Diggins, M; Pant, H C

    1999-07-01

    In mammalian and squid nervous systems, the phosphorylation of neurofilament proteins (NFs) seems to be topographically regulated. Although NFs and relevant kinases are synthesized in cell bodies, phosphorylation of NFs, particularly in the lys-ser-pro (KSP) repeats in NF-M and NF-H tail domains, seem to be restricted to axons. To explore the factors regulating the cellular compartmentalization of NF phosphorylation, we separated cell bodies (GFL) from axons in the squid stellate ganglion and compared the kinase activity in the respective lysates. Although total kinase activity was similar in each lysate, the profile of endogenous phosphorylated substrates was strikingly different. Neurofilament protein 220 (NF220), high-molecular-weight NF protein (HMW), and tubulin were the principal phosphorylated substrates in axoplasm, while tubulin was the principal GFL phosphorylated substrate, in addition to highly phosphorylated low-molecular-weight proteins. Western blot analysis showed that whereas both lysates contained similar kinases and cytoskeletal proteins, phosphorylated NF220 and HMW were completely absent from the GFL lysate. These differences were highlighted by P13(suc1) affinity chromatography, which revealed in axoplasm an active multimeric phosphorylation complex(es), enriched in cytoskeletal proteins and kinases; the equivalent P13 GFL complex exhibited six to 20 times less endogenous and exogenous phosphorylation activity, respectively, contained fewer cytoskeletal proteins and kinases, and expressed a qualitatively different cdc2-like kinase epitope, 34 kDa rather than 49 kDa. Cell bodies and axons share a similar repertoire of molecular consitutents; however, the data suggest that the cytoskeletal/kinase phosphorylation complexes extracted from each cellular compartment by P13 are fundamentally different. Copyright 1999 John Wiley & Sons, Inc.

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

    PubMed

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

    2016-02-12

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

    SciTech Connect

    Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

    1981-11-01

    Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

  19. Run-and-pause dynamics of cytoskeletal motor proteins

    NASA Astrophysics Data System (ADS)

    Hafner, Anne E.; Santen, Ludger; Rieger, Heiko; Shaebani, M. Reza

    2016-11-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. When attached to cytoskeletal filaments, the motor exhibits distinct states of motility: active motion along the filaments, and pause phase in which it remains stationary for a finite time interval. The transition probabilities between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of individual non-interacting molecular motors on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition probabilities between the two states of motility drastically changes the dynamics: multiple transitions between different types of anomalous diffusive dynamics occur and the crossover time to the asymptotic diffusive or ballistic motion varies by several orders of magnitude. We map out the phase diagrams in the space of transition probabilities, and address the role of initial conditions of motion on the resulting dynamics.

  20. Run-and-pause dynamics of cytoskeletal motor proteins

    PubMed Central

    Hafner, Anne E.; Santen, Ludger; Rieger, Heiko; Shaebani, M. Reza

    2016-01-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. When attached to cytoskeletal filaments, the motor exhibits distinct states of motility: active motion along the filaments, and pause phase in which it remains stationary for a finite time interval. The transition probabilities between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of individual non-interacting molecular motors on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition probabilities between the two states of motility drastically changes the dynamics: multiple transitions between different types of anomalous diffusive dynamics occur and the crossover time to the asymptotic diffusive or ballistic motion varies by several orders of magnitude. We map out the phase diagrams in the space of transition probabilities, and address the role of initial conditions of motion on the resulting dynamics. PMID:27849013

  1. Whirlin, a cytoskeletal scaffolding protein, stabilizes the paranodal region and axonal cytoskeleton in myelinated axons.

    PubMed

    Green, James A; Yang, Jun; Grati, M'hamed; Kachar, Bechara; Bhat, Manzoor A

    2013-09-06

    Myelinated axons are organized into distinct subcellular and molecular regions. Without proper organization, electrical nerve conduction is delayed, resulting in detrimental physiological outcomes. One such region is the paranode where axo-glial septate junctions act as a molecular fence to separate the sodium (Na+) channel-enriched node from the potassium (K+) channel-enriched juxtaparanode. A significant lack of knowledge remains as to cytoskeletal proteins which stabilize paranodal domains and underlying cytoskeleton. Whirlin (Whrn) is a PDZ domain-containing cytoskeletal scaffold whose absence in humans results in Usher Syndromes or variable deafness-blindness syndromes. Mutant Whirlin (Whrn) mouse model studies have linked such behavioral deficits to improper localization of critical transmembrane protein complexes in the ear and eye. Until now, no reports exist about the function of Whrn in myelinated axons. RT-PCR and immunoblot analyses revealed expression of Whrn mRNA and Whrn full-length protein, respectively, in several stages of central and peripheral nervous system development. Comparing wild-type mice to Whrn knockout (Whrn-/-) mice, we observed no significant differences in the expression of standard axonal domain markers by immunoblot analysis but observed and quantified a novel paranodal compaction phenotype in 4 to 8 week-old Whrn-/- nerves. The paranodal compaction phenotype and associated cytoskeletal disruption was observed in Whrn-/- mutant sciatic nerves and spinal cord fibers from early (2 week-old) to late (1 year-old) stages of development. Light and electron microscopic analyses of Whrn knockout mice reveal bead-like swellings in cerebellar Purkinje axons containing mitochondria and vesicles by both. These data suggest that Whrn plays a role in proper cytoskeletal organization in myelinated axons. Domain organization in myelinated axons remains a complex developmental process. Here we demonstrate that loss of Whrn disrupts proper axonal

  2. The time course of activity-regulated cytoskeletal (ARC) gene and protein expression in the whisker-barrel circuit using two paradigms of whisker stimulation.

    PubMed

    Khodadad, Aida; Adelson, P David; Lifshitz, Jonathan; Thomas, Theresa Currier

    2015-05-01

    Immediate early genes have previously demonstrated a rapid increase in gene expression after various behavioral paradigms. The main focus of this article is to identify a molecular marker of circuit activation after manual whisker stimulation or exploration of a novel environment. To this end, we investigated the dynamics of ARC transcription in adult male rats during whisker somatosensation throughout the whisker barrel circuit. At various time points, tissue was biopsied from the ventral posterior medial nucleus (VPM) of the thalamus, primary somatosensory barrel field (S1BF) cortex and hippocampus for quantification using real-time PCR and western blot. Our results show that there were no significant differences in ARC gene or protein expression in the VPM after both types of stimulation. However, manual whisker stimulation resulted in increased ARC gene expression at 15, 30, 60 and 300 min in the S1BF, and 15 min in the hippocampus (p<0.05). Also, exploration of a novel environment resulted in increased ARC mRNA expression at 15 and 30 min in the S1BF and at 15 min in the hippocampus (p<0.05). The type of stimulation (manual versus exploration of a novel environment) influenced the magnitude of ARC gene expression in the S1BF (p<0.05). These data are the first to demonstrate that ARC is a specific, quantifiable and input dependent molecular marker of circuit activation which can serve to quantify the impact of brain injury and subsequent rehabilitation on whisker sensation. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Mapping cytoskeletal protein function in cells by means of nanobodies.

    PubMed

    Van Audenhove, Isabel; Van Impe, Katrien; Ruano-Gallego, David; De Clercq, Sarah; De Muynck, Kevin; Vanloo, Berlinda; Verstraete, Hanne; Fernández, Luis Á; Gettemans, Jan

    2013-10-01

    Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection.

  4. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  5. A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula.

    PubMed

    Ivanov, Sergey; Harrison, Maria J

    2014-12-01

    Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis-specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub-cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens-shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  6. Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovich, J. G.; Zhambalova, A. P.; Romanov, Yu. A.; Buravkova, L. B.

    2012-01-01

    Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of "stem cells markers" were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10-20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

  7. Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABA(A) receptor subunits.

    PubMed

    Zepeda, Angelica; Sengpiel, Frank; Guagnelli, Miguel Angel; Vaca, Luis; Arias, Clorinda

    2004-02-25

    Reorganization of cortical representations after focal visual cortex lesions has been documented. It has been suggested that functional reorganization may rely on cellular mechanisms involving modifications in the excitatory/inhibitory neurotransmission balance and on morphological changes of neurons peripheral to the lesion. We explored functional reorganization of cortical retinotopic maps after a focal ischemic lesion in primary visual cortex of kittens using optical imaging of intrinsic signals. After 1, 2, and 5 weeks postlesion (wPL), we addressed whether functional reorganization correlated in time with changes in the expression of MAP-2, GAP-43, GFAP, GABA(A) receptor subunit alpha1 (GABA(A)alpha1), subunit 1 of the NMDA receptor (NMDAR1), and in neurotransmitter levels at the border of the lesion. Our results show that: (1) retinotopic maps reorganize with time after an ischemic lesion; (2) MAP-2 levels increase gradually from 1wPL to 5wPL; (3) MAP-2 upregulation is associated with an increase in dendritic-like structures surrounding the lesion and a decrease in GFAP-positive cells; (4) GAP-43 levels reach the highest point at 2wPL; (5) NMDAR1 and glutamate contents increase in parallel from 1wPL to 5wPL; (6) GABA(A)alpha1 levels increase from 1wPL to 2wPL but do not change after this time point; and (7) GABA contents remain low from 1wPL to 5wPL. This is a comprehensive study showing for the first time that functional reorganization correlates in time with dendritic sprouting and with changes in the excitatory/inhibitory neurotransmission systems previously proposed to participate in cortical remodeling and suggests mechanisms by which plasticity of cortical representations may occur.

  8. Regulation of protein kinase C by the cytoskeletal protein calponin.

    PubMed

    Leinweber, B; Parissenti, A M; Gallant, C; Gangopadhyay, S S; Kirwan-Rhude, A; Leavis, P C; Morgan, K G

    2000-12-22

    Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

  9. Force profiles of protein pulling with or without cytoskeletal links studied by AFM

    SciTech Connect

    Afrin, Rehana; Ikai, Atsushi . E-mail: aikai@bio.titech.ac.jp

    2006-09-15

    To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.

  10. PDZD8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1.

    PubMed

    Henning, Matthew S; Stiedl, Patricia; Barry, Denis S; McMahon, Robert; Morham, Scott G; Walsh, Derek; Naghavi, Mojgan H

    2011-07-05

    The host cytoskeleton plays a central role in the life cycle of many viruses yet our knowledge of cytoskeletal regulators and their role in viral infection remains limited. Recently, moesin and ezrin, two members of the ERM (Ezrin/Radixin/Moesin) family of proteins that regulate actin and plasma membrane cross-linking and microtubule (MT) stability, have been shown to inhibit retroviral infection. To further understand how ERM proteins function and whether they also influence infection by other viruses, we identified PDZD8 as a novel moesin-interacting protein. PDZD8 is a poorly understood protein whose function is unknown. Exogenous expression of either moesin or PDZD8 reduced the levels of stable MTs, suggesting that these proteins functioned as part of a cytoskeletal regulatory complex. Additionally, exogenous expression or siRNA-mediated knockdown of either factor affected Herpes Simplex Virus type 1 (HSV-1) infection, identifying a cellular function for PDZD8 and novel antiviral properties for these two cytoskeletal regulatory proteins.

  11. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

    PubMed Central

    Bonin, Sawyer R.; Gibeault, Sabrina; De Repentigny, Yves; Kothary, Rashmi

    2016-01-01

    Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic

  12. Cocaine affects the dynamics of cytoskeletal proteins via sigma(1) receptors.

    PubMed

    Su, T P; Hayashi, T

    2001-09-01

    Cytoskeletal proteins are important in protein trafficking, membrane protein clustering, dendrite growth and the morphological maintenance of neurons. Sigma(1) receptors are unique endoplasmic reticular (ER) proteins that bind (+)benzomorphans, neurosteroids and psychotropic drugs such as cocaine. Cocaine, via sigma(1) receptors, can cause the dissociation of a cytoskeletal adaptor protein ankyrin from inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptors on the ER as a sigma(1)-receptor-ankyrin complex, which then translocates to the plasma membrane and nucleus. The dissociation of sigma(1)-receptor-ankyrin from Ins(1,4,5)P(3) receptors also increases the intracellular Ca(2+) concentration [[Ca(2+)](i)], which affects the activity of cytoskeletal proteins. Furthermore, cocaine might increase [Ca(2+)](i) via phospholipase C (PLC)-linked dopamine D1 receptors. We hypothesize that cocaine might cause life-long changes in neurons via cytoskeletal proteins by interacting with both D1 receptors and sigma(1) receptors.

  13. p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

    PubMed

    Kunimoto, M; Shibata, K; Miura, T

    1987-12-11

    Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

  14. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.

    PubMed Central

    Ornelles, D A; Fey, E G; Penman, S

    1986-01-01

    Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements. Images PMID:3785175

  15. The cytoskeletal protein talin contains at least two distinct vinculin binding domains

    PubMed Central

    1993-01-01

    We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2- 6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin. PMID:8320257

  16. Post mortem development of meat quality as related to changes in cytoskeletal proteins of chicken muscles.

    PubMed

    Tomaszewska-Gras, J; Schreurs, F J G; Kijowski, J

    2011-04-01

    1. A procedure was developed to separate high and medium molecular weight myofibrillar proteins from chicken muscular tissue with a high resolution by flat bed sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent detection by either a general protein stain or Western blotting. These procedures were used to analyse the degradation process of cytoskeletal proteins in chicken breast and leg muscles during meat ageing. 2. This study demonstrates the degradation of all the examined cytoskeletal proteins: titin, nebulin and desmin as well as vinculin, a protein component of the costamere structure. All the examined proteins were found to be degraded during ageing of chicken breast and leg muscles. 3. Degradation of titin, nebulin and desmin started at 3 h post mortem in breast muscle. Intact titin and nebulin disappeared within 1 d. Intact desmin and vinculin were not detectable after 3 d post mortem. In leg muscle, the degradation process of all the examined proteins evolved much more slowly than in breast chicken muscles. 4. The changes observed in shear force, myofibrillar fragmentation and cooking loss were related to changes in cytoskeletal proteins and used to identify marker proteins or degradation products for the purpose of monitoring the development of meat ageing. The ageing process was faster in breast muscle than in leg muscle. 5. Significant correlations were found between degradation processes of titin, nebulin, and desmin and shear force, as well as myofibril fragmentation index of breast and leg muscles.

  17. Homology Modeling Procedures for Cytoskeletal Proteins of Tetrahymena and Other Ciliated Protists.

    PubMed

    Pagano, Giovanni J; Hufnagel, Linda A; King, Roberta S

    2016-01-01

    In recent years there has been an explosive increase in the number of annotated protein sequences available through genome sequencing, as well as an accumulation of published protein structural data based on crystallographic and NMR methods. When taken together with the development of computational methods for the prediction of protein structural and functional properties through homology modeling, an opportunity exists for prediction of properties of cytoskeletal proteins in a suitable model organism, such as Tetrahymena thermophila and its ciliated protist relatives. In particular, the recently sequenced genome of T. thermophila, long a model for cytoskeletal studies, provides a good starting point for undertaking such homology modeling studies. Homology modeling can produce functional predictions, for example regarding potential molecular interactions, that are of great interest to the drug industry and Tetrahymena is an attractive model system in which to follow up computational predictions with experimental analyses. We provide here procedures that can be followed to gain entry into this promising avenue of analysis.

  18. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

    PubMed Central

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-01-01

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

  19. Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion

    PubMed Central

    Berens, Eric B.; Sharif, Ghada M.; Schmidt, Marcel O.; Yan, Gai; Shuptrine, Casey W.; Weiner, Louis M.; Glasgow, Eric; Riegel, Anna T.; Wellstein, Anton

    2016-01-01

    Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression. PMID:27375028

  20. Cytoskeletal scaffolding proteins interact with Lynch-Syndrome associated mismatch repair protein MLH1.

    PubMed

    Brieger, Angela; Adryan, Boris; Wolpert, Fabian; Passmann, Sandra; Zeuzem, Stefan; Trojan, Jörg

    2010-09-01

    The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.

  1. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    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

  2. Membrane protein carboxyl methylation does not appear to be involved in the response of erythrocytes to cytoskeletal stress.

    PubMed

    Barber, J R; Clarke, S

    1984-08-30

    We have investigated the effect of changes of human erythrocyte cell shape on the degree of covalent modification by carboxyl methylation of membrane cytoskeletal proteins. The results indicate that the cell probably does not utilize carboxyl methylation to respond to cytoskeletal perturbations caused by such agents as A23187, 2,4-dinitrophenol, and chlorpromazine, all of which are known to cause large changes in cell shape. Protein carboxyl methylation also remained unchanged in the presence of cytochalasin B, which prevents such changes in cell shape. These results are not consistent with a cytoskeletal regulatory role for protein methylation reactions in the intact erythrocyte.

  3. Espin cytoskeletal proteins in the sensory cells of rodent taste buds.

    PubMed

    Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R

    2005-09-01

    Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 +/- 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 +/- 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP(3)R(3))(,) alpha-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP(3)R(3), PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP(3)R(3), alpha-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a

  4. {alpha}-Actinin-2, a cytoskeletal protein, binds to angiogenin

    SciTech Connect

    Hu Huajun; Gao Xiangwei; Sun Yishan; Zhou Jiliang; Yang Min; Xu Zhengping . E-mail: zpxu@zju.edu.cn

    2005-04-08

    Angiogenin is an angiogenic factor which is involved in tumorigenesis. However, no particular intracellular protein is known to interact directly with angiogenin. In the present study, we reported the identification of {alpha}-actinin-2, an actin-crosslinking protein, as a potential angiogenin-interacting partner by yeast two-hybrid screening. This interaction was confirmed by different approaches. First, angiogenin was pulled down together with His-tagged {alpha}-actinin-2 by Ni{sup 2+}-agarose resins. Second, {alpha}-actinin-2 was coimmunoprecipitated with angiogenin by anti-angiogenin monoclonal antibody. Third, the in vivo interaction of these two proteins was revealed by fluorescence resonance energy transfer analysis. Since members of {alpha}-actinin family play pivotal roles in cell proliferation, migration, and invasion, the interaction between {alpha}-actinin-2 and angiogenin may underline one possible mechanism of angiogenin in angiogenesis. Our finding presents the first evidence of an interaction of a cytosolic protein with angiogenin, which might be a novel interference target for anti-angiogenesis and anti-tumor therapy.

  5. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

    PubMed

    Li, Aiqing; Benoit, Joshua B; Lopez-Martinez, Giancarlo; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L

    2009-05-01

    Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

  6. Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

    PubMed

    Berens, E B; Sharif, G M; Schmidt, M O; Yan, G; Shuptrine, C W; Weiner, L M; Glasgow, E; Riegel, A T; Wellstein, A

    2017-02-02

    Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

  7. Attenuation of LDHA expression in cancer cells leads to redox-dependent alterations in cytoskeletal structure and cell migration.

    PubMed

    Arseneault, Robert; Chien, Andrew; Newington, Jordan T; Rappon, Tim; Harris, Richard; Cumming, Robert C

    2013-09-28

    Aerobic glycolysis, the preferential use of glycolysis even in the presence of oxygen to meet cellular metabolic demands, is a near universal feature of cancer. This unique type of metabolism is thought to protect cancer cells from damaging reactive oxygen species (ROS) produced in the mitochondria. Using the cancer cell line MDA-MB-435 it is shown that shRNA mediated knockdown of lactate dehydrogenase A (LDHA), a key mediator of aerobic glycolysis, results in elevated mitochondrial ROS production and a concomitant decrease in cell proliferation and motility. Redox-sensitive proteins affected by oxidative stress associated with LDHA knockdown were identified by Redox 2D-PAGE and mass spectrometry. In particular, tropomyosin (Tm) isoforms Tm4, Tm5NM1 and Tm5NM5, proteins involved in cell migration and cytoskeletal dynamics, exhibited changes in disulfide bonding and co-localized with peri-nuclear actin aggregates in LDHA knockdown cells. In contrast, treatment with the thiol-based antioxidant N-acetylcysteine promoted the relocalization of Tms to cortical actin microfilaments and partially rescued the migration defects associated with attenuated LDHA expression. These results suggest that aerobic glycolysis and reduced mitochondrial ROS production create an environment conducive to cytoskeletal remodeling; key events linked to the high cell motility associated with cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

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

  9. Differential impact of REM sleep deprivation on cytoskeletal proteins of brain regions involved in sleep regulation.

    PubMed

    Rodríguez-Vázquez, Jennifer; Camacho-Arroyo, Ignacio; Velázquez-Moctezuma, Javier

    2012-01-01

    Rapid eye movement (REM) sleep is involved in memory consolidation, which implies synaptic plasticity. This process requires protein synthesis and the reorganization of the neural cytoskeleton. REM sleep deprivation (REMSD) has an impact on some neuronal proteins involved in synaptic plasticity, such as glutamate receptors and postsynaptic density protein 95, but its effects on cytoskeletal proteins is unknown. In this study, the effects of REMSD on the content of the cytoskeletal proteins MAP2 and TAU were analyzed. Adult female rats were submitted to selective REMSD by using the multiple platform technique. After 24, 48 or 72 h of REMSD, rats were decapitated and the following brain areas were dissected: pons, preoptic area, hippocampus and frontal cortex. Protein extraction and Western blot were performed. Results showed an increase in TAU content in the pons, preoptic area and hippocampus after 24 h of REMSD, while in the frontal cortex a significant increase in TAU content was observed after 72 h of REMSD. A TAU content decrease was observed in the hippocampus after 48 h of REMSD. Interestingly, a marked increase in TAU content was observed after 72 h of REMSD. MAP2 content only increased in the preoptic area at 24 h, and in the frontal cortex after 24 and 72 h of REMSD, without significant changes in the pons and hippocampus. These results support the idea that REM sleep plays an important role in the organization of neural cytoskeleton, and that this effect is tissue-specific.

  10. Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces

    PubMed Central

    Bagchi, Sonchita; Tomenius, Henrik; Belova, Lyubov M; Ausmees, Nora

    2008-01-01

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks. PMID:18976278

  11. Modulation of cytoskeletal dynamics by mammalian nucleoside diphosphate kinase (NDPK) proteins.

    PubMed

    Snider, Natasha T; Altshuler, Peter J; Omary, M Bishr

    2015-02-01

    Nucleoside diphosphate kinase (NDPK) proteins comprise a family of ten human isoforms that participate in the regulation of multiple cellular processes via enzymatic and nonenzymatic functions. The major enzymatic function of NDPKs is the generation of nucleoside triphosphates, such as guanosine triphosphate (GTP). Mechanisms behind the nonenzymatic NDPK functions are not clear but likely involve context-dependent signaling roles of NDPK within multi-protein complexes. This is most evident for NDPK-A, which is encoded by the human NME1 gene, the first tumor metastasis suppressor gene to be identified. Understanding which protein interactions are most relevant for the biological and metastasis-related functions of NDPK will be important in the potential utilization of NDPK as a disease target. Accumulating evidence suggests that NDPK interacts with and affects various components and regulators of the cytoskeleton, including actin-binding proteins, intermediate filaments, and cytoskeletal attachment structures (adherens junctions, desmosomes, and focal adhesions). We review the existing literature on this topic and highlight outstanding questions and potential future directions that should clarify the impact of NDPK on the different cytoskeletal systems.

  12. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system.

    PubMed

    Segura-Uribe, Julia J; Pinto-Almazán, Rodolfo; Coyoy-Salgado, Angélica; Fuentes-Venado, Claudia E; Guerra-Araiza, Christian

    2017-08-01

    Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1), MAP2, neurofilament 38 (NF38) by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects.

  13. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.

  14. Cytoskeletal proteins in thymic epithelial cells of the Australian lungfish Neoceratodus forsteri

    PubMed Central

    Mohammad, Mohammad G; Raftos, David A; Joss, Jean

    2009-01-01

    The vertebrate thymus consists of distinctive subpopulations of epithelial cells that contain a diverse repertoire of cytoskeletal proteins. In this study of the thymus in the Australian lungfish, Neoceratodus forsteri, immunohistochemistry was used to distinguish the cytoskeletal proteins present in each class of thymic epithelial cell. A panel of antibodies (Abs), each specific for a different cytoskeletal polypeptide (keratins, vimentin, desmin, actin and tubulins), was used on paraffin and ultrathin resin sections of thymus. Ab AE I (reactive against human type I cytokeratins (CK) 14, 16 and 19) selectively stained the cytoplasm of capsular, trabecular and the outermost epithelial cells of Hassall's corpuscles. Anti-CK 10 Abs strongly labelled the capsular epithelial cells and less than 20% of cortical and medullary epithelial cells. The anti-50-kDa desmin Ab did not react with any thymic cells, whereas the anti-53-kDa desmin Ab labelled some capsular, cortical and medullary thymic epithelial cells. The anti-vimentin Ab stained most of the capsular and ∼60% of the cortical epithelium. Thymic nurse cells and Hassall's corpuscles were found to be devoid of actin, which was strongly detected in medullary and perivascular epithelium. Both α and β tubulins were detected in all thymic cells. This study extends the concept of thymic epithelial heterogeneity. The complexity of thymic epithelium in N. forsteri may indicate a relationship between thymic epithelial subpopulations and the thymic microenvironment. These data identify anti-keratin Abs as a valuable tool for studying differentiation and ontogeny of the thymic epithelium in N. forsteri. PMID:19166477

  15. Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush

    SciTech Connect

    McQuarrie, I.G.; Jacob, J.M. )

    1991-03-01

    To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting {sup 35}S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative 'cytomatrix' proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with {sup 3}H-proline 24 hours before nerve retrieval.

  16. A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

    SciTech Connect

    Pradhan, D.; Williamson, P.; Schlegel, R.A. )

    1989-08-22

    A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho({sup 14}C)ethanolamine(({sup 14}C)AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of ({sup 14}C)AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. ({sup 14}C)AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.

  17. Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G

    PubMed Central

    2005-01-01

    The cell-surface localization of GPCRs (G-protein-coupled receptors) has emerged as one of critical factors of the GPCR-mediated signal transduction. It has been reported that the C-termini of GPCRs contain the sequences for sorting the receptors to cell surface. In the present study, we have searched for proteins that interact with the C-terminus of PTH (parathyroid hormone)/PTH-related protein receptor (PTHR) by using the yeast two-hybrid system, and identified a cytoskeletal protein 4.1G (generaltype 4.1 protein) as an interactant with the C-terminus. Immunohistochemical study revealed that both PTHR and 4.1G were co-localized on plasma membranes, when they were transiently expressed in COS-7 cells. When 4.1G or the C-terminal domain of 4.1G (4.1G-CTD), a dominant-negative form of 4.1G, was co-expressed with PTHR in COS-7 cells, 4.1G, but not 4.1G-CTD, facilitated the cell-surface localization of PTHR, determined by cell-surface biotinylation assay. PTH-(1–34) caused phosphorylation of ERK (extracellular-signal-regulated kinase) 1/2 in PTHR-expressed cells mainly mediated through EGF (epidermal growth factor) receptor. The phosphorylation was enhanced by the expression of 4.1G, but not 4.1G-CTD. PTH-(1–34) elevated [Ca2+]i (intracellular Ca2+ concentration) independent of EGF receptor activation, and the elevation was enhanced by the expression of 4.1G, but not 4.1G-CTD. These data indicate that 4.1G facilitates the cell-surface localization of PTHR through its interaction with the C-terminus of the receptor, resulting in the potentiation of PTHR-mediated signal transduction. PMID:16029167

  18. Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

    PubMed

    Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

    2016-10-07

    The directional movement towards extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

  19. Effect of long-term monotherapy with the aldosterone receptor blocker eplerenone on cytoskeletal proteins and matrix metalloproteinases in dogs with heart failure.

    PubMed

    Rastogi, Sharad; Mishra, Sudhish; Zacà, Valerio; Alesh, Issa; Gupta, Ramesh C; Goldstein, Sidney; Sabbah, Hani N

    2007-12-01

    Long-term monotherapy with the aldosterone receptor blocker eplerenone in dogs with HF was previously shown to improve LV systolic and diastolic function. This study examined the effects of long-term monotherapy with the aldosterone receptor blocker eplerenone on mRNA and protein expression of the cytoskeletal proteins titin, tubulin, fibronectin and vimentin, the matrix metalloproteinases (MMPs)-1, -2 and -9, and the tissue inhibitors of MMPs (TIMPs)-1 and -2 in left ventricular (LV) myocardium of dogs with heart failure (HF). HF was produced in 12 dogs by intracoronary microembolizations. Dogs were randomized to 3 months oral therapy with eplerenone (10 mg/kg twice daily, n = 6) or to no therapy at all (HF-control, n = 6). LV tissue from six normal dogs was used for comparison. mRNA expression was measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and protein expression using Western blots. Compared to NL dogs, control dogs showed upregulation of mRNA and protein expression for tubulin, fibronectin, MMP-1, -2 and -9, and down-regulation of mRNA and protein expression for total titin. Normalization of mRNA and protein expression for all these genes was seen after treatment with eplerenone. N2BA/N2B-titin mRNA expression ratio increased significantly in dogs with HF treated with eplerenone. No differences in expression for vimentin, TIMP-1 and -2 were observed among groups. In dogs with HF, long-term eplerenone therapy normalizes mRNA and protein expression of key cytoskeletal proteins and MMPs. Reversal of these molecular maladaptations may partly explain the improvement in LV diastolic function seen after long-term therapy with eplerenone.

  20. Methylphenidate regulates activity regulated cytoskeletal associated but not brain-derived neurotrophic factor gene expression in the developing rat striatum.

    PubMed

    Chase, T; Carrey, N; Soo, E; Wilkinson, M

    2007-02-09

    Methylphenidate (MPH) is a psychostimulant drug used to treat attention deficit hyperactivity disorder in children. To explore the central effects of chronic MPH, we investigated the expression of an effector immediate early gene, activity regulated cytoskeletal associated (arc), and the neurotrophin, brain-derived neurotrophic factor (bdnf) in the brain of immature and adult rats following repeated MPH. Prepubertal (postnatal day (PD) 25-38) and adult (PD 53-66) male rats were injected once daily for: a) 14 days with saline or MPH (2 or 10 mg/kg; s.c.) or b) 13 days with saline followed by a single dose of MPH (2 or 10 mg/kg; s.c.). To determine possible long-term effects of MPH, prepubertal rats were allowed a drug-free period of 4 weeks following the 14 days of treatment, and then were given a challenge dose of MPH. We demonstrated, for the first time, that an acute injection of MPH increased levels of activity-regulated cytoskeletal protein (ARC) and arc mRNA in the prepubertal rat striatum and cingulate/frontal cortex. This response was significantly attenuated by chronic MPH. The desensitization in arc expression observed in prepubertal rats persisted in the adult striatum following a later MPH challenge. In contrast to these data we observed little effect of MPH on bdnf expression. We also developed an effective, non-stressful technique to treat freely moving immature rats with oral MPH. Consistent with the results described above, we observed that oral MPH (7.5 and 10 mg/kg) also increased arc expression in the prepubertal rat striatum. However, unlike the effects of injected MPH, repeated oral MPH (7.5 mg/kg) did not alter the normal arc response. This result raises the important possibility that oral doses of MPH that reproduce clinically relevant blood levels of MPH may not down-regulate gene expression, at least in the short term (14 days). We confirmed, using mass spectrometry, that the oral doses of MPH used in our experiments yielded blood levels

  1. EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-09-01

    DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.

  2. HTLV-I Tax and Cytokeratin: Tax-Expressing Cells Show Morphological Changes in Keratin-Containing Cytoskeletal Networks.

    PubMed

    Trihn, D.; Jeang, K.-T.; Semmes, O.J.

    1997-01-01

    Human T cell leukemia virus type I (HTLV-I) has been linked to the development of an aggressive lymphoproliferative disorder (adult T cell leukemia), a chronic neurodegenerative presentation (HTLV-I-associated myelopathy/tropical spastic paraparesis) and numerous less well-defined inflammatory conditions. The viral regulatory protein Tax has been implicated in cellular transformation events leading to the onset of adult T cell leukemia. Details on the stepwise processes through which Tax induces morphological changes in cells are poorly understood. We show here that Tax can bind to a class of intermediate filaments, the cytokeratins (Ker). Tax interacts with the 1B helical coil of keratin 8, a domain critical for higher-order intermediate filament matrix formation. Expression of Tax in epithelial cells visibly altered the structural pattern of the Ker network. In a T lymphocyte cell line, induction of Tax expression resulted in increased cellular adherence/invasion of Matrigel filters. We propose that one aspect of Tax function is the induction of morphological changes in cellular cytoskeletal structures. This finding for Tax-expressing cells might be one factor contributing directly to the pathogenesis of HTLV-I disease(s). Copyright 1997 S. Karger AG, Basel

  3. Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence.

    PubMed

    Pich, Oscar Q; Burgos, Raul; Ferrer-Navarro, Mario; Querol, Enrique; Piñol, Jaume

    2008-10-01

    The terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, mg218(-) and mg317(-) mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the mg218(-) mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of mg317(-) mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.

  4. Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt.

    PubMed

    Tom, Moshe; Manfrin, Chiara; Chung, Sook J; Sagi, Amir; Gerdol, Marco; De Moro, Gianluca; Pallavicini, Alberto; Giulianini, Piero G

    2014-12-01

    The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure.

  5. Dematin, a human erythrocyte cytoskeletal protein, is a substrate for a recombinant FIKK kinase from Plasmodium falciparum.

    PubMed

    Brandt, Gabriel S; Bailey, Scott

    2013-09-01

    P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction.

  6. Utilizing Ultrasound to Transiently Increase Blood-Brain Barrier Permeability, Modulate of the Tight Junction Proteins, and Alter Cytoskeletal Structure.

    PubMed

    Bae, Mi Jung; Lee, Young Mi; Kim, Yeoun Hee; Han, Hyung Soo; Lee, Hak Jong

    2015-01-01

    The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

  7. Coronin1 Proteins Dictate Rac1 Intracellular Dynamics and Cytoskeletal Output

    PubMed Central

    Ojeda, Virginia; Castro-Castro, Antonio

    2014-01-01

    Rac1 regulates lamellipodium formation, myosin II-dependent contractility, and focal adhesions during cell migration. While the spatiotemporal assembly of those processes is well characterized, the signaling mechanisms involved remain obscure. We report here that the cytoskeleton-related Coronin1A and -1B proteins control a myosin II inactivation-dependent step that dictates the intracellular dynamics and cytoskeletal output of active Rac1. This step is signaling-branch specific, since it affects the functional competence of active Rac1 only when forming complexes with downstream ArhGEF7 and Pak proteins in actomyosin-rich structures. The pathway is used by default unless Rac1 is actively rerouted away from the structures by upstream activators and signals from other Rho GTPases. These results indicate that Coronin1 proteins are at the center of a regulatory hub that coordinates Rac1 activation, effector exchange, and the F-actin organization state during cell signaling. Targeting this route could be useful to hamper migration of cancer cells harboring oncogenic RAC1 mutations. PMID:24980436

  8. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription

    PubMed Central

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A.; Shore, Paul

    2012-01-01

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  9. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines?

  10. [Contractile properties of fibers and cytoskeletal proteins of gerbil's hindlimb muscles after space flight].

    PubMed

    Lipets, E N; Ponomareva, E V; Ogneva, I V; Vikhliantsev, I M; Karaduleva, E V; Kratashkina, N L; Kuznetsov, S L; Podlubnaia, Z A; Shenkman, B S

    2009-01-01

    The work had the goal to compare the microgravity effects on gerbil's muscles-antagonists, m. soleus and m. tibialis anterior. The animals were exposed in 12-d space microgravity aboard Earth's artificial satellite "Foton-M3". Findings of the analysis of single skinned fibers contractility are 19.7% diminution of the diameter and 21.8% loss of the total contractive force of m. soleus fibers post flight. However, there was no significant difference in calcium sensitivity which agrees with the absence of changes in the relative content of several major cytoskeletal proteins (titin and nebulin ratios to heavy chains of myosin were identical in the flight and control groups) and a slight shifting of the myosin phenotype toward the "fast type" (9%, p < 0.05). These parameters were mostly unaffected by the space flight in m. tibialis anterior. To sum up, the decline of contractility and diminution of gerbil's myofibers after the space flight were less significant as compared with rats and did not impact the sytoskeletal protein ratios.

  11. Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

    PubMed

    Gascard, P; Nunomura, W; Lee, G; Walensky, L D; Krauss, S W; Takakuwa, Y; Chasis, J A; Mohandas, N; Conboy, J G

    1999-06-01

    The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

  12. Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture.

    PubMed

    Ciesielski-Treska, J; Ulrich, G; Mensch, C; Aunis, D

    1984-01-01

    One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

  13. Methamphetamine enhances the cleavage of the cytoskeletal protein tau in the rat brain.

    PubMed

    Wallace, T L; Vorhees, C V; Zemlan, F P; Gudelsky, G A

    2003-01-01

    The view that methamphetamine is neurotoxic to dopaminergic and serotonergic axon terminals has been based largely on biochemical and histological studies. In the present study, methamphetamine-induced structural damage to axons was quantified using a sensitive sandwich enzyme-linked immunosorbent assay developed for the detection of the cleaved form of the cytoskeletal protein tau. The administration of a monoamine-depleting regimen of methamphetamine (4 x 10 mg/kg, i.p. every 2 hours for a total of four injections) produced a time-dependent increase in the concentration of cleaved tau in the striatum. Maximal concentrations of cleaved tau were detected 3 days following methamphetamine administration. Cleaved tau concentrations also were significantly elevated in the dorsal hippocampus and, to a lesser extent, in the prefrontal cortex of methamphetamine-treated rats. Maintenance of rats in a cold (4 degrees C) environment not only prevented the methamphetamine-induced depletion of striatal dopamine and serotonin but also prevented the methamphetamine-induced increase in striatal cleaved tau concentrations. The novel findings from this study are supportive of the view that methamphetamine produces acute structural damage to neurons that may lead to the long-term neurotoxic effects of repeated, high-dose administration of the drug and that cleaved tau reliably quantifies the time-dependent neurotoxic effects of methamphetamine.

  14. Cytoskeletal proteins in gastric H/sup +/ secretion: cAMP dependent phosphorylation, immunolocalization, and protein blotting

    SciTech Connect

    Cuppoletti, J.; Sachs, G.; Malinowska, D.H.

    1986-05-01

    The rabbit gastric parietal cell is an excellent model for the study of regulation of secretion and the role of cytoskeleton in secretion. Changes in morphology (appearance of expanded secretory canaliculi lined with microvilli) accompany H/sup +/ secretion stimulated by histamine (cAMP mediated). Parietal cells contain immunoreactive tubulin and are highly enriched in F-actin at secretory canaliculi, detected with fluorescently labelled phallacidin. They have previously shown increased protein phosphorylation in histamine-stimulated purified parietal cells concommitant with increases in H/sup +/ secretion. They report here possible functions of the phosphoproteins. Four of these proteins of apparent size on SDS PAGE of 24, 30, 48 and 130 Kd were membrane associated. /sup 125/I-actin binding to three proteins (24, 30 and 48 Kd) was shown using overlays. A 130 Kd protein reacted with anti-vinculin monoclonal antibody on immunoblots, and was immunolocalized at secretory canaliculi. As a working hypothesis, parietal cells possess membrane-associated proteins which change their state of phosphorylation upon stimulation of H/sup +/. These proteins may be cytoskeletal elements involved in regulation of H/sup +/ secretion. The 130 Kd vinculin-like protein may serve a microfilament-membrane linking role.

  15. The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

    PubMed Central

    Walensky, Loren D.; Gascard, Philippe; Field, Michael E.; Blackshaw, Seth; Conboy, John G.; Mohandas, Narla; Snyder, Solomon H.

    1998-01-01

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds. PMID:9531554

  16. The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Gascard, P; Fields, M E; Blackshaw, S; Conboy, J G; Mohandas, N; Snyder, S H

    1998-04-06

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

  17. RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability.

    PubMed

    Grau, Marijke; Pauly, Sebastian; Ali, Jamal; Walpurgis, Katja; Thevis, Mario; Bloch, Wilhelm; Suhr, Frank

    2013-01-01

    Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold. Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects. This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological

  18. Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

    SciTech Connect

    Wang, Daojing; Jang, Deok-Jin

    2009-08-21

    Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We demonstrated that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between day 3 and day 6. This was confirmed by senescence-associated beta-galactosidase (SA-{beta}-gal) staining, protein expression profiles of key cell cycle regulators (retinoblastoma (Rb) protein, p53, p21{sup waf1/Cip1}, and p16{sup INK4A}), and senescence-associated secretory phenotypes (SASPs) (IL-8, IL-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9, and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser1943, coinciding with its redistribution. Importantly, through treatment with cell permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)), and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser1943, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2{alpha} and CK2{alpha}{prime} induced hMSC senescence. However, only knockdown of CK2{alpha} resulted in morphological phenotypes resembling those of radiation-induced senescence. These results suggest that CK2{alpha} and CK2{alpha}{prime} play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.

  19. Effect of Rapid Chilling on Beef Quality and Cytoskeletal Protein Degradation in M. longissimus of Chinese Yellow Crossbred Bulls

    PubMed Central

    Mao, Yanwei; Zhang, Yimin; Liang, Rongrong; Ren, Lulu; Zhu, He; Li, Ke; Zhu, Lixian; Luo, Xin

    2012-01-01

    The objective of this study was to investigate the effect of rapid chilling (RC) on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC) were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES) treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM). The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness. PMID:25049681

  20. A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.

    PubMed

    Helyer, R; Cacciabue-Rivolta, D; Davies, D; Rivolta, M N; Kros, C J; Holley, M C

    2007-02-01

    We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.

  1. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  2. Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

    PubMed Central

    Stout, A. L.; Axelrod, D.

    1994-01-01

    removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:7811947

  3. E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures.

    PubMed Central

    Montine, T. J.; Amarnath, V.; Martin, M. E.; Strittmatter, W. J.; Graham, D. G.

    1996-01-01

    Lipid peroxidation increases with age in brain and is elevated further in Alzheimer's disease. E-4-hydroxy-2-nonenal and malondialdehyde are products of lipid peroxidation that can adduct and cross-link protein. Neurofibrillary tangles, a feature of Alzheimer's disease composed mostly of tau protein, contain cross-linked and ubiquitin-conjugated protein. In P19 neuroglial cultures, E-4-hydroxy-2-nonenal was a potent cytotoxin that cross-linked cytoskeletal proteins, including tau into high molecular weight species that were conjugated with ubiquitin. Malondialdehyde formed monoadducts with cell protein but did not cross-link and was not cytotoxic. A non-crosslinking analogue of E-4-hydroxy-2-nonenal was not cytotoxic. E-4-Hydroxy-2-nonenal may contribute to neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease. Images Figure 2 Figure 3 PMID:8546230

  4. Dystrophin- and MLP-deficient mouse hearts: marked differences in morphology and function, but similar accumulation of cytoskeletal proteins.

    PubMed

    Wilding, James R; Schneider, Jürgen E; Sang, A Elizabeth; Davies, Kay E; Neubauer, Stefan; Clarke, Kieran

    2005-01-01

    In humans, cytoskeletal dystrophin and muscle LIM protein (MLP) gene mutations can cause dilated cardiomyopathy, yet these mutations may have different effects in mice, owing to increased accumulation of other, compensatory cytoskeletal proteins. Consequently, we characterized left-ventricular (LV) morphology and function in vivo using high-resolution cine-magnetic resonance imaging (MRI) in 2- to 3-month old dystrophin-deficient (mdx) and MLP-null mice, and their respective controls. LV passive stiffness was assessed in isolated, perfused hearts, and cytoskeletal protein levels were determined using Western blot analyses. In mdx mouse hearts, LV-to-body weight ratio, cavity volume, ejection fraction, stroke volume, and cardiac output were normal. However, MLP-null mouse hearts had 1.2-fold higher LV-to-body weight ratios (P<0.01), 1.5-fold higher end-diastolic volumes (P<0.01), and decreased ejection fraction compared with controls (25% vs. 66%, respectively, P<0.01), indicating dilated cardiomyopathy and heart failure. In both models, isolated, perfused heart end-diastolic pressure-volume relationships and passive left-ventricular stiffness were normal. Hearts from both models accumulated desmin and beta-tubulin, mdx mouse hearts accumulated utrophin and MLP, and MLP-null mouse hearts accumulated dystrophin and syncoilin. Although the increase in MLP and utrophin in the mdx mouse heart was able to compensate for the loss of dystrophin, accumulation of desmin, syncoilin and dystrophin were unable to compensate for the loss of MLP, resulting in heart failure.

  5. Early Cytoskeletal Protein Modifications Precede Overt Structural Degeneration in the DBA/2J Mouse Model of Glaucoma

    PubMed Central

    Wilson, Gina N.; Smith, Matthew A.; Inman, Denise M.; Dengler-Crish, Christine M.; Crish, Samuel D.

    2016-01-01

    Axonal transport deficits precede structural loss in glaucoma and other neurodegenerations. Impairments in structural support, including modified cytoskeletal proteins, and microtubule-destabilizing elements, could be initiating factors in glaucoma pathogenesis. We investigated the time course of changes in protein levels and post-translational modifications in the DBA/2J mouse model of glaucoma. Using anterograde tract tracing of the retinal projection, we assessed major cytoskeletal and transported elements as a function of transport integrity in different stages of pathological progression. Using capillary-based electrophoresis, single- and multiplex immunosorbent assays, and immunofluorescence, we quantified hyperphosphorylated neurofilament-heavy chain, phosphorylated tau (ptau), calpain-mediated spectrin breakdown product (145/150 kDa), β–tubulin, and amyloid-β42 proteins based on age and transport outcome to the superior colliculus (SC; the main retinal target in mice). Phosphorylated neurofilament-heavy chain (pNF-H) was elevated within the optic nerve (ON) and SC of 8–10 month-old DBA/2J mice, but was not evident in the retina until 12–15 months, suggesting that cytoskeletal modifications first appear in the distal retinal projection. As expected, higher pNF-H levels in the SC and retina were correlated with axonal transport deficits. Elevations in hyperphosphorylated tau (ptau) occurred in ON and SC between 3 and 8 month of age while retinal ptau accumulations occurred at 12–15 months in DBA/2J mice. In vitro co-immunoprecipitation experiments suggested increased affinity of ptau for the retrograde motor complex protein dynactin. We observed a transport-related decrease of β-tubulin in ON of 10–12 month-old DBA/2J mice, suggesting destabilized microtubule array. Elevations in calpain-mediated spectrin breakdown product were seen in ON and SC at the earliest age examined, well before axonal transport loss is evident. Finally, transport

  6. Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

    SciTech Connect

    Kumeta, Masahiro; Hirai, Yuya; Yoshimura, Shige H.; Horigome, Tsuneyoshi; Takeyasu, Kunio

    2013-12-10

    To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

  7. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

    Mofrad, Mohammad R. K.; Kamm, Roger D.

    2011-08-01

    1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

  8. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

    Mofrad, Mohammad R. K.; Kamm, Roger D.

    2006-10-01

    1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

  9. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  10. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

    PubMed Central

    1987-01-01

    The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000- 74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane. PMID:3308904

  11. Molecular dissection of the mechanism by which EWS/FLI expression compromises actin cytoskeletal integrity and cell adhesion in Ewing sarcoma

    PubMed Central

    Chaturvedi, Aashi; Hoffman, Laura M.; Jensen, Christopher C.; Lin, Yi-Chun; Grossmann, Allie H.; Randall, R. Lor; Lessnick, Stephen L.; Welm, Alana L.; Beckerle, Mary C.

    2014-01-01

    Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised. PMID:25057021

  12. ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

    PubMed

    Nakagawa, N; Hoshijima, M; Oyasu, M; Saito, N; Tanizawa, K; Kuroda, S

    2000-06-07

    The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

  13. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  14. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin

    PubMed Central

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-01-01

    ABSTRACT Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation. PMID:25315833

  15. Changes of cytoskeletal proteins in nerve tissues and serum of rats treated with 2,5-hexanedione.

    PubMed

    Wang, Qing-Shan; Hou, Li-Yan; Zhang, Cui-Li; Song, Fu-Yong; Xie, Ke-Qin

    2008-02-28

    To investigate the mechanisms and biomarker of the neuropathy induced by 2,5-hexanedione (HD), male Wistar rats were administrated HD at dosage of 200 or 400mg/kg for 8 weeks (five-times per week). All rats were sacrificed after 8 weeks of treatment and the cerebrum cortex (CC), spinal cord (SC) and sciatic nerves (SN) were dissected, homogenized and used for the determination of cytoskeletal proteins by western blotting. The levels of neurofilaments (NFs) subunits (NF-L, NF-M and NF-H) in nerve tissues of 200 and 400mg/kg HD rats significantly decreased in both the supernatant and pellet fractions. Furthermore, significant negative correlations between NFs levels and gait abnormality were observed. As for microtubule (MT) and microfilament (MF) proteins, the levels of alpha-tubulin, beta-tubulin and beta-actin in the supernatant and pellet fraction of SN significantly decreased in 200 and 400mg/kg HD rats and correlated negatively with gait abnormality. However, the contents of MT and MF proteins in CC and SC were inconsistently affected and had no significant correlation with gait abnormality. The levels of NF-L and NF-H in serum significantly increased, while NF-M, alpha-tubulin, beta-tubulin and beta-actin contents remain unchanged. A significant positive correlation (R=0.9427, P<0.01) was observed between gait abnormality and NF-H level in serum as the intoxication went on. These findings suggested that HD intoxication resulted in a progressive decline of cytoskeletal protein contents, which might be relevant to the mechanisms of HD-induced neuropathy. NF-H was the most sensitive index, which may serve as a good indicator for neurotoxicity of n-hexane or HD.

  16. Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations.

    PubMed

    Clark, Paul R; Manes, Thomas D; Pober, Jordan S; Kluger, Martin S

    2007-04-01

    Tumor necrosis factor (TNF)-induced ICAM-1 in endothelial cells (EC) promotes leukocyte adhesion. Here we report that ICAM-1 also effects EC barrier function. Control- or E-selectin-transduced human dermal microvascular EC (HDMEC) form a barrier to flux of proteins and to passage of current (measured as transendothelial electrical resistance or TEER). HDMEC transduced with ICAM-1 at levels comparable to that induced by TNF show reduced TEER, but do so without overtly changing their cell junctions, cell shape, or cytoskeleton organization. Higher levels of ICAM-1 further reduce TEER, increase F/G-actin ratios, rearrange the actin cytoskeleton to cause cell elongation, and alter junctional zona occludens 1 and vascular endothelial-cadherin staining. Transducing with ICAM-1 lacking an intracellular region also reduces TEER. TNF-induced changes in TEER and shape follow a similar time course as ICAM-1 induction; however, the fall in TEER occurs at lower TNF concentrations. Inhibiting NF-kappaB activation blocks ICAM-1 induction; TEER reduction, and shape change. Specific small-interfering RNA knockdown of ICAM-1 partially inhibits TNF-induced shape change. We conclude that moderately elevated ICAM-1 expression reduces EC barrier function and that expressing higher levels of ICAM-1 affects cell junctions and the cytoskeleton. Induction of ICAM-1 may contribute to but does not fully account for TNF-induced vascular leak and EC shape change.

  17. Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

    PubMed

    Gabai, V L; Kabakov, A E; Mosin, A F

    1992-01-01

    ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.

  18. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-08-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism.

  19. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed Central

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-01-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism. Images PMID:8336735

  20. Kingdom Chromista and its eight phyla: a new synthesis emphasising periplastid protein targeting, cytoskeletal and periplastid evolution, and ancient divergences.

    PubMed

    Cavalier-Smith, Thomas

    2017-09-05

    In 1981 I established kingdom Chromista, distinguished from Plantae because of its more complex chloroplast-associated membrane topology and rigid tubular multipartite ciliary hairs. Plantae originated by converting a cyanobacterium to chloroplasts with Toc/Tic translocons; most evolved cell walls early, thereby losing phagotrophy. Chromists originated by enslaving a phagocytosed red alga, surrounding plastids by two extra membranes, placing them within the endomembrane system, necessitating novel protein import machineries. Early chromists retained phagotrophy, remaining naked and repeatedly reverted to heterotrophy by losing chloroplasts. Therefore, Chromista include secondary phagoheterotrophs (notably ciliates, many dinoflagellates, Opalozoa, Rhizaria, heliozoans) or walled osmotrophs (Pseudofungi, Labyrinthulea), formerly considered protozoa or fungi respectively, plus endoparasites (e.g. Sporozoa) and all chromophyte algae (other dinoflagellates, chromeroids, ochrophytes, haptophytes, cryptophytes). I discuss their origin, evolutionary diversification, and reasons for making chromists one kingdom despite highly divergent cytoskeletons and trophic modes, including improved explanations for periplastid/chloroplast protein targeting, derlin evolution, and ciliary/cytoskeletal diversification. I conjecture that transit-peptide-receptor-mediated 'endocytosis' from periplastid membranes generates periplastid vesicles that fuse with the arguably derlin-translocon-containing periplastid reticulum (putative red algal trans-Golgi network homologue; present in all chromophytes except dinoflagellates). I explain chromist origin from ancestral corticates and neokaryotes, reappraising tertiary symbiogenesis; a chromist cytoskeletal synapomorphy, a bypassing microtubule band dextral to both centrioles, favoured multiple axopodial origins. I revise chromist higher classification by transferring rhizarian subphylum Endomyxa from Cercozoa to Retaria; establishing retarian

  1. Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Chen, N. X.; Ryder, K. D.; Pavalko, F. M.; Turner, C. H.; Burr, D. B.; Qiu, J.; Duncan, R. L.

    2000-01-01

    Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

  2. Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Chen, N. X.; Ryder, K. D.; Pavalko, F. M.; Turner, C. H.; Burr, D. B.; Qiu, J.; Duncan, R. L.

    2000-01-01

    Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

  3. Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

    PubMed

    Vagin, Olga; Tokhtaeva, Elmira; Garay, Patton E; Souda, Puneet; Bassilian, Sara; Whitelegge, Julian P; Lewis, Ramilla; Sachs, George; Wheeler, Larry; Aoki, Roger; Fernandez-Salas, Ester

    2014-08-01

    Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity. © 2014. Published by The Company of Biologists Ltd.

  4. Loss of the Cytoskeletal Protein Pdlim7 Predisposes Mice to Heart Defects and Hemostatic Dysfunction

    PubMed Central

    Krcmery, Jennifer; Gupta, Rajesh; Sadleir, Rudyard W.; Ahrens, Molly J.; Misener, Sol; Kamide, Christine; Fitchev, Philip; Losordo, Douglas W.; Crawford, Susan E.; Simon, Hans-Georg

    2013-01-01

    The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult Pdlim7-/- mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in Pdlim7-/- mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in Pdlim7+/- (11/60) and Pdlim7-/- (19/35) mice. In line with a prothrombotic phenotype, adult Pdlim7-/- mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice. PMID:24278323

  5. The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala.

    PubMed

    Ploski, Jonathan E; Pierre, Vicki J; Smucny, Jason; Park, Kevin; Monsey, Melissa S; Overeem, Kathie A; Schafe, Glenn E

    2008-11-19

    The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene that has been widely implicated in hippocampal-dependent learning and memory and is believed to play an integral role in synapse-specific plasticity. Here, we examined the role of Arc/Arg3.1 in amygdala-dependent Pavlovian fear conditioning. We first examined the regulation of Arc/Arg3.1 mRNA and protein after fear conditioning and LTP-inducing stimulation of thalamic inputs to the lateral amygdala (LA). Quantitative real-time PCR, in situ hybridization, Western blotting and immunohistochemistry revealed a significant upregulation of Arc/Arg3.1 mRNA and protein in the LA relative to controls. In behavioral experiments, intra-LA infusion of an Arc/Arg3.1 antisense oligodeoxynucleotide (ODN) was observed to be anatomically restricted to the LA, taken up by LA cells, and to promote significant knockdown of Arc/Arg3.1 protein. Rats given intra-LA infusions of multiple doses of the Arc/Arg3.1 ODN showed an impairment of LTM (tested approximately 24 later), but no deficit in STM (tested 3 h later) relative to controls infused with scrambled ODN. Finally, to determine whether upregulation of Arc/Arg3.1 occurs downstream of ERK/MAPK activation, we examined Arc/Arg3.1 expression in rats given intra-LA infusion of the MEK inhibitor U0126. Relative to vehicle controls, infusion of U0126 impaired training-induced increases in Arc/Arg3.1 expression. These findings suggest that Arc/Arg3.1 expression in the amygdala is required for fear memory consolidation, and further suggest that Arc/Arg3.1 regulation in the LA is downstream of the ERK/MAPK signaling pathway.

  6. The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin.

    PubMed

    Flick, M J; Konieczny, S F

    2000-05-01

    Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein betaI-spectrin was isolated. Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril.

  7. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns.

    PubMed

    Loose, Martin; Mitchison, Timothy J

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other's assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.

  8. The Repetitive Cytoskeletal Protein H49 of Trypanosoma cruzi Is a Calpain-Like Protein Located at the Flagellum Attachment Zone

    PubMed Central

    Galetović, Alexandra; Souza, Renata T.; Santos, Marcia R. M.; Cordero, Esteban M.; Bastos, Izabela M. D.; Santana, Jaime M.; Ruiz, Jeronimo C.; Lima, Fabio M.; Marini, Marjorie M.; Mortara, Renato A.; da Silveira, José Franco

    2011-01-01

    Background Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. In the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins. Methods and Findings All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. The H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. The H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body. Conclusions H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell

  9. The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone.

    PubMed

    Galetović, Alexandra; Souza, Renata T; Santos, Marcia R M; Cordero, Esteban M; Bastos, Izabela M D; Santana, Jaime M; Ruiz, Jeronimo C; Lima, Fabio M; Marini, Marjorie M; Mortara, Renato A; da Silveira, José Franco

    2011-01-01

    Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. In the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins. All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. The H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. The H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body. H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell body remains attached to the flagellum by

  10. Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease

    PubMed Central

    Otey, Carol A.; Dixon, Richard; Stack, Christianna; Goicoechea, Silvia M.

    2009-01-01

    Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. PMID:19466753

  11. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    PubMed Central

    Bhargava, Amol; Cotton, James A.; Dixon, Brent R.; Gedamu, Lashitew; Yates, Robin M.; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. PMID:26334299

  12. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    PubMed

    Bhargava, Amol; Cotton, James A; Dixon, Brent R; Gedamu, Lashitew; Yates, Robin M; Buret, Andre G

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  13. Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4. 1, ezrin, and talin

    SciTech Connect

    Qing Yang; Tonks, N.K. )

    1991-07-15

    The polymerase chain reaction (PCR), from primers corresponding to conserved sequences within the catalytic domains of the protein-tyrosine phosphatases, was used to amplify protein-tyrosine phosphatase-related cDNAs from a HeLa cell library. After probing the same cDNA library with one of the PCR products, 10 positive clones were identified. The longest of these clones (3984 base pairs) contained 2739 base pairs of open reading frame and, after a stop codon, a 3{prime} nontranslated segment of 1222 base pairs. A 4.3-kilobase transcript was detected by Northern blot analysis of HeLa cell poly(A){sup +} RNA. The open reading frame predicts a protein of 913 amino acids ({approx} 104 kDa), termed PTPH1. The sequence of PTPH1 can be described in terms of three segments. (1) The N-terminal segment displays homology to the domains in the cytoskeletal-associated proteins band 4.1, erzin, and talin that direct their association with proteins at the interface between the plasma membrane and the cytoskeleton in structures such as focal adhesions. (2) There is a central segment bearing putative phosphorylation sites for protein-serine/threonine kinases. (3) A segment that is homologous to the members of the protein-tyrosine phosphatase family is located at the C terminus. The structure is discussed in the light of the potential role of PTPH1 in controlling cytoskeletal integrity and the possibility that overexpression of PTPH1 may reverse transformation induced by oncogenic protein-tyrosine kinases, such as the members of the src family.

  14. Kakapo, a novel cytoskeletal-associated protein is essential for the restricted localization of the neuregulin-like factor, vein, at the muscle-tendon junction site.

    PubMed

    Strumpf, D; Volk, T

    1998-11-30

    In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype.

  15. Kakapo, a Novel Cytoskeletal-associated Protein Is Essential for the Restricted Localization of the Neuregulin-like Factor, Vein, at the Muscle–Tendon Junction Site

    PubMed Central

    Strumpf, Dan; Volk, Talila

    1998-01-01

    In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle–tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype. PMID:9832554

  16. Cytoskeletal protein Flightless (Flii) is elevated in chronic and acute human wounds and wound fluid: neutralizing its activity in chronic but not acute wound fluid improves cellular proliferation.

    PubMed

    Ruzehaji, Nadira; Grose, Randall; Krumbiegel, Doreen; Zola, Heddy; Dasari, Pallave; Wallace, Hilary; Stacey, Michael; Fitridge, Robert; Cowin, Allison J

    2012-01-01

    Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.

  17. Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading.

    PubMed

    Chao, Pen-Hsiu Grace; West, Alan C; Hung, Clark T

    2006-10-01

    While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (-200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli.

  18. Active components of ginger potentiate β-agonist-induced relaxation of airway smooth muscle by modulating cytoskeletal regulatory proteins.

    PubMed

    Townsend, Elizabeth A; Zhang, Yi; Xu, Carrie; Wakita, Ryo; Emala, Charles W

    2014-01-01

    β-Agonists are the first-line therapy to alleviate asthma symptoms by acutely relaxing the airway. Purified components of ginger relax airway smooth muscle (ASM), but the mechanisms are unclear. By elucidating these mechanisms, we can explore the use of phytotherapeutics in combination with traditional asthma therapies. The objectives of this study were to: (1) determine if 6-gingerol, 8-gingerol, or 6-shogaol potentiate β-agonist-induced ASM relaxation; and (2) define the mechanism(s) of action responsible for this potentiation. Human ASM was contracted in organ baths. Tissues were relaxed dose dependently with β-agonist, isoproterenol, in the presence of vehicle, 6-gingerol, 8-gingerol, or 6-shogaol (100 μM). Primary human ASM cells were used for cellular experiments. Purified phosphodiesterase (PDE) 4D or phospholipase C β enzyme was used to assess inhibitory activity of ginger components using fluorescent assays. A G-LISA assay was used to determine the effects of ginger constituents on Ras homolog gene family member A activation. Significant potentiation of isoproterenol-induced relaxation was observed with each of the ginger constituents. 6-Shogaol showed the largest shift in isoproterenol half-maximal effective concentration. 6-Gingerol, 8-gingerol, or 6-shogaol significantly inhibited PDE4D, whereas 8-gingerol and 6-shogaol also inhibited phospholipase C β activity. 6-Shogaol alone inhibited Ras homolog gene family member A activation. In human ASM cells, these constituents decreased phosphorylation of 17-kD protein kinase C-potentiated inhibitory protein of type 1 protein phosphatase and 8-gingerol decreased myosin light chain phosphorylation. Isolated components of ginger potentiate β-agonist-induced relaxation in human ASM. This potentiation involves PDE4D inhibition and cytoskeletal regulatory proteins. Together with β-agonists, 6-gingerol, 8-gingerol, or 6-shogaol may augment existing asthma therapy, resulting in relief of symptoms through

  19. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    PubMed

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  1. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells.

    PubMed Central

    Morita, T; Kurihara, H; Maemura, K; Yoshizumi, M; Yazaki, Y

    1993-01-01

    Hemodynamic shear stress alters the architecture and functions of vascular endothelial cells. We have previously shown that the synthesis of endothelin-1 (ET-1) in endothelial cells is increased by exposure to shear stress. Here we examined whether shear stress-induced alterations in cytoskeletal structures are responsible for increases in ET-1 synthesis in cultured porcine aortic endothelial cells. Exposure of endothelial cells to 5 dyn/cm2 of low shear stress rapidly increased monomeric G-actin contents within 5 min without changing total actin contents. The ratio of G- to total actin, 54 +/- 0.8% in quiescent endothelial cells, increased to 87 +/- 4.2% at 6 h and then decreased. Following the disruption of filamentous (F)-actin into G-actin, ET-1 mRNA levels in endothelial cells also increased within 30 min and reached a peak at 6 h. The F-actin stabilizer, phalloidin, abolished shear stress-induced increases in ET-1 mRNA; however, it failed to inhibit increases in ET-1 mRNA secondary to other stimulants. This indicates that shear stress-induced increases in ET-1 mRNA levels may be mediated by the disruption of actin fibers. Furthermore, increases in ET-1 gene expression can be induced by actin-disrupting agents, cytochalasin B and D. Another cytoskeleton-disrupting agent, colchicine, which inhibits dimerization of tubulin, did not affect the basal level of ET-1 mRNA. However, colchicine completely inhibited shear stress- and cytochalasin B-induced increases in ET-1 mRNA levels. These results suggest that shear stress-induced ET-1 gene expression in endothelial cells is mediated by the disruption of actin cytoskeleton and this induction is dependent on the integrity of microtubules. Images PMID:8408624

  2. Alternative cytoskeletal landscapes: cytoskeletal novelty and evolution in basal excavate protists

    PubMed Central

    Dawson, Scott C.; Paredez, Alexander R.

    2016-01-01

    Microbial eukaryotes encompass the majority of eukaryotic evolutionary and cytoskeletal diversity. The cytoskeletal complexity observed in multicellular organisms appears to be an expansion of components present in genomes of diverse microbial eukaryotes such as the basal lineage of flagellates, the Excavata. Excavate protists have complex and diverse cytoskeletal architectures and life cycles – essentially alternative cytoskeletal “landscapes” – yet still possess conserved microtubule- and actin-associated proteins. Comparative genomic analyses have revealed that a subset of excavates, however, lack many canonical actin-binding proteins central to actin cytoskeleton function in other eukaryotes. Overall, excavates possess numerous uncharacterized and “hypothetical” genes, and may represent an undiscovered reservoir of novel cytoskeletal genes and cytoskeletal mechanisms. The continued development of molecular genetic tools in these complex microbial eukaryotes will undoubtedly contribute to our overall understanding of cytoskeletal diversity and evolution. PMID:23312067

  3. Effects of Ca2+- binding agent EGTA on fiber contractility and content of sarcomeric cytoskeletal proteins of hindlimb suspended rats

    NASA Astrophysics Data System (ADS)

    Litvinova, K. S.; Vikhlyantsev, I. M.; Podlubnaya, Z. A.; Shenkman, B. S.

    2005-08-01

    The study was purposed to analyze the effects of binding agent (10% EGTA) on soleus fiber contractile characteristics and cytoskeletal sarcomeric proteins in hindlimb suspended rats. Cage control and hindlimb- suspended rats were administered with saline or EGTA i.p. injections. Whereas the fiber diameter of unloaded rat soleus was 17% less than in control group and there were no significant differences between EGTA-treated rats and saline-treated animals. Values of fiber tension in saline- treated unloaded rats were less than in the control group by 45%, and in EGTA-treated rats - by 28%. The Ca/tension curve in hindlimb suspended saline-treated rats shifted to the right, which indicated to the less Ca2+ sensitivity of myofibrils of unloaded soleus. At the same time Ca2+ sensitivity in EGTA-treated suspended rats even exceeded the control levels. The hindlimb- suspended EGTA-treated rats tended to decrease their titin content to less degree (17%) than group of suspended rats injected with physiological saline (51%). The relative nebulin content decreased significantly (more than 50%) after hindlimb suspension but was not changed in EGTA-treated unloaded rats.

  4. EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

    PubMed Central

    Clore, A. M.; Dannenhoffer, J. M.; Larkins, B. A.

    1996-01-01

    By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei. PMID:12239373

  5. Biochemical and structural properties of the integrin-associated cytoskeletal protein talin.

    PubMed

    Critchley, David R

    2009-01-01

    Interaction of cells with the extracellular matrix is fundamental to a wide variety of biological processes, such as cell proliferation, cell migration, embryogenesis, and organization of cells into tissues, and defects in cell-matrix interactions are an important element in many diseases. Cell-matrix interactions are frequently mediated by the integrin family of cell adhesion molecules, transmembrane alphabeta-heterodimers that are typically linked to the actin cytoskeleton by one of a number of adaptor proteins including talin, alpha-actinin, filamin, tensin, integrin-linked kinase, melusin, and skelemin. The focus of this review is talin, which appears unique among these proteins in that it also induces a conformational change in integrins that is propagated across the membrane, and increases the affinity of the extracellular domain for ligand. Particular emphasis is given to recent progress on the structure of talin, its interaction with binding partners, and its mode of regulation.

  6. JWA loss promotes cell migration and cytoskeletal rearrangement by affecting HER2 expression and identifies a high-risk subgroup of HER2-positive gastric carcinoma patients

    PubMed Central

    Qian, Jing; Zhu, Weiyou; Wang, Keming; Ma, Lin; Xu, Jin; Xu, Tongpeng; Røe, Oluf Dimitri; Li, Aiping; Zhou, Jianwei; Shu, Yongqian

    2016-01-01

    Background and Aims JWA, a microtubule-associated protein (MAP) involved in apoptosis, has been identified as a suppressor of metastasis, and it affects cell migration in melanoma and its downregulation in tumor is an idependent negative prognostic factor in resectable gastric cancer. HER2 overexpression has been observed in gastric cancer (GC) cells and implicated in the metastatic phenotype. However, the biological role of JWA in migration and its clinical value in HER2-positive GC remain elusive. Results JWA suppresses EGF-induced cell migration and actin cytoskeletal rearrangement by abrogating HER2 expression and downstream PI3K/AKT signaling in HER2-overexpressing GC cell lines. The modulation of HER2 by JWA is dependent on ERK activation and consequent PEA3 upregulation and activation. Reduced JWA expression is associated with high HER2 expression and with poor survival in patients with AGC, whereas HER2 expression alone is not associated with survival. However, concomitant low JWA and high HER2 expression is associated with unfavorable outcomes. Additionally, when patients were stratified by JWA expression, those with higher HER2 expression in the low JWA expression subgroup exhibited worse survival. Methods The impact of JWA on the EGF-induced migration of HER2-positive GC cells was studied using transwell assays and G-LISA assays. Western blotting, real-time PCR, electrophoretic mobility shift assays and luciferase assays were utilized to investigate the mechanisms by which JWA affects HER2. The association of JWA with HER2 and its clinical value were further analyzed by IHC in 128 pairs of advanced gastric cancer (AGC) and adjacent normal tissue samples. Conclusions This study characterizes a novel mechanism for regulating cell motility in HER2-overexpressing GC cells involving JWA-mediated MEK/ERK/PEA3 signaling activation and HER2 downregulation. Furthermore, JWA may be a useful prognostic indicator for advanced GC and may help stratify HER2-positive

  7. Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

    PubMed

    Hirokawa, N; Takemura, R; Hisanaga, S

    1985-11-01

    We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic

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

    SciTech Connect

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

    1988-04-01

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

  9. Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

    PubMed Central

    1993-01-01

    Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis. PMID:8380174

  10. Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

    PubMed

    Loesser, K E; Doane, K J; Wilson, F J; Roisen, F J; Malamed, S

    1986-11-01

    We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

  11. The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

    PubMed

    Hägerstrand, H; Danieluk, M; Bobrowska-Hägerstrand, M; Holmström, T; Kralj-Iglic, V; Lindqvist, C; Nikinmaa, M

    1999-01-01

    The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.

  12. The Cytoskeletal Adapter Protein Spinophilin Regulates Invadopodia Dynamics and Tumor Cell Invasion in Glioblastoma.

    PubMed

    Cheerathodi, Mujeeburahiman; Avci, Naze G; Guerrero, Paola A; Tang, Leung K; Popp, Julia; Morales, John E; Chen, Zhihua; Carnero, Amancio; Lang, Frederick F; Ballif, Bryan A; Rivera, Gonzalo M; McCarty, Joseph H

    2016-12-01

    Glioblastoma is a primary brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to invasive cancer cells that disperse from the main tumor site, escape surgical resection, and contribute to recurrent secondary lesions. The adhesion and signaling mechanisms that drive glioblastoma cell invasion remain enigmatic, and as a result there are no effective anti-invasive clinical therapies. Here we have characterized a novel adhesion and signaling pathway comprised of the integrin αvβ8 and its intracellular binding partner, Spinophilin (Spn), which regulates glioblastoma cell invasion in the brain microenvironment. We show for the first time that Spn binds directly to the cytoplasmic domain of β8 integrin in glioblastoma cells. Genetically targeting Spn leads to enhanced invasive cell growth in preclinical models of glioblastoma. Spn regulates glioblastoma cell invasion by modulating the formation and dissolution of invadopodia. Spn-regulated invadopodia dynamics are dependent, in part, on proper spatiotemporal activation of the Rac1 GTPase. Glioblastoma cells that lack Spn showed diminished Rac1 activities, increased numbers of invadopodia, and enhanced extracellular matrix degradation. Collectively, these data identify Spn as a critical adhesion and signaling protein that is essential for modulating glioblastoma cell invasion in the brain microenvironment.

  13. Activated ADF/cofilin sequesters phosphorylated microtubule-associated-protein during the assembly of Alzheimer-like neuritic cytoskeletal striations

    PubMed Central

    Whiteman, Ineka T.; Gervasio, Othon L.; Cullen, Karen M.; Guillemin, Gilles J.; Jeong, Erica V.; Witting, Paul K.; Antao, Shane T.; Minamide, Laurie S.; Bamburg, James R.; Goldsbury, Claire

    2009-01-01

    In Alzheimer disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads) and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence Resonance Energy Transfer (FRET) analysis revealed co-localization of cofilin-GFP and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and siRNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous Aβ peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. PMID:19828813

  14. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  15. Smoking correlates with increased cytoskeletal protein-related coding region mutations in the lung and head and neck datasets of the cancer genome atlas.

    PubMed

    Yavorski, John M; Blanck, George

    2016-12-01

    Cancer from smoking tobacco is considered dependent on mutagens, but significant molecular aspects of smoking-specific, cancer development remain unknown. We defined sets of coding regions for oncoproteins, tumor suppressor proteins, and cytoskeletal-related proteins that were compared between nonsmokers and smokers, for mutation occurrences, in the lung adenocarcinoma (LUAD), head and neck squamous carcinoma (HNSC), bladder carcinoma (BLCA), and pancreatic adenocarcinoma ( PAAD) datasets from the cancer genome atlas (TCGA). We uncovered significant differences in overall mutation rates, and in mutation rates in cytoskeletal protein-related coding regions (CPCRs, including extracellular matrix protein coding regions), between nonsmokers and smokers in LUAD and HNSC (P < 0.001), raising the question of whether the CPCR mutation differences lead to different clinical courses for nonsmoker and smoker cancers. Another important question inspired by these results is, whether high smoker cancer mutation rates would facilitate genotoxicity or neoantigen-based therapies. No significant, mutation-based differences were found in the BLCA or PAAD datasets, between nonsmokers and smokers. However, a significant difference was uncovered for the average number of overall cancer mutations, in LUAD, for persons who stopped smoking more than 15 years ago, compared with more recent smokers (P < 0.032).

  16. Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

    PubMed Central

    Welch, W J; Feramisco, J R

    1985-01-01

    Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements. Images PMID:4040602

  17. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

    PubMed Central

    Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

    2016-01-01

    African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

  18. Apo and calcium-bound crystal structures of cytoskeletal protein alpha-14 giardin (annexin E1) from the intestinal protozoan parasite Giardia lamblia.

    PubMed

    Pathuri, Puja; Nguyen, Emily Tam; Ozorowski, Gabriel; Svärd, Staffan G; Luecke, Hartmut

    2009-01-30

    Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca(2+)-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca(2+)-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 A, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca(2+)-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to

  19. Kinetic Proofreading of Cytoskeletal Structures

    NASA Astrophysics Data System (ADS)

    Swanson, Douglas; Wingreen, Ned

    2010-03-01

    Cytoskeletal polymer dynamics play a role in cellular processes as varied as reproduction, locomotion, and intracellular transport. Microtubules are cytoskeletal biopolymers that grow by accumulating tubulin subunits bound to guanosine triphosphate (GTP). The subunits hydrolyze GTP to guanosine diphosphate (GDP), causing a conformational change in the protein that destabilizes the microtubule. GDP-bound subunits tend to depolymerize, leading to stochastic microtubule disassembly in a process known as dynamic instability. Over time polymerization and depolymerization come to steady state, leading to a local steady-state concentration of tubulin subunits. This may be viewed as a kind of ``kinetic proofreading,'' in which the system consumes energy actively to ``proofread'' the steady-state subunit concentration. We suggest that the same mechanism could also ``proofread'' between different cytoskeletal structures. For example, we show that a small free-energy difference between two polymer orientations, combined with dynamic instability, can strongly drive the system towards the lower free-energy state. This might help to explain the long-time stability of many cytoskeletal structures despite the short-time rapid turnover of the individual subunits.

  20. Toxaphene, but not beryllium, induces human neutrophil chemotaxis and apoptosis via reactive oxygen species (ROS): involvement of caspases and ROS in the degradation of cytoskeletal proteins.

    PubMed

    Lavastre, Valérie; Roberge, Charles J; Pelletier, Martin; Gauthier, Marc; Girard, Denis

    2002-07-01

    Chemicals of environmental concern are known to alter the immune system. Recent data indicate that some contaminants possess proinflammatory properties by activating neutrophils, an area of research that is still poorly investigated. We have previously documented that toxaphene activates human neutrophils to produce reactive oxygen species (ROS) and accelerates apoptosis by a yet unknown mechanism. In this study, we found that toxaphene induces another neutrophil function, chemotaxis. Furthermore, we found that toxaphene induces both chemotaxis and apoptosis via a ROS-dependent mechanism, since these responses were blocked by the addition of catalase to the culture. In addition, toxaphene was found to induce the degradation of the cytoskeletal proteins gelsolin, paxillin, and vimentin during apoptosis, and this was reversed by the addition of z-VAD-FMK (caspase inhibitor) or catalase, demonstrating the importance of caspases and ROS in this process. In contrast to toxaphene, we found that beryllium does not induce superoxide production, and, this correlates with its inability to induce chemotaxis and apoptosis. We conclude that toxaphene induces chemotaxis and apoptosis via ROS and that caspases and ROS are involved in the degradation of cytoskeletal proteins.

  1. Autoinduction of Protein Expression

    PubMed Central

    Fox, Brian G.; Blommel, Paul G.

    2017-01-01

    This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included. PMID:19365792

  2. The cytoskeletal protein alpha-actinin regulates acid-sensing ion channel 1a through a C-terminal interaction.

    PubMed

    Schnizler, Mikael K; Schnizler, Katrin; Zha, Xiang-Ming; Hall, Duane D; Wemmie, John A; Hell, Johannes W; Welsh, Michael J

    2009-01-30

    The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that alpha-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an alpha-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for alpha-actinin-1 and -4. Co-expressing alpha-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing alpha-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that alpha-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.

  3. Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria

    PubMed Central

    Ray, Sandipan; Patel, Sandip K.; Venkatesh, Apoorva; Bhave, Amruta; Kumar, Vipin; Singh, Vaidhvi; Chatterjee, Gangadhar; Shah, Veenita G.; Sharma, Sarthak; Renu, Durairaj; Nafis, Naziya; Gandhe, Prajakta; Gogtay, Nithya; Thatte, Urmila; Sehgal, Kunal; Verma, Sumit; Karak, Avik; Khanra, Dibbendhu; Talukdar, Arunansu; Kochar, Sanjay K.; S. B, Vijeth; Kochar, Dhanpat K.; Rojh, Dharmendra; Varma, Santosh G.; Gandhi, Mayuri N.; Srikanth, Rapole; Patankar, Swati; Srivastava, Sanjeeva

    2016-01-01

    In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity. PMID:27090372

  4. Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria.

    PubMed

    Ray, Sandipan; Patel, Sandip K; Venkatesh, Apoorva; Bhave, Amruta; Kumar, Vipin; Singh, Vaidhvi; Chatterjee, Gangadhar; Shah, Veenita G; Sharma, Sarthak; Renu, Durairaj; Nafis, Naziya; Gandhe, Prajakta; Gogtay, Nithya; Thatte, Urmila; Sehgal, Kunal; Verma, Sumit; Karak, Avik; Khanra, Dibbendhu; Talukdar, Arunansu; Kochar, Sanjay K; S B, Vijeth; Kochar, Dhanpat K; Rojh, Dharmendra; Varma, Santosh G; Gandhi, Mayuri N; Srikanth, Rapole; Patankar, Swati; Srivastava, Sanjeeva

    2016-04-19

    In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.

  5. RNA Helicase DDX5 Regulates MicroRNA Expression and Contributes to Cytoskeletal Reorganization in Basal Breast Cancer Cells

    SciTech Connect

    Wang, Daojing; Huang, Jing; Hu, Zhi

    2011-11-15

    RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was upregulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by upregulation of tumor suppressor PDCD4 (a known miR-21 target); as well as upregulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 downregulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.

  6. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

    PubMed Central

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  7. short stop is allelic to kakapo, and encodes rod-like cytoskeletal-associated proteins required for axon extension.

    PubMed

    Lee, S; Harris, K L; Whitington, P M; Kolodziej, P A

    2000-02-01

    short stop (shot) is required for sensory and motor axons to reach their targets in the Drosophila embryo. Growth cones in shot mutants initiate at the normal times, and they appear normal with respect to overall morphology and their abilities to orient and fasciculate. However, sensory axons are unable to extend beyond a short distance from the cell body, and motor axons are unable to reach target muscles. The shot gene encodes novel actin binding proteins that are related to plakins and dystrophin and expressed in axons during development. The longer isoforms identified are predicted to contain an N-terminal actin binding domain, a long central triple helical coiled-coil domain, and a C-terminal domain that contains two EF-hand Ca(2+) binding motifs and a short stretch of homology to the growth arrest-specific 2 protein. Other isoforms lack all or part of the actin binding domains or are truncated and contain a different C-terminal domain. Only the isoforms containing full-length actin binding domains are detectably expressed in the nervous system. shot is allelic to kakapo, a gene that may function in integrin-mediated adhesion in the wing and embryo. We propose that Shot's interactions with the actin cytoskeleton allow sensory and motor axons to extend.

  8. Immediate Early Gene Activity-Regulated Cytoskeletal-Associated Protein Regulates Estradiol-Induced Lordosis Behavior in Female Rats

    PubMed Central

    Christensen, Amy; Dewing, Phoebe; Micevych, Pavel

    2016-01-01

    Sensory feedback is an important component of any behavior, with each instance influencing subsequent activity. Female sexual receptivity is mediated both by the steroid hormone milieu and interaction with the male. We tested the influence of repeated mating on the level of sexual receptivity in ovariectomized rats treated with estradiol benzoate (EB) once every fourth day to mimic the normal phasic changes of circulating estradiol. Females were divided into two groups: naïve, which were tested for lordosis behavior once, and experienced rats, which were tested for lordosis after each EB injection. To monitor the effect of mating, the number of neurons expressing the immediate early gene activity-regulated cytoskeleton-associated protein (Arc) were counted in the mediobasal hypothalamus. Females were unreceptive following the first EB treatment, but the mating induced Arc expression. In naïve rats, each subsequent EB injection increased the levels of sexual receptivity. This ramping was not observed in experienced rats, which achieved only a moderate level of sexual receptivity. However, experienced females treated with EB and progesterone were maximally receptive and did not have Arc expresion. To test whether the expression of Arc attenuated lordosis, Arc antisense oligodeoxynucleotides (asODN) were microinjected into experienced females’ arcuate nuclei. Arc expression was attenuated, and the experienced EB-treated females achieved maximal sexual receptivity. These results demonstrate that Arc expression in the hypothalamus might influence future sexual receptivity and provides evidence of learning in the arcuate nucleus. The loss of Arc results in unrestrained sexual receptivity. PMID:25088303

  9. SNAIL-induced epithelial-to-mesenchymal transition produces concerted biophysical changes from altered cytoskeletal gene expression.

    PubMed

    McGrail, Daniel J; Mezencev, Roman; Kieu, Quang Minh N; McDonald, John F; Dawson, Michelle R

    2015-04-01

    A growing body of evidence suggests that the developmental process of epithelial-to-mesenchymal transition (EMT) is co-opted by cancer cells to metastasize to distant sites. This transition is associated with morphologic elongation and loss of cell-cell adhesions, though little is known about how it alters cell biophysical properties critical for migration. Here, we use multiple-particle tracking (MPT) microrheology and traction force cytometry to probe how genetic induction of EMT in epithelial MCF7 breast cancer cells changes their intracellular stiffness and extracellular force exertion, respectively, relative to an empty vector control. This analysis demonstrated that EMT alone was sufficient to produce dramatic cytoskeletal softening coupled with increases in cell-exerted traction forces. Microarray analysis revealed that these changes corresponded with down-regulation of genes associated with actin cross-linking and up-regulation of genes associated with actomyosin contraction. Finally, we show that this loss of structural integrity to expedite migration could inhibit mesenchymal cell proliferation in a secondary tumor as it accumulates solid stress. This work demonstrates that not only does EMT enable escape from the primary tumor through loss of cell adhesions but it also induces a concerted series of biophysical changes enabling enhanced migration of cancer cells after detachment from the primary tumor. © FASEB.

  10. The cytoskeletal protein septin 11 is associated with human obesity and is involved in adipocyte lipid storage and metabolism.

    PubMed

    Moreno-Castellanos, Natalia; Rodríguez, Amaia; Rabanal-Ruiz, Yoana; Fernández-Vega, Alejandro; López-Miranda, José; Vázquez-Martínez, Rafael; Frühbeck, Gema; Malagón, María M

    2017-02-01

    Septins are newly identified members of the cytoskeleton that have been proposed as biomarkers of a number of diseases. However, septins have not been characterised in adipose tissue and their relationship with obesity and insulin resistance remains unknown. Herein, we characterised a member of this family, septin 11 (SEPT11), in human adipose tissue and analysed its potential involvement in the regulation of adipocyte metabolism. Gene and protein expression levels of SEPT11 were analysed in human adipose tissue. SEPT11 distribution was evaluated by immunocytochemistry, electron microscopy and subcellular fractionation techniques. Glutathione S-transferase (GST) pull-down, immunoprecipitation and yeast two-hybrid screening were used to identify the SEPT11 interactome. Gene silencing was used to assess the role of SEPT11 in the regulation of insulin signalling and lipid metabolism in adipocytes. We demonstrate the expression of SEPT11 in human adipocytes and its upregulation in obese individuals, with SEPT11 mRNA content positively correlating with variables of insulin resistance in subcutaneous adipose tissue. SEPT11 content was regulated by lipogenic, lipolytic and proinflammatory stimuli in human adipocytes. SEPT11 associated with caveolae in mature adipocytes and interacted with both caveolin-1 and the intracellular fatty acid chaperone, fatty acid binding protein 5 (FABP5). Lipid loading of adipocytes caused the association of the three proteins with the surface of lipid droplets. SEPT11 silencing impaired insulin signalling and insulin-induced lipid accumulation in adipocytes. Our findings support a role for SEPT11 in lipid traffic and metabolism in adipocytes and open new avenues for research on the control of lipid storage in obesity and insulin resistance.

  11. Protein expression in liposomes.

    PubMed

    Oberholzer, T; Nierhaus, K H; Luisi, P L

    1999-08-02

    Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. Copyright 1999 Academic Press.

  12. Preliminary identification of differentially expressed tear proteins in keratoconus

    PubMed Central

    Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

    2013-01-01

    Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. PMID:24194634

  13. Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity.

    PubMed

    Corydon, Thomas J; Mann, Vivek; Slumstrup, Lasse; Kopp, Sascha; Sahana, Jayashree; Askou, Anne Louise; Magnusson, Nils E; Echegoyen, David; Bek, Toke; Sundaresan, Alamelu; Riwaldt, Stefan; Bauer, Johann; Infanger, Manfred; Grimm, Daniela

    2016-01-01

    Microgravity (µg) has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19) cells is unknown. In this study we investigated the influence of simulated µg (s-µg; 5 and 10 days (d)), using a Random Positioning Machine (RPM), on ARPE-19 cells. We performed phase-contrast/fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis. Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS), whereas the majority of the cells remained adherent (AD). After 5d, alterations of F-actin and fibronectin were observed which reverted after 10d-exposure, suggesting a time-dependent adaptation to s-µg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a 10d-RPM-exposure. 11 genes were down-regulated in AD and MCS 10d-RPM-samples compared to 1g, whereas FLK1 was up-regulated in 5d- and 10d-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, β-actin, laminin and fibronectin of 5d-RPM-samples compared to 10d, whereas different alterations of β-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin β-1. These findings clearly show that s-µg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis. © 2016 The Author(s) Published by S. Karger AG, Basel.

  14. Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.

    PubMed

    Cao, Xu; Zhang, Ying; Zou, Liangyu; Xiao, Haibing; Chu, Yinghao; Chu, Xiaofan

    2010-07-26

    Astrocytes are thought to play a role in the maintenance of homeostasis and the provision of metabolic substrates for neurons as well as the coupling of cerebral blood flow to neuronal activity. Accordingly, astrocytic death due to various types of injury can critically influence neuronal survival. The exact pathway of cell death after brain ischemia is under debate. In the present study, we used astrocytes from rat primary culture treated with persistent oxygen-glucose-deprivation (OGD) as a model of ischemia to examine the pathway of cell death and the relevant mechanisms. We observed changes in the cellular morphology, the energy metabolism of astrocytes, and the percentage of apoptosis or oncosis of the astrocytes induced by OGD. Electron microscopy revealed the co-existence of ultrastructural features in both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased and the percentages of apoptotic and oncotic cells were increased during OGD. After 4h of OGD, ATP depletion to less than 35% of the control was observed, and oncosis became the primary pathway for astrocytic death. Increased plasma membrane permeability due to oncosis was associated with increased calpain-mediated degradation of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP. Pre-treatment with the calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) could delay the OGD-induced astrocytic oncosis. These results suggest that there is a narrow range of ATP that determines astrocytic oncotic death induced by persistent OGD and that calpain-mediated hydrolysis of the cytoskeletal-associated proteins may contribute to astrocytes oncosis.

  15. Cytoskeletal protein flightless I inhibits apoptosis, enhances tumor cell invasion and promotes cutaneous squamous cell carcinoma progression

    PubMed Central

    Kopecki, Zlatko; Yang, Gink N.; Jackson, Jessica E.; Melville, Elizabeth L.; Cal1ey, Matthew P.; Murrell, Dedee F.; Darby, Ian A.; O'Toole, Edel A.; Samuel, Michael S.; Cowin, Allison J.

    2015-01-01

    Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous (Flii+/−), wild-type and Flii overexpressing (FliiTg/Tg) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. FliiTg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii+/− mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro, decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs. PMID:26497552

  16. Tenotomy of m.soleus antagonists prevents the changes in fiber type characteristics and sarcomeric cytoskeletal proteins in unloaded rats

    NASA Astrophysics Data System (ADS)

    Moukhina, Alexandra; Ardabievskaya, Anna; Vikhlyantsev, Ivan; Podlubnaya, Zoya; Nemirovskaya, Tatiana; Shenkman, Boris

    2005-08-01

    It is known that activity of postural extensors (m. soleus) decreases and activity of flexors (m. tibialis anterior) increases under unloading conditions. We have tested the hypothesis supposing that increased flexor activities during unloading exert suppressive influence on postural extensor activities and thus lead to dramatic changes in fiber size, MHC expression, sarcomeric proteins content in m.soleus. We have inactivated hindlimb flexor muscles (m.soleus antagonists) by bilateral tenotomy. 20 male Wistar rats were divided on 3 groups: cage control (C), hindlimb suspension for 14 days (HS), tenotomy of hindlimb flexor muscles with 14 days hindlimb suspension afterwards (HST). Several soleus muscle fiber characteristics decreased significantly in HS group (p<0.05) as compared with C group: cross sectional area (CSA) of type I muscle fibers, titin/MyHC ratio and nebulin/MyHC ratio. MyHC isoform pattern shifted slow-to-fast significantly. NFATc1 content increased in nuclear protein extract of m. soleus in HS group. None of these parameters was significantly different in HST group from those of C group. It has been concluded that the tenotomy of flexors under hindlimb suspension prevents atrophy of type I muscle fibers, decrease the degradation of titin and nebulin and prevent slow-to-fast shift of fiber MyHC isoform pattern, possibly through prevention of increase NFATc1 content in muscle fiber nuclear protein extract. Therefore, suppressive influence of increased flexor activity could be one of mechanisms that lead to the changes in m. soleus under unloading conditions. The work was supported by RFBR grants: 02-04-50025, 03- 04-48487 and the special program of RAS "Integration mechanisms of functional control in the living system".

  17. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    NASA Technical Reports Server (NTRS)

    Melhado, C. D.; Sanford, G. L.; Bosah, F.; Harris-Hooker, S.

    1998-01-01

    Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal

  18. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    NASA Technical Reports Server (NTRS)

    Melhado, C. D.; Sanford, G. L.; Bosah, F.; Harris-Hooker, S.

    1998-01-01

    Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal

  19. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  20. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed Central

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-01-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin. PMID:8552082

  1. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

    PubMed

    Turner, C E; Brown, M C; Perrotta, J A; Riedy, M C; Nikolopoulos, S N; McDonald, A R; Bagrodia, S; Thomas, S; Leventhal, P S

    1999-05-17

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

  2. Leptospira Protein Expression During Infection

    USDA-ARS?s Scientific Manuscript database

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  3. Identification of a new subtilisin-like protease NbSLP2 interacting with cytoskeletal protein septin in Microsporidia Nosema bombycis.

    PubMed

    Liu, Fangyan; Ma, Qiang; Dang, Xiaoqun; Wang, Ying; Song, Yue; Meng, Xianzhi; Bao, Jialing; Chen, Jie; Pan, Guoqing; Zhou, Zeyang

    2017-09-01

    Nosema bombycis is the pathogen of pébrine which brings heavy losses to sericulture every year. As a member of serine proteases, subtilisin-like protease (SLP) is related to the pathogenicity in fungi. In this study, we characterized a novel 63.8kDa subtilisin-like protease NbSLP2 with a predicted transmembrane domain from Microsporidia, N. bombycis. RT-PCR showed that the transcript of NbSLP2 was detected from third day post infection. Immunofluorescence assay (IFA) indicated that NbSLP2 mainly scattered around the spore wall of N. bombycis. Co-immunoprecipitation data and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed that NbSLP2 directly interacts with septin2 of N. bombycis, which is a cytoskeletal protein. IFA showed that NbSLP2 and Nbseptin2 co-localized beneath the spore wall. NbSLP2 can be pulled down by Nbseptin2, further confirming the interaction between NbSLP2 and Nbseptin2. As an important serine protease with a transmembrane domain, NbSLP2 interacting with Nbseptin2, a scaffold protein adjacent to the membrane may provide advantages to stabilize the NbSLP2 for its hydrolysis function. Copyright © 2017. Published by Elsevier Inc.

  4. Differential carbonylation of cytoskeletal proteins in blood group O erythrocytes: potential role in protection against severe malaria.

    PubMed

    Méndez, Darío; Hernáez, María L; Kamali, Ali N; Diez, Amalia; Puyet, Antonio; Bautista, José M

    2012-12-01

    The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC-MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.

  5. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

  6. Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells

    PubMed Central

    1981-01-01

    The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the

  7. Biotechnological aspects of cytoskeletal regulation in plants.

    PubMed

    Komis, George; Luptovciak, Ivan; Doskocilova, Anna; Samaj, Jozef

    2015-11-01

    The cytoskeleton is a protein-based intracellular superstructure that evolved early after the appearance of bacterial prokaryotes. Eventually cytoskeletal proteins and their macromolecular assemblies were established in eukaryotes and assumed critical roles in cell movements, intracellular organization, cell division and cell differentiation. In biomedicine the small-molecules targeting cytoskeletal elements are in the frontline of anticancer research with plant-derived cytoskeletal drugs such as Vinca alkaloids and toxoids, being routinely used in the clinical practice. Moreover, plants are also major material, food and energy resources for human activities ranging from agriculture, textile industry, carpentry, energy production and new material development to name some few. Most of these inheritable traits are associated with cell wall synthesis and chemical modification during primary and secondary plant growth and inevitably are associated with the dynamics, organization and interactions of the plant cytoskeleton. Taking into account the vast intracellular spread of microtubules and actin microfilaments the cytoskeleton collectively assumed central roles in plant growth and development, in determining the physical stance of plants against the forces of nature and becoming a battleground between pathogenic invaders and the defense mechanisms of plant cells. This review aims to address the role of the plant cytoskeleton in manageable features of plants including cellulose biosynthesis with implications in wood and fiber properties, in biofuel production and the contribution of plant cytoskeletal elements in plant defense responses against pathogens or detrimental environmental conditions. Ultimately the present work surveys the potential of cytoskeletal proteins as platforms of plant genetic engineering, nominating certain cytoskeletal proteins as vectors of favorable traits in crops and other economically important plants.

  8. Arsenic trioxide (AT) is a novel human neutrophil pro-apoptotic agent: effects of catalase on AT-induced apoptosis, degradation of cytoskeletal proteins and de novo protein synthesis.

    PubMed

    Binet, François; Cavalli, Hélène; Moisan, Eliane; Girard, Denis

    2006-02-01

    The anti-cancer drug arsenic trioxide (AT) induces apoptosis in a variety of transformed or proliferating cells. However, little is known regarding its ability to induce apoptosis in terminally differentiated cells, such as neutrophils. Because neutropenia has been reported in some cancer patients after AT treatment, we hypothesised that AT could induce neutrophil apoptosis, an issue that has never been investigated. Herein, we found that AT-induced neutrophil apoptosis and gelsolin degradation via caspases. AT did not increase neutrophil superoxide production and did not induce mitochondrial generation of reactive oxygen species. AT-induced apoptosis in PLB-985 and X-linked chronic granulomatous disease (CGD) cells (PLB-985 cells deficient in gp91(phox) mimicking CGD) at the same potency. Addition of catalase, an inhibitor of H2O2, reversed AT-induced apoptosis and degradation of the cytoskeletal proteins gelsolin, alpha-tubulin and lamin B1. Unexpectedly, AT-induced de novo protein synthesis, which was reversed by catalase. Cycloheximide partially reversed AT-induced apoptosis. We conclude that AT induces neutrophil apoptosis by a caspase-dependent mechanism and via de novo protein synthesis. H2O2 is of major importance in AT-induced neutrophil apoptosis but its production does not originate from nicotinamide adenine dinucleotide phosphate dehydrogenase activation and mitochondria. Cytoskeletal structures other than microtubules can now be considered as novel targets of AT.

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

    USDA-ARS?s Scientific Manuscript database

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

  10. A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.

    PubMed

    Yu, J; Wang, C; Palmieri, S J; Haarer, B K; Field, J

    1999-07-09

    In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.

  11. Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black-Backed Jackal (Canis mesomelas).

    PubMed

    Madekurozwa, M-C; Booyse, D

    2017-02-01

    Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black-backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.

  12. Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

    PubMed

    Egbowon, Biola F; Harris, Wayne; Arnott, Gordon; Mills, Chris Lloyd; Hargreaves, Alan J

    2016-04-01

    The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl2 for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12 μM Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.

  13. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  14. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  15. Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling.

    PubMed

    Zipfel, Patricia A; Bunnell, Stephen C; Witherow, D Scott; Gu, Jing Jin; Chislock, Elizabeth M; Ring, Colleen; Pendergast, Ann Marie

    2006-01-10

    The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.

  16. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  17. Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells

    SciTech Connect

    Flaskos, J.; Nikolaidis, E.; Harris, W.; Sachana, M.; Hargreaves, A.J.

    2011-11-15

    protein are reduced Black-Right-Pointing-Pointer Neurofilament heavy chain forms aggregates in cell bodies Black-Right-Pointing-Pointer Thus at least two axon-associated cytoskeletal proteins are disrupted by this agent.

  18. Rate and extent of pH decline affect proteolysis of cytoskeletal proteins and water-holding capacity in pork.

    PubMed

    Bee, Giuseppe; Anderson, Abbey L; Lonergan, Steven M; Huff-Lonergan, Elisabeth

    2007-06-01

    The objective of this study was to determine the extent to which early postmortem (PM) pH decline influences proteolysis of the intermediate filament protein desmin, the costameric proteins vinculin and talin and autolysis of μ-calpain in the longissimus muscle (LM) of pigs from two genetic lines. Based on the LM 3h pH (H=3h pH of LM>6.0; L=3h pH of LM pH<5.7) PM, 10 carcasses per line and pH group were selected. The average 3h pH within pH group was 6.23 (H) and 5.44 (L). The LM samples were collected 24, 48, 72, and 120h PM and percent drip loss was measured after 1, 2, and 4d of storage. Samples collected at 24, 48, 72, and 120h PM were used to monitor desmin, vinculin, and talin degradation and samples collected at 24h PM were used to determine the extent of μ-calpain autolysis by immunoblotting. Higher (P<0.01) pH values at 45min, 6h, and 24h PM and lower (P<0.01) drip losses after 1, 2, and 4d of storage were recorded in the H-compared to the L-group. Abundance of the 76kDa μ-calpain autolysis product was greater (P<0.01), proteolysis of talin at all measured time points and proteolysis of desmin after 24 and 48h PM was greater (P⩽0.03) in the H-group than in the L-group. The current findings indicate activation rate of μ-calpain may be associated with proteolysis of desmin and talin and could play a role in the development of drip loss. The rate of early PM pH decline can partly explain the variation of desmin and talin degradation by affecting the activation of μ-calpain.

  19. Nonsteroidal anti-inflammatory drugs attenuate amyloid-β protein-induced actin cytoskeletal reorganization through Rho signaling modulation.

    PubMed

    Ferrera, Patricia; Zepeda, Angélica; Arias, Clorinda

    2017-01-25

    Amyloid-β protein (Aβ) neurotoxicity occurs along with the reorganization of the actin-cytoskeleton through the activation of the Rho GTPase pathway. In addition to the classical mode of action of the non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin, and ibuprofen have Rho-inhibiting effects. In order to evaluate the role of the Rho GTPase pathway on Aβ-induced neuronal death and on neuronal morphological modifications in the actin cytoskeleton, we explored the role of NSAIDS in human-differentiated neuroblastoma cells exposed to Aβ. We found that Aβ induced neurite retraction and promoted the formation of different actin-dependent structures such as stress fibers, filopodia, lamellipodia, and ruffles. In the presence of Aβ, both NSAIDs prevented neurite collapse and formation of stress fibers without affecting the formation of filopodia and lamellipodia. Similar results were obtained when the downstream effector, Rho kinase inhibitor Y27632, was applied in the presence of Aβ. These results demonstrate the potential benefits of the Rho-inhibiting NSAIDs in reducing Aβ-induced effects on neuronal structural alterations.

  20. Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

    PubMed Central

    Kühn, Juliane; Briegel, Ariane; Mörschel, Erhard; Kahnt, Jörg; Leser, Katja; Wick, Stephanie; Jensen, Grant J; Thanbichler, Martin

    2010-01-01

    The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways. PMID:19959992

  1. Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

    PubMed

    Dewey, Evan B; Johnston, Christopher A

    2017-09-15

    Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. 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).

  2. Early and Late Loss of the Cytoskeletal Scaffolding Protein, Ankyrin G Reveals its Role in Maturation and Maintenance of Nodes of Ranvier in Myelinated Axons.

    PubMed

    Saifetiarova, Julia; Taylor, Anna M; Bhat, Manzoor A

    2017-02-01

    The mechanisms that govern node of Ranvier organization, stability and long-term maintenance remain to be fully elucidated. One of the molecular components of the node is the cytoskeletal scaffolding protein, Ankyrin G (AnkG), which interacts with multiple members of the nodal complex. The role of AnkG in nodal organization and maintenance is still not clearly defined, as to whether AnkG functions as an initial nodal organizer or whether it functions as a nodal stabilizer after the nodal complex has been assembled. Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. Early loss of AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid NaV channel and βIV Spectrin loss with reduced effects on Neurofascin 186. On the other hand, late ablation of AnkG from established nodal complexes leads to slow but progressive nodal destabilization over 10 months, primarily affecting βIV Spectrin, followed by NaV channels, with modest impact on Neurofascin 186. We also show that Ankyrin R and βI Spectrin are not sufficient to prevent nodal disorganization after AnkG ablation. Additionally, nodal disorganization in both early and late AnkG mutants is accompanied by axonal pathology and neurological dysfunction. Together, our results suggest that AnkG plays an indispensable role in maturation and long-term stabilization of the newly assembled nodal complex, and that loss of AnkG after nodal stabilization does not lead to rapid nodal disassembly but loss of specific nodal components in a time-dependent manner.

  3. Effects of maternal separation and methamphetamine exposure on protein expression in the nucleus accumbens shell and core.

    PubMed

    Dimatelis, J J; Russell, V A; Stein, D J; Daniels, W M

    2012-09-01

    Early life adversity has been suggested to predispose an individual to later drug abuse. The core and shell sub-regions of the nucleus accumbens are differentially affected by both stressors and methamphetamine. This study aimed to characterize and quantify methamphetamine-induced protein expression in the shell and core of the nucleus accumbens in animals exposed to maternal separation during early development. Isobaric tagging (iTRAQ) which enables simultaneous identification and quantification of peptides with tandem mass spectrometry (MS/MS) was used. We found that maternal separation altered more proteins involved in structure and redox regulation in the shell than in the core of the nucleus accumbens, and that maternal separation and methamphetamine had differential effects on signaling proteins in the shell and core. Compared to maternal separation or methamphetamine alone, the maternal separation/methamphetamine combination altered more proteins involved in energy metabolism, redox regulatory processes and neurotrophic proteins. Methamphetamine treatment of rats subjected to maternal separation caused a reduction of cytoskeletal proteins in the shell and altered cytoskeletal, signaling, energy metabolism and redox proteins in the core. Comparison of maternal separation/methamphetamine to methamphetamine alone resulted in decreased cytoskeletal proteins in both the shell and core and increased neurotrophic proteins in the core. This study confirms that both early life stress and methamphetamine differentially affect the shell and core of the nucleus accumbens and demonstrates that the combination of early life adversity and later methamphetamine use results in more proteins being affected in the nucleus accumbens than either treatment alone.

  4. Cytoskeletal proteins regulate chromatin access of BR-C transcription factor and Rpd3-Sin3A histone deacetylase complex in Drosophila salivary glands.

    PubMed

    Farkaš, Robert; Kuchárová-Mahmood, Silvia; Mentelová, Lucia; Juda, Pavel; Raška, Ivan; Mechler, Bernard M

    2011-01-01

    At the onset of Drosophila metamorphosis the steroid hormone ecdysone induces a process leading to a rapid degeneration of the larval salivary glands (SGs). Ecdysone acts through the ecdysone receptor heterodimer, which activates primary response genes. In particular these genes include the Broad-Complex (BR-C) gene encoding a set of BTB/POZ-transcription factors, among which the Z1 isoform is critical for SG cell death. The timing of SG disappearance depends upon of p127 (l(2)gl) , a cytoskeletal tumor suppressor that interacts with nonmuscle myosin II heavy chain (nmMHC) encoded by the zipper (zip) gene. Reduced l(2)gl expression delays SG histolysis whereas over-expression accelerates this process without affecting larval and pupal development. However, the mechanism by which l(2)gl controls SG histolysis remains yet unknown. Here we analyze the regulation controlled by p127 (l(2)gl) and nmMHC in the cytoplasm on the association of BR-C Z1 with chromatin and remodeling factors, such as Rpd3, Sin3A, and Smrter. In wild-type SGs these factors bind to chromatin but in l(2)gl SGs they accumulate in the cytoplasm and the cortical nuclear zone (CNZ). Similar chromatin exclusion occurs in SGs of developmentally delayed zip (E(br)) /+ larvae or can be achieved by high levels of nmMHC synthesis. The present data show that p127 (l(2)gl) and nmMHC regulate the access of BR-C Z1, Rpd3, Sin3A, and Smrter to chromatin. As the interaction between p127 (l(2)gl) and nmMHC occurs in the cytoplasm, we propose that these nuclear factors are processed by p127 (l(2)gl) and then released from p127 (l(2)gl) by nmMHC to allow their binding to chromatin. This process may constitute a novel mechanism of gene regulation, which in the absence of p127 (l(2)gl) , or excessive amounts of nmMHC, could lead to a fixed configuration in the pattern of gene expression that prevents further progression of SG differentiation, and programmed cell death (PCD). Such a transcriptional block could play a

  5. Differential dissolved protein expression throughout the life cycle of Giardia lamblia.

    PubMed

    Lingdan, Li; Pengtao, Gong; Wenchao, Li; Jianhua, Li; Ju, Yang; Chengwu, Liu; He, Li; Guocai, Zhang; Wenzhi, Ren; Yujiang, Chen; Xichen, Zhang

    2012-12-01

    Giardia lamblia (G. lamblia) has a simple life cycle that alternates between a cyst and a trophozoite, and this parasite is an important human and animal pathogen. To increase our understanding of the molecular basis of the G. lamblia encystment, we have analyzed the soluble proteins expressed by trophozoites and cysts extracted from feces by quantitative proteomic analysis. A total of 63 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and were categorized as cytoskeletal proteins, a cell-cycle-specific kinase, metabolic enzymes and stress resistance proteins. Importantly, we demonstrated that the expression of seven proteins differed significantly between trophozoites and cysts. In cysts, the expression of three proteins (one variable surface protein (VSP), ornithine carbamoyltransferase (OTC), β-tubulin) increased, whereas the expression of four proteins (14-3-3 protein, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein disulfide isomerase 2 (PDI-2)) decreased significantly when compared with the levels of these proteins in trophozoites. The mRNA expression patterns of four of these proteins (OTC, α-tubulin, GAPDH, VSP) were similar to the expression levels of the proteins. These seven proteins appear to play an important role in the completion of the life cycle of G. lamblia. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

    PubMed

    Klonk, S; Deuticke, B

    1992-04-29

    Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain

  7. Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

    PubMed Central

    Bailleul, P A; Newnam, G P; Steenbergen, J N; Chernoff, Y O

    1999-01-01

    Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process. PMID:10471702

  8. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  9. Intracellular cytoskeletal elements and cytoskeletons in bacteria.

    PubMed

    Madkour, Mohamed H F; Mayer, Frank

    2007-01-01

    Within a short period of time after the discovery of bacterial cytoskletons, major progress had been made in areas such as general spatial layout of cytoskeletons, their involvement in a variety of cellfunctions (shape control, cell division, chromosome segregation, cell motility). This progress was achieved by application of advanced investigation techniques. Homologs of eukaryotic actin, tubulin, and intermediate filaments were found in bacteria; cytoskeletal proteins not closely or not at all related to any of these major cytoskeletal proteins were discovered in a number of bacteria such as Mycoplasmas, Spiroplasmas, Spirochetes, Treponema, Caulobacter. A structural role for bacterial elongation factor Tu was indicated. On the basis of this new thinking, new approaches in biotechnology and new drugs are on the way.

  10. Mechanical Response of Cytoskeletal Networks

    PubMed Central

    Gardel, Margaret L.; Kasza, Karen E.; Brangwynne, Clifford P.; Liu, Jiayu; Weitz, David A.

    2015-01-01

    The cellular cytoskeleton is a dynamic network of filamentous proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. However, these networks are not simple linear, elastic solids; they can exhibit highly nonlinear elasticity and athermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing complex cellular behaviors, it is necessary to understand the underlying physical principles that regulate force transmission and dynamics within these networks. In this chapter, we review our current understanding of the physics of networks of cytoskeletal proteins formed in vitro. We introduce rheology, the technique used to measure mechanical response. We discuss our current understanding of the mechanical response of F-actin networks, and how the biophysical properties of F-actin and actin cross-linking proteins can dramatically impact the network mechanical response. We discuss how incorporating dynamic and rigid microtubules into F-actin networks can affect the contours of growing microtubules and composite network rigidity. Finally, we discuss the mechanical behaviors of intermediate filaments. PMID:19118688

  11. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  12. The cytoskeletal proteins in the contractile tissues of the testis and its excurrent ducts of the passerine bird, Masked Weaver (Ploceus velatus).

    PubMed

    Ozegbe, P C; Aire, T A; Deokar, M S

    2012-02-01

    The cellular composition of the testicular capsule, seminiferous peritubular tissue, the epithelia as well as periductal muscle cell layers of the excurrent ducts was studied, in sexually mature and active Masked Weaver (Ploceus velatus) birds of the passerine family, Ploceidae. Ultrastructure of the contractile cells in the testicular capsule, peritubular and periductal tissues showed that these cells were smooth muscles of typical morphological characteristics. Variability in the immunohistochemical co-expression of microfilaments and intermediate filaments in the different tissues was evident. Actin and desmin proteins were co-expressed immunohistochemically in the testicular capsule and seminiferous peritubular smooth muscle layer. Actin was singly and very weakly expressed in the rete testis epithelium while cytokeratins and desmin were co-expressed in the epithelium of the excurrent ducts. The periductal muscle layer of all ducts of the epididymis, the ductus deferens as well as the seminal glomus, strongly co-expressed actin and desmin. Vimentin was absent in all cells and tissue types studied. There is clear evidence that the tissues of the male gonad and its excurrent ducts in the Masked Weaver, as has been reported for members of the Galloanserae and Ratitae, contain well-formed contractile tissues whose function would include the transportation of luminal through-flow from the testis into, and through, its excurrent ducts. The microtubule helix in the head and of the mid-piece, of elongating spermatids, as well as of the mature spermatozoa in the various excurrent ducts, including some spermatozoa in the seminal glomus, also co-expressed these three proteins.

  13. Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ

    PubMed Central

    Schumacher, Maria A.; Zeng, Wenjie

    2016-01-01

    Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called “nucleoid occlusion” (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator–CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA–DNA–FtsZ CTD regulatory interaction and provide insight into FtsZ–regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA–DNA–FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA–DNA–FtsZ interaction with implications for SlmA’s NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins. PMID:27091999

  14. kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin.

    PubMed

    Gregory, S L; Brown, N H

    1998-11-30

    Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.

  15. kakapo, a Gene Required for Adhesion Between and Within Cell Layers in Drosophila, Encodes a Large Cytoskeletal Linker Protein Related to Plectin and Dystrophin

    PubMed Central

    Gregory, Stephen L.; Brown, Nicholas H.

    1998-01-01

    Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000–amino acid region, and contains within this region an α-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest–specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules. PMID:9832555

  16. Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

    PubMed Central

    Álvarez-González, Begoña; Bastounis, Effie; Meili, Ruedi; del Álamo, Juan C.; Firtel, Richard; Lasheras, Juan C.

    2014-01-01

    Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper, we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during

  17. Altered expression of glomerular heat shock protein 27 in experimental nephrotic syndrome.

    PubMed Central

    Smoyer, W E; Gupta, A; Mundel, P; Ballew, J D; Welsh, M J

    1996-01-01

    Although nephrotic syndrome is a very common kidney disease, little is known about the molecular changes occurring within glomerular capillary loops during development of disease. The characteristic histologic change is retraction (effacement) of the distal "foot" processes of glomerular epithelial cells (GEC) which surround the capillary loops. The GEC foot processes are an essential part of the kidney's filtration barrier, and their structure is regulated primarily by actin microfilaments, cytoskeletal proteins present in high concentrations in foot processes. Actin polymerization has been reported to be regulated via phosphorylation of the low molecular weight heat shock protein, hsp27. We localized hsp27 within normal rat GECs using immunofluorescence and immunoelectron microscopy. Induction of nephrotic syndrome and GEC foot process effacement using the puromycin aminonucleoside rat model resulted in significant increases in: (a) renal cortical hsp27 mRNA expression (826 +/- 233%, x +/- SEM, P < 0.01 vs. control); (b) glomerular hsp27 protein expression (87 +/- 2%, P < 0.001 vs. control); and (c) glomerular hsp27 phosphorylation (101 +/- 32%, P < 0.05 vs. control). These findings support the hypothesis that hsp27, by regulating GEC foot process actin polymerization, may be important in maintaining normal foot process structure, and regulating pathophysiologic GEC cytoskeletal changes during development of nephrotic syndrome. PMID:8675679

  18. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  19. Sporadic inclusion body myositis: morphology, regeneration, and cytoskeletal structure of muscle fibres

    PubMed Central

    Arnardottir, S; Borg, K; Ansved, T

    2004-01-01

    Methods: 14 muscle biopsies from 11 patients with s-IBM were characterised for morphological abnormalities and fibre type composition as well as muscle fibre regeneration and cytoskeletal structure, using histochemical and immunohistochemical techniques. Results: Morphological abnormalities included inflammatory infiltrates and "rimmed vacuoles," and pronounced variation in fibre size. There were no significant differences in fibre type composition between s-IBM patients and controls based on the myofibrillar ATPase staining. A differential effect on muscle fibre sizes was noted, type II fibres being smaller in the s-IBM patients than in the controls. Conversely, the mean type I muscle fibre diameter in the s-IBM patients was larger than in the controls, though this difference was not significant. An ongoing intense regeneration process was present in s-IBM muscle, as indicated by the expression of neonatal myosin heavy chain, vimentin, and CD56 (Leu-19) in most of the muscle fibres. The cytoskeletal proteins dystrophin and desmin were normally expressed in s-IBM muscle fibres that were not undergoing degeneration or regeneration. Conclusions: There are extensive morphological and morphometric alterations in s-IBM, affecting different muscle fibre types in different ways. The cytoskeletal structure of type I and II muscle fibres remains unaffected in different stages of the disease. PMID:15146016

  20. Proteolysis of submembrane cytoskeletal proteins ankyrin-G and alpha-II-spectrin following diffuse brain injury: A role in white matter vulnerability at Nodes of Ranvier

    PubMed Central

    Reeves, Thomas M.; Greer, John E.; Vanderveer, Andrew S.; Phillips, Linda L.

    2012-01-01

    A high membrane-to-cytoplasm ratio makes axons particularly vulnerable to traumatic injury. Posttraumatic shifts in ionic homeostasis promote spectrin cleavage, disrupt ankyrin linkages, and destabilize axolemmal proteins. This study contrasted ankyrin-G and αII-spectrin degradation in cortex and corpus callosum following diffuse axonal injury produced by fluid percussion insult. Ankyrin-G lysis occurred preferentially in white matter, with acute elevation of all fragments and long term reduction of a low kD form. Calpain-generated αII-spectrin fragments increased in both regions. Caspase-3 lysis of αII-spectrin showed a small, acute rise in cortex, but was absent in callosum. White matter displayed nodal damage, with horseradish peroxidase permeability into the submyelin space. Ankyrin-G binding protein neurofascin and spectrin binding protein ankyrin-B showed acute alterations in expression. These results support ankyrin-G vulnerability in white matter following trauma and suggest that ankyrin-G and αII-spectrin proteolysis disrupts Node of Ranvier integrity. The time course of such changes were comparable to previously observed functional deficits in callosal fibers. PMID:20557305

  1. Changes in morphology, gene expression and protein content in chondrocytes cultured on a random positioning machine.

    PubMed

    Aleshcheva, Ganna; Sahana, Jayashree; Ma, Xiao; Hauslage, Jens; Hemmersbach, Ruth; Egli, Marcel; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

    2013-01-01

    Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β1, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  2. Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

    PubMed Central

    Aleshcheva, Ganna; Sahana, Jayashree; Ma, Xiao; Hauslage, Jens; Hemmersbach, Ruth; Egli, Marcel; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

    2013-01-01

    Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β1, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates. PMID:24244418

  3. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

  4. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  5. Espin actin-cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice-isoforms with a functional nuclear localization signal.

    PubMed

    Sekerková, Gabriella; Zheng, Lili; Mugnaini, Enrico; Bartles, James R

    2008-08-20

    The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience.

  6. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

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

    PubMed

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

    2005-01-01

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

  8. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  9. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    SciTech Connect

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-10-01

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes.

  10. The TRPM7 interactome defines a cytoskeletal complex linked to neuroblastoma progression.

    PubMed

    Middelbeek, Jeroen; Vrenken, Kirsten; Visser, Daan; Lasonder, Edwin; Koster, Jan; Jalink, Kees; Clark, Kristopher; van Leeuwen, Frank N

    2016-11-01

    Neuroblastoma is the second-most common solid tumor in children and originates from poorly differentiated neural crest-derived progenitors. Although most advanced stage metastatic neuroblastoma patients initially respond to treatment, a therapy resistant pool of poorly differentiated cells frequently arises, leading to refractory disease. A lack of insight into the molecular mechanisms that underlie neuroblastoma progression hampers the development of effective new therapies for these patients. Normal neural crest development and maturation is guided by physical interactions between the cell and its surroundings, in addition to soluble factors such as growth factors. This mechanical crosstalk is mediated by actin-based adhesion structures and cell protrusions that probe the cellular environment to modulate migration, proliferation, survival and differentiation. Whereas such signals preserve cellular quiescence in non-malignant cells, perturbed adhesion signaling promotes de-differentiation, uncontrolled cell proliferation, tissue invasion and therapy resistance. We previously reported that high expression levels of the channel-kinase TRPM7, a protein that maintains the progenitor state of embryonic neural crest cells, are closely associated with progenitor-like features of tumor cells, accompanied by extensive cytoskeletal reorganization and adhesion remodeling. To define mechanisms by which TRPM7 may contribute to neuroblastoma progression, we applied a proteomics approach to identify TRPM7 interacting proteins. We show that TRPM7 is part of a large complex of proteins, many of which function in cytoskeletal organization, cell protrusion formation and adhesion dynamics. Expression of a subset of these TRPM7 interacting proteins strongly correlates with neuroblastoma progression in independent neuroblastoma patient datasets. Thus, TRPM7 is part of a large cytoskeletal complex that may affect the malignant potential of tumor cells by regulating actomyosin dynamics

  11. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  12. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions.

    PubMed

    McReynolds, Jayme R; Donowho, Kyle; Abdi, Amin; McGaugh, James L; Roozendaal, Benno; McIntyre, Christa K

    2010-03-01

    Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a beta-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of beta-adrenoceptors with propranolol (0.5 microg/0.2 microL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA beta-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.

  13. Cytoskeletal control of plant cell shape: getting the fine points.

    PubMed

    Smith, Laurie G

    2003-02-01

    The shapes of plant cells, which are defined by their surrounding walls, are often important for cell function. The cytoskeleton plays key roles in determining plant cell shape, mainly by influencing the patterns in which wall materials are deposited in expanding cells. Studies employing cytoskeleton-disrupting drugs, together with studies of mutants with cytoskeletal defects, have demonstrated that both microtubules and actin filaments are critical for all modes of cell expansion, although their precise roles remain poorly understood. In recent years, however, significant progress has been made in understanding the contributions of a variety of proteins that influence cell shape by regulating the organization and polymerization of cytoskeletal filaments in expanding cells.

  14. Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Chang, Tsong-Min; Shi, Guey-Yueh; Wu, Hua-Lin; Wu, Chieh-Hsi; Su, Yan-Di; Wang, Hui-Lin; Wen, Hsin-Yun; Huang, Huey-Chun

    2011-01-01

    Salvianolic acid B (Sal B), a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs) were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B. PMID:21423689

  15. Design Principles of Length Control of Cytoskeletal Structures

    PubMed Central

    Mohapatra, Lishibanya; Goode, Bruce L.; Jelenkovic, Predrag; Phillips, Rob; Kondev, Jane

    2017-01-01

    Cells contain elaborate and interconnected networks of protein polymers, which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles and controls dynamic changes in cell polarity, shape, and movement. Many of these processes require tight control of the size and shape of cytoskeletal structures, which is achieved despite rapid turnover of their molecular components. Here we review mechanisms by which cells control the size of filamentous cytoskeletal structures, from the point of view of simple quantitative models that take into account stochastic dynamics of their assembly and disassembly. Significantly, these models make experimentally testable predictions that distinguish different mechanisms of length control. Although the primary focus of this review is on cytoskeletal structures, we believe that the broader principles and mechanisms discussed herein will apply to a range of other subcellular structures whose sizes are tightly controlled and are linked to their functions. PMID:27145876

  16. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  17. Protein expression profile in the striatum of rats with methamphetamine-induced behavioral sensitization.

    PubMed

    Iwazaki, Takeshi; McGregor, Iain S; Matsumoto, Izuru

    2007-04-01

    Repeated administration of methamphetamine (MAP) results in an increased behavioral response to the drug during subsequent exposure. This phenomenon is called behavioral sensitization. Sensitization is an enduring phenomenon, and suggests chronic alterations in neuronal plasticity. MAP-induced sensitization has been proposed and widely investigated as an animal model of MAP psychosis and schizophrenia. However, little is known about the molecular mechanisms underlying MAP-induced sensitization. 2-DE-based proteomics allows us to examine global changes in protein expression in complex biological systems and to propose hypotheses concerning the mechanisms underlying various pathological conditions. In the present study, we examined protein expression profiles in the striatum of MAP-sensitized rats using 2-DE-based proteomics. Repeated administration of MAP (4.0 mg/kg, once a day, intraperitoneal (i.p.)) for 10 days significantly augmented the locomotor response to an MAP challenge injection (1.0 mg/kg, i.p.) on day 11. This enhanced activity was maintained even after a week of drug abstinence. 2-DE analysis revealed 42 protein spots were differentially regulated in the striatum of MAP-sensitized rats compared to control. Thirty-one protein spots were identified using MALDI-TOF, including synapsin II, synaptosomal-associated protein 25 (SNAP-25), adenylyl cyclase-associated protein 1 (CAP1), and dihydropyrimidinase-related protein 2 (DRP2). These proteins can be related to underlying mechanisms of MAP-induced behavioral sensitization, indicating cytoskeletal modification, and altered synaptic function.

  18. The role of cytoskeletal elements in shaping bacterial cells.

    PubMed

    Cho, Hongbaek

    2015-03-01

    Beginning from the recognition of FtsZ as a bacterial tubulin homolog in the early 1990s, many bacterial cytoskeletal elements have been identified, including homologs to the major eukaryotic cytoskeletal elements (tubulin, actin, and intermediate filament) and the elements unique in prokaryotes (ParA/MinD family and bactofilins). The discovery and functional characterization of the bacterial cytoskeleton have revolutionized our understanding of bacterial cells, revealing their elaborate and dynamic subcellular organization. As in eukaryotic systems, the bacterial cytoskeleton participates in cell division, cell morphogenesis, DNA segregation, and other important cellular processes. However, in accordance with the vast difference between bacterial and eukaryotic cells, many bacterial cytoskeletal proteins play distinct roles from their eukaryotic counterparts; for example, control of cell wall synthesis for cell division and morphogenesis. This review is aimed at providing an overview of the bacterial cytoskeleton, and discussing the roles and assembly dynamics of bacterial cytoskeletal proteins in more detail in relation to their most widely conserved functions, DNA segregation and coordination of cell wall synthesis.

  19. Heterologous and cell free protein expression systems.

    PubMed

    Farrokhi, Naser; Hrmova, Maria; Burton, Rachel A; Fincher, Geoffrey B

    2009-01-01

    In recognition of the fact that a relatively small percentage of 'named' genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

  20. Neuronal Expression of Muscle LIM Protein in Postnatal Retinae of Rodents

    PubMed Central

    Levin, Evgeny; Leibinger, Marco; Andreadaki, Anastasia; Fischer, Dietmar

    2014-01-01

    Muscle LIM protein (MLP) is a member of the cysteine rich protein family and has so far been regarded as a muscle-specific protein that is mainly involved in myogenesis and the organization of cytoskeletal structure in myocytes, respectively. The current study demonstrates for the first time that MLP expression is not restricted to muscle tissue, but is also found in the rat naive central nervous system. Using quantitative PCR, Western blot and immunohistochemical analyses we detected MLP in the postnatal rat retina, specifically in the somas and dendritic arbors of cholinergic amacrine cells (AC) of the inner nuclear layer and the ganglion cell layer (displaced AC). Induction of MLP expression started at embryonic day 20 and peaked between postnatal days 7 and 14. It subsequently decreased again to non-detectable protein levels after postnatal day 28. MLP was identified in the cytoplasm and dendrites but not in the nucleus of AC. Thus, retinal MLP expression correlates with the morphologic and functional development of cholinergic AC, suggesting a potential role of this protein in postnatal maturation and making MLP a suitable marker for these neurons. PMID:24945278

  1. Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

    PubMed Central

    Morgan, Joshua T.; Pfeiffer, Emily R.; Thirkill, Twanda L.; Kumar, Priyadarsini; Peng, Gordon; Fridolfsson, Heidi N.; Douglas, Gordon C.; Starr, Daniel A.; Barakat, Abdul I.

    2011-01-01

    Changes in blood flow regulate gene expression and protein synthesis in vascular endothelial cells, and this regulation is involved in the development of atherosclerosis. How mechanical stimuli are transmitted from the endothelial luminal surface to the nucleus is incompletely understood. The linker of nucleus and cytoskeleton (LINC) complexes have been proposed as part of a continuous physical link between the plasma membrane and subnuclear structures. LINC proteins nesprin-1, -2, and -4 have been shown to mediate nuclear positioning via microtubule motors and actin. Although nesprin-3 connects intermediate filaments to the nucleus, no functional consequences of nesprin-3 mutations on cellular processes have been described. Here we show that nesprin-3 is robustly expressed in human aortic endothelial cells (HAECs) and localizes to the nuclear envelope. Nesprin-3 regulates HAEC morpho­logy, with nesprin-3 knockdown inducing prominent cellular elongation. Nesprin-3 also organizes perinuclear cytoskeletal organization and is required to attach the centrosome to the nuclear envelope. Finally, nesprin-3 is required for flow-induced polarization of the centrosome and flow-induced migration in HAECs. These results represent the most complete description to date of nesprin-3 function and suggest that nesprin-3 regulates vascular endothelial cell shape, perinuclear cytoskeletal architecture, and important aspects of flow-mediated mechanotransduction. PMID:21937718

  2. Proteomic analysis reveals differentially expressed proteins in the rat frontal cortex after methamphetamine treatment.

    PubMed

    Faure, J J; Hattingh, S M; Stein, D J; Daniels, W M

    2009-12-01

    Methamphetamine (MA) is an addictive psycho-stimulant and the illicit use of the drug is escalating. In the present study, we examined protein expression profiles in the rat frontal cortex exposed to a total of eight MA injections (1 mg/kg, intraperitoneal) using 2-DE based proteomics. We investigated protein changes occurring in both the cytosolic fraction and the membrane fraction. 2-DE analysis resulted in 62 cytosolic and 44 membrane protein spots that were differentially regulated in the frontal cortex of rats exposed to MA when compared to control animals. Of these spots, 47 cytosolic and 42 membrane proteins were identified respectively, using ESI-Quad-TOF, which included ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), beta-synuclein, 78 kDa glucose-regulated protein (GRP 78), gamma-enolase, dihydropyrimidase-related protein 2 (DRP 2), complexin 2 and synapsin II. These proteins are associated with protein degradation, redox regulation, energy metabolism, cellular growth, cytoskeletal modifications and synaptic function. Proteomic research may be useful in exploring the complex underlying molecular mechanisms of MA dependence.

  3. Solution NMR structures of the C-domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium-induced structural rearrangements.

    PubMed

    Kilpatrick, Adina M; Honts, Jerry E; Sleister, Heidi M; Fowler, C Andrew

    2016-11-01

    Tcb2 is a calcium-binding protein that localizes to the membrane-associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium-triggered, ATP-independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2's structure-function relationship and contractile properties, we determined solution NMR structures of its C-terminal domain in the calcium-free and calcium-bound states. The overall architecture is similar to other calcium-binding proteins, with paired EF-hand calcium-binding motifs. Comparison of the two structures reveals that Tcb2-C's calcium-induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full-length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748-1756. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Structure of Cytoskeletal Supramolecular Assemblies in the Nerve Cell Axon

    NASA Astrophysics Data System (ADS)

    Ojeda-López, Miguel A.; Case, Ryan; Miller, Herb P.; Wilson, Leslie; Safinya, Cyrus R.

    2001-03-01

    The cytoskeleton of eucaryotic cells is an intricate network of supramolecular assemblies of protein filaments, e.g., actin, intermediate filaments (IFs), tubulin, and a multi-associated family of cross-linking proteins. Most of its multiple functions rely on its structural stability, which depends on a variety of specific interactions of the subunit proteins and its local physico-chemical environment. In neurodegenerative diseases the cytoskeletal supramolecular structure is almost universally altered. Preliminary results on the supramolecular structure of cytoskeletal filaments in isolated axons from bovine white matter will be presented as obtained using synchrotron small angle x-ray diffraction. These results will be compared to our ongoing cell-free studies on the structures formed by neurofilaments in vitro. Supported by NIH GM59288, NSF-DMR-9972246, and University of California Biotechnology Grant 99-14 & UC.

  5. Cytoskeletal reorganizations in human umbilical vein endothelial cells as a result of cytokine exposure

    SciTech Connect

    Molony, L.; Armstrong, L. )

    1991-09-01

    Treatment of HUVECs in culture with several cytokines and phorbol esters caused reorganizations of the actin and microtubule networks, as well as a redistribution of focal contract proteins. However, expression of the cytoskeletal proteins which link cells, via integrins, to the substrate, was not significantly affected. Indirect immunofluorescence microscopy of endothelial cells after treatment with interleukin-1 alpha and beta, gamma-interferon, tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, and phorbol 12,13-dibutyrate allowed us to observe reductions in the areas of cell-cell contact, redistribution of the stress fiber network, and concomitant changes in focal contacts. Microtubule arrays in TNF-treated cells became bundled. Phorbol esters induced formation of microtubule organizing centers not seen in resting or TNF-treated HUVECs. Talin was distributed along stress fibers and not exclusively in focal contacts. Vitronectin receptor was observed in focal contacts, occasionally at cell-cell contacts, and in vesicular structures close to the lumenal surface, after both types of treatment. Although these morphological changes were easily observed by indirect immunofluorescence, no quantitative differences in specific cytoskeletal proteins were detected by immunoblots and (35S)cysteine metabolic labeling experiments.

  6. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

    PubMed Central

    Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T.; Martin, James F.

    2015-01-01

    The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair. PMID:25943351

  7. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice.

    PubMed

    Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T; Martin, James F

    2015-05-05

    The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex, a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near the scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation, after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling. Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to mechanical changes associated with heart injury to promote repair. Copyright © 2015, American Association for the Advancement of Science.

  8. Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation.

    PubMed

    Shojaei Saadi, Habib A; van Riemsdijk, Evine; Dance, Alysha L; Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

    2013-04-26

    The objective was to investigate expression patterns of proteins in pyriform sperm, a common morphological abnormality in bull sperm. Ejaculates were collected from sexually mature Holstein bulls (n=3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during week 2. Total sperm proteins were extracted from pre- and post-thermal insult sperm samples and subjected to two-dimensional gel electrophoresis. Among the protein spots detected, 131 spots were significantly expressed (False Detection Rate <0.01) with ≥ 2 fold changes between normal and pyriform sperm. Among them, 25 spots with ≥ 4 fold difference in expression patterns were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins regulating antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. To our knowledge, this study is the first report on differential expression of proteins in pyriform bovine sperm versus morphologically normal sperm. We report that expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins which regulate antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding

  9. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  10. Expression of clock proteins in developing tooth.

    PubMed

    Zheng, Li; Papagerakis, Silvana; Schnell, Santiago D; Hoogerwerf, Willemijntje A; Papagerakis, Petros

    2011-01-01

    Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  12. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  13. Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

    PubMed

    Evans, S S; Schleider, D M; Bowman, L A; Francis, M L; Kansas, G S; Black, J D

    1999-03-15

    L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

  14. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  15. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  16. Actin cytoskeletal defects in immunodeficiency

    PubMed Central

    Moulding, Dale A; Record, Julien; Malinova, Dessislava; Thrasher, Adrian J

    2013-01-01

    The importance of the cytoskeleton in mounting a successful immune response is evident from the wide range of defects that occur in actin-related primary immunodeficiencies (PIDs). Studies of these PIDs have revealed a pivotal role for the actin cytoskeleton in almost all stages of immune system function, from hematopoiesis and immune cell development, through to recruitment, migration, intercellular and intracellular signaling, and activation of both innate and adaptive immune responses. The major focus of this review is the immune defects that result from mutations in the Wiskott-Aldrich syndrome gene (WAS), which have a broad impact on many different processes and give rise to clinically heterogeneous immunodeficiencies. We also discuss other related genetic defects and the possibility of identifying new genetic causes of cytoskeletal immunodeficiency. PMID:24117828

  17. Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

    PubMed Central

    Schindl, M; Wallraff, E; Deubzer, B; Witke, W; Gerisch, G; Sackmann, E

    1995-01-01

    Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex. Images FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 FIGURE 12 PMID:7756537

  18. Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells.

    PubMed

    Flaskos, J; Nikolaidis, E; Harris, W; Sachana, M; Hargreaves, A J

    2011-11-01

    Previous work in our laboratory has shown that sub-lethal concentrations (1-10 μM) of chlorpyrifos (CPF), diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon (CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions similar to our previous work, sub-lethal concentrations (1-10 μM) of CPO were found to inhibit N2a cell differentiation. However, unlike previous studies with DZ and DZO, there was a high level of sustained inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly, further work will help to determine whether the persistent inhibition of AChE by CPO can account for the different effects induced by CPO and DZO on the levels of total and phosphorylated NFH.

  19. An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo

    SciTech Connect

    Jeffery, W.R. )

    1990-09-01

    Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by (35S)methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg.

  20. N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases.

    PubMed Central

    Miki, H; Miura, K; Takenawa, T

    1996-01-01

    Here we identify a 65 kDa protein (N-WASP) from brain that binds the SH3 domains of Ash/Grb2. The sequence is homologous to Wiskott-Aldrich syndrome protein (WASP). N-WASP has several functional motifs, such as a pleckstrin homology (PH) domain and cofilin-homologous region, through which N-WASP depolymerizes actin filaments. When overexpressed in COS 7 cells, the wild-type N-WASP causes several surface protrusions where N-WASP co-localizes with actin filaments. Epidermal growth factor (EGF) treatment induces the complex formation of EGF receptors and N-WASP, and produces microspikes. On the other hand, two mutants, C38W (a point mutation in the PH domain) and deltaVCA (deletion of the actin binding domain), localize predominantly in the nucleus and do not cause a change in the cytoskeleton, irrespective of EGF treatment. Interestingly, the C38W PH domain binds less effectively to phosphatidylinositol 4,5-bisphosphate (PIP2) than the wild-type PH domain. These results suggest the importance of the PIP2 binding ability of the PH domain and the actin binding for retention in membranes. Collectively, we conclude that N-WASP transmits signals from tyrosine kinases to cause a polarized rearrangement of cortical actin filaments dependent on PIP2. Images PMID:8895577

  1. Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

    PubMed

    Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

    2015-10-01

    Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

  2. Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin

    PubMed Central

    Govind, Sheila; Kozma, Robert; Monfries, Clinton; Lim, Louis; Ahmed, Sohail

    2001-01-01

    Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin. PMID:11157984

  3. Transcriptional networks regulating the costamere, sarcomere, and other cytoskeletal structures in striated muscle.

    PubMed

    Estrella, Nelsa L; Naya, Francisco J

    2014-05-01

    Structural abnormalities in striated muscle have been observed in numerous transcription factor gain- and loss-of-function phenotypes in animal and cell culture model systems, indicating that transcription is important in regulating the cytoarchitecture. While most characterized cytoarchitectural defects are largely indistinguishable by histological and ultrastructural criteria, analysis of dysregulated gene expression in each mutant phenotype has yielded valuable information regarding specific structural gene programs that may be uniquely controlled by each of these transcription factors. Linking the formation and maintenance of each subcellular structure or subset of proteins within a cytoskeletal compartment to an overlapping but distinct transcription factor cohort may enable striated muscle to control cytoarchitectural function in an efficient and specific manner. Here we summarize the available evidence that connects transcription factors, those with established roles in striated muscle such as MEF2 and SRF, as well as other non-muscle transcription factors, to the regulation of a defined cytoskeletal structure. The notion that genes encoding proteins localized to the same subcellular compartment are coordinately transcriptionally regulated may prompt rationally designed approaches that target specific transcription factor pathways to correct structural defects in muscle disease.

  4. α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

    PubMed Central

    Sousa, Vítor L.; Bellani, Serena; Giannandrea, Maila; Yousuf, Malikmohamed; Valtorta, Flavia; Meldolesi, Jacopo

    2009-01-01

    The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration. PMID:19553474

  5. Pattern formation in actin gels: A study in the mechanics of gels formed by the important cytoskeletal protein actin, especially as applied to cellular motility

    NASA Astrophysics Data System (ADS)

    Balter, Ariel

    We have studied pattern formation in actin gels to better understand how they function in biological systems, especially in the motility mechanism used by some pathogenic bacteria such as Listeria. By coating themselves with certain enzymes, these bacteria appropriate actin (a protein) from the surrounding host cell's cytoplasm and cause a network or "gel" of actin filaments to grow on their outer surface. As the resulting "comet tail" shaped protrusion grows, it pushes the bacterium away. In experiments, polystyrene beads coated with the same enzymes will also generate comet tails and swim in a very similar manner. However, these bead experiments have also generated anomalous results such as the formation of many comet tails. In some experiments, when two comet tails formed they systematically grew into regular, oppositely handed helices. The formation of any comet tails on a bead poses a physical conundrum. The bacterial enzyme coating is asymmetrical so the comet tail forms in a particular place. But the beads are symmetrical, so comet tails formation constitutes symmetry breaking and spontaneous pattern formation. We have modeled this process as a competition between elastic energy (which favors many tails) and chemical energy (which favors few tails). Our analytical model explains the factors that experimentally determine the number of tails, and numerical simulations confirm these predictions. To understand the helical tails, we did extensive data analysis involving image processing, statistical analysis and mathematical modeling of images of the helical tails. We identified some important features of how the twin tails form. For instance, the tail growth rate is independent of drag force, and bead rotation must accompany helical tail formation. We also created a physical model for helical growth. Numerical simulations of our model show that at very low Reynolds number, a cylindrical object growing under the conditions of an actin comet tail can spontaneously

  6. Neurotrophic effects of GnRH on neurite outgrowth and neurofilament protein expression in cultured cerebral cortical neurons of rat embryos.

    PubMed

    Quintanar, J Luis; Salinas, Eva

    2008-06-01

    The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.

  7. ESPRESSO: a system for estimating protein expression and solubility in protein expression systems.

    PubMed

    Hirose, Shuichi; Noguchi, Tamotsu

    2013-05-01

    Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we present a computational approach to estimate the probability of protein expression and solubility for two different protein expression systems: in vivo Escherichia coli and wheat germ cell-free, from only the sequence information. It implements two kinds of methods: a sequence/predicted structural property-based method that uses both the sequence and predicted structural features, and a sequence pattern-based method that utilizes the occurrence frequencies of sequence patterns. In the benchmark test, the proposed methods obtained F-scores of around 70%, and outperformed publicly available servers. Applying the proposed methods to genomic data revealed that proteins associated with translation or transcription have a strong tendency to be expressed as soluble proteins by the in vivo E. coli expression system. The sequence pattern-based method also has the potential to indicate a candidate region for modification, to increase protein solubility. All methods are available for free at the ESPRESSO server (http://mbs.cbrc.jp/ESPRESSO). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita

    PubMed Central

    Kopecki, Zlatko; Ludwig, Ralf J.; Cowin, Allison J.

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

  9. Spectraplakins: Master orchestrators of cytoskeletal dynamics

    PubMed Central

    Suozzi, Kathleen C.; Wu, Xiaoyang

    2012-01-01

    The dynamics of different cytoskeletal networks are coordinated to bring about many fundamental cellular processes, from neuronal pathfinding to cell division. Increasing evidence points to the importance of spectraplakins in integrating cytoskeletal networks. Spectraplakins are evolutionarily conserved giant cytoskeletal cross-linkers, which belong to the spectrin superfamily. Their genes consist of multiple promoters and many exons, yielding a vast array of differential splice forms with distinct functions. Spectraplakins are also unique in their ability to associate with all three elements of the cytoskeleton: F-actin, microtubules, and intermediate filaments. Recent studies have begun to unveil their role in a wide range of processes, from cell migration to tissue integrity. PMID:22584905

  10. Cytoskeletal and membrane dynamics during higher plant cytokinesis.

    PubMed

    McMichael, Colleen M; Bednarek, Sebastian Y

    2013-03-01

    Following mitosis, cytoplasm, organelles and genetic material are partitioned into daughter cells through the process of cytokinesis. In somatic cells of higher plants, two cytoskeletal arrays, the preprophase band and the phragmoplast, facilitate the positioning and de novo assembly of the plant-specific cytokinetic organelle, the cell plate, which develops across the division plane and fuses with the parental plasma membrane to yield distinct new cells. The coordination of cytoskeletal and membrane dynamics required to initiate, assemble and shape the cell plate as it grows toward the mother cell cortex is dependent upon a large array of proteins, including molecular motors, membrane tethering, fusion and restructuring factors and biosynthetic, structural and regulatory elements. This review focuses on the temporal and molecular requirements of cytokinesis in somatic cells of higher plants gleaned from recent studies using cell biology, genetics, pharmacology and biochemistry.

  11. Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda)

    PubMed Central

    Cortés, Alba; Sotillo, Javier; Muñoz-Antoli, Carla; Fried, Bernard; Esteban, J. Guillermo; Toledo, Rafael

    2015-01-01

    Background Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. Methodology/Principal Findings To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. Conclusion/Significance Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection. PMID:26390031

  12. Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda).

    PubMed

    Cortés, Alba; Sotillo, Javier; Muñoz-Antoli, Carla; Fried, Bernard; Esteban, J Guillermo; Toledo, Rafael

    2015-01-01

    Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection.

  13. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  14. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    PubMed

    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.

  15. EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.

    PubMed

    Katschnig, A M; Kauer, M O; Schwentner, R; Tomazou, E M; Mutz, C N; Linder, M; Sibilia, M; Alonso, J; Aryee, D N T; Kovar, H

    2017-07-03

    Ewing sarcoma (EWS) is a paediatric bone cancer with high metastatic potential. Cellular plasticity resulting from dynamic cytoskeletal reorganization, typically regulated via the Rho pathway, is a prerequisite for metastasis initiation. Here, we interrogated the role of the Ewing sarcoma driver oncogene EWS-FLI1 in cytoskeletal reprogramming. We report that EWS-FLI1 strongly represses the activity of the Rho-F-actin signal pathway transcriptional effector MRTFB, affecting the expression of a large number of EWS-FLI1-anticorrelated genes including structural and regulatory cytoskeletal genes. Consistent with this finding, chromatin immunoprecipitation sequencing (ChIP-seq) revealed strong overlaps in myocardin-related transcription factor B (MRTFB) and EWS-FLI1 chromatin occupation, especially for EWS-FLI1-anticorrelated genes. Binding of the transcriptional co-activator Yes-associated protein (YAP)-1, enrichment of TEAD-binding motifs in these shared genomic binding regions and overlapping transcriptional footprints of MRTFB and TEAD factors led us to propose synergy between MRTFB and the YAP/TEAD complex in the regulation of EWS-FLI1-anticorrelated genes. We propose that EWS-FLI1 suppresses the Rho-actin pathway by perturbation of a MRTFB/YAP-1/TEAD transcriptional module, which directly affects the actin-autoregulatory feedback loop. As spontaneous fluctuations in EWS-FLI1 levels of Ewing sarcoma cells in vitro and in vivo, associated with a switch between a proliferative, non-migratory EWS-FLI1-high and a non-proliferative highly migratory EWS-FLI1-low state, were recently described, our data provide a mechanistic basis for the underlying EWS-FLI1-dependent reversible cytoskeletal reprogramming of Ewing sarcoma cells.Oncogene advance online publication, 3 July 2017; doi:10.1038/onc.2017.202.

  16. c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts.

    PubMed

    Hesketh, J E; Campbell, G P; Whitelaw, P F

    1991-03-01

    3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

  17. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  18. MIEN1 drives breast tumor cell migration by regulating cytoskeletal-focal adhesion dynamics

    PubMed Central

    Van Treuren, Timothy; Vishwanatha, Jamboor K.

    2016-01-01

    Migration and invasion enhancer 1 (MIEN1) is an important regulator of cell migration and invasion. MIEN1 overexpression represents an oncogenic event that promotes tumor cell dissemination and metastasis. The underlying mechanism by which MIEN1 regulates migration and invasion has yet to be deciphered. Here, we demonstrate that MIEN1 acts as a cytoskeletal-signaling adapter protein to drive breast cancer cell migration. MIEN1 localization is concentrated underneath the actin-enriched protrusive structures of the migrating breast cancer cells. Depletion of MIEN1 led to the loss of actin-protrusive structures whereas the over-expression of MIEN1 resulted in rich and thick membrane extensions. Knockdown of MIEN1 also decreased the cell-substratum adhesion, suggesting a role for MIEN1 in actin cytoskeletal dynamics. Our results show that MIEN1 supports the transition of G-actin to F-actin polymerization and stabilizes F-actin polymers. Additionally, MIEN1 promotes cellular adhesion and actin dynamics by inducing phosphorylation of FAK at Tyr-925 and reducing phosphorylation of cofilin at Ser-3, which results in breast cancer cell migration. Collectively, our data show that MIEN1 plays an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, which leads to an increase in cell motility. Hence, targeting MIEN1 might represent a promising means to prevent breast tumor metastasis. PMID:27462783

  19. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  20. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  1. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  2. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

    PubMed

    Moll, R; Schiller, D L; Franke, W W

    1990-08-01

    A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

  3. MicroRNA-203 mimics age-related aortic smooth muscle dysfunction of cytoskeletal pathways.

    PubMed

    Nicholson, Christopher J; Seta, Francesca; Lee, Sophie; Morgan, Kathleen G

    2017-01-01

    Increased aortic stiffness is a biomarker for subsequent adverse cardiovascular events. We have previously reported that vascular smooth muscle Src-dependent cytoskeletal remodelling, which contributes to aortic plasticity, is impaired with ageing. Here, we use a multi-scale approach to determine the molecular mechanisms behind defective Src-dependent signalling in an aged C57BL/6 male mouse model. Increased aortic stiffness, as measured in vivo by pulse wave velocity, was found to have a comparable time course to that in humans. Bioinformatic analyses predicted several miRs to regulate Src-dependent cytoskeletal remodelling. qRT-PCR was used to determine the relative levels of predicted miRs in aortas and, notably, the expression of miR-203 increased almost twofold in aged aorta. Increased miR-203 expression was associated with a decrease in both mRNA and protein expression of Src, caveolin-1 and paxillin in aged aorta. Probing with phospho-specific antibodies confirmed that overexpression of miR-203 significantly attenuated Src and extracellular signal regulated kinase (ERK) signalling, which we have previously found to regulate vascular smooth muscle stiffness. In addition, transfection of miR-203 into aortic tissue from young mice increased phenylephrine-induced aortic stiffness ex vivo, mimicking the aged phenotype. Upstream of miR-203, we found that DNA methyltransferases (DNMT) 1, 3a, and 3b are also significantly decreased in the aged mouse aorta and that DNMT inhibition significantly increases miR-203 expression. Thus, the age-induced increase in miR-203 may be caused by epigenetic promoter hypomethylation in the aorta. These findings indicate that miR-203 promotes a re-programming of Src/ERK signalling pathways in vascular smooth muscle, impairing the regulation of stiffness in aged aorta. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  4. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization.

    PubMed

    Hummel, T; Leifker, K; Klämbt, C

    2000-04-01

    In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding.

  5. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  6. Effects of typical (haloperidol) and atypical (risperidone) antipsychotic agents on protein expression in rat neural stem cells.

    PubMed

    Kashem, Mohammed A; Ummehany, Rahnuma; Ukai, Wataru; Hashimoto, Eri; Saito, Toshikazu; Mcgregor, Iain S; Matsumoto, Izuru

    2009-12-01

    Neural stem cells (NSCs) play a crucial role in the development and maturation of the central nervous system. Recently studies suggest that antipsychotic drugs regulate the activities of NSCs. However, the molecular mechanisms underlying antipsychotic-induced changes of the activity of NSCs, particularly protein expression, are still unknown. We studied the growth and protein expression in haloperidol (HD) and risperidone (RS) treated rat NSCs. The treatment with RS (3microM) or HD (3microM) had no effect on morphology of NSCs after 24h, but significantly promotes or inhibits the differentiation of NSCs after a 96h of treatment. 2-DE based proteomics was performed at 24h, a stage before phenotypic expression of NSCs. Gel image analysis revealed that 30 protein spots in HD- and 60 spots in RS-treated groups were differentially regulated in their expression compared to control group (p<0.05; ANOVA). When these spots were compared between the two drug-treated groups, 23 spots overlapped leaving 7 HD-specific and 37 RS-specific spots. Of these 67 spots, 32 different proteins were identified. The majority of the differentially regulated proteins were classified into several functional groups, such as cytoskeletal, calcium regulating protein, metabolism, signal transduction and proteins related to oxidative stress. Our data shows that atypical RS expressed more proteins than typical HD, and these results might explain the molecular mechanisms underlying the different effects of both drugs on NSCs activities as described above. Identified proteins in this experiment may be useful in future studies of NSCs differentiation and/or understanding in molecular mechanisms of different neural diseases including schizophenia.

  7. Androgen Control of Cell Proliferation and Cytoskeletal Reorganization in Human Fibrosarcoma Cells

    PubMed Central

    Chauhan, Sanjay; Kunz, Susan; Davis, Kelli; Roberts, Jordan; Martin, Greg; Demetriou, Manolis C.; Sroka, Thomas C.; Cress, Anne E.; Miesfeld, Roger L.

    2009-01-01

    We recently generated an HT-1080-derived cell line called HT-AR1 that responds to dihydrotestosterone (DHT) treatment by undergoing cell growth arrest in association with cytoskeletal reorganization and induction of neuroendocrine-like cell differentiation. In this report, we show that DHT induces a dose-dependent increase in G0/G1 growth-arrested cells using physiological levels of hormone. The arrested cells increase in cell size and contain a dramatic redistribution of desmoplakin, keratin 5, and chromogranin A proteins. DHT-induced cytoskeletal changes were also apparent from time lapse video microscopy that showed that androgen treatment resulted in the rapid appearance of neuronal-like membrane extensions. Expression profiling analysis using RNA isolated from DHT-treated HT-AR1 cells revealed that androgen receptor activation leads to the coordinate expression of numerous cell signaling genes including RhoB, PTGF-β, caveolin-2, Egr-1, myosin 1B, and EHM2. Because RhoB has been shown to have a role in tumor suppression and neuronal differentiation in other cell types, we investigated RhoB signaling functions in the HT-AR1 steroid response. We found that steroid induction of RhoB was DHT-specific and that newly synthesized RhoB protein was post-translationally modified and localized to endocytic vesicles. Moreover, treatment with a farnesyl transferase inhibitor reduced DHT-dependent growth arrest, suggesting that prenylated RhoB might function to inhibit HT-AR1 cell proliferation. This was directly shown by transfecting HT-AR1 cells with RhoB coding sequences containing activating or dominant negative mutations. PMID:14576147

  8. Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor.

    PubMed

    Grasis, Juris A; Browne, Cecille D; Tsoukas, Constantine D

    2003-04-15

    The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.

  9. Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

    PubMed

    Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

    2017-01-01

    The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

  10. The correlation between morphology and the expression of TGF-β signaling pathway proteins and epithelial-mesenchymal transition-related proteins in synovial sarcomas

    PubMed Central

    Qi, Yan; Wang, Cui-Cui; He, Yong-Lai; Zou, Hong; Liu, Chun-Xia; Pang, Li-Juan; Hu, Jian-Ming; Jiang, Jin-fang; Zhang, Wen-Jie; Li, Feng

    2013-01-01

    Synovial sarcoma (SS) is a malignant tumor of soft tissue and is noted for late local recurrence and metastasis. Aberrant epithelial-mesenchymal transition (EMT) has been implicated in the pathogenesis of diverse human malignancies. Immunohistochemical techniques were used to assess EMT-related proteins (E-cadherin, N-cadherin, β-catenin, Snail, and Slug) and the TGF-β1 pathway (TGF-β1 and Smad2/3) proteins expression in different histological subtypes and epithelial mesenchymal compositions of SS. The expression of cell-surface (E-cadherin) and cytoskeletal proteins (β-catenin) were higher significantly in biphasic SSs (BSSs) (70.4%, 51.9%) than MFSSs (both for 10%). Among monophasic fibrous SSs (MFSSs) samples, E-cadherin protein expression was negatively correlated with expression Snail, Slug, TGF-β1, and Smad2/3. The expression levels of Snail and Smad2/3 were correlated with the pTNM stage (I-II vs. III-IV; P=0.047, P=0.021) and TGF-β1 exhibited a tendency toward a positive correlation with pTNM stage (I-II vs. III-IV; P=0.052), but did not correlate with the histological grade (p>0.05). Interestingly, our data showed that expression of E-cadherin protein correlated with greater survival in SS patients. Overexpression of Snail, and TGF-β1 is associated with suppressed expression of E-cadherin in MFSSs, which supports the hypothesis that the MFSS subtype may have developed via neoplastic EMT. PMID:24294365

  11. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  12. Supramolecular Assembly in Cytoskeletal Filaments and their Associated Biomolecules

    NASA Astrophysics Data System (ADS)

    Safinya, Cyrus R.

    2002-03-01

    With the completion of the Human Genome Project and the emerging proteomics era, the biosciences community is beginning the daunting task of understanding the functions of a large number of interacting proteins. Cellular activity, which is usually tightly regulated, results from protein-protein and protein-nucleic acid interactions, which often lead to the formation of very large assemblies of biomolecules for distinct functions. Examples include DNA condensation states during the cell cycle, and bundle and network formation of filamentous proteins in cell attachment, motility, and cytokinesis. We present recent synchrotron x-ray diffraction and optical imaging data, in cell-free systems of cytoskeletal filaments and their associated biomolecules, which reveal novel supramolecular assemblies, spanning lengths from the nanometer to the micrometer scale. Supported by NSF DMR-9972246 and NIH GM59288.

  13. Biophysical models of length control of cytoskeletal structures

    NASA Astrophysics Data System (ADS)

    Mohapatra, Lishibanya

    Cells contain elaborate and interconnected networks of protein polymers which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles, and controls dynamic changes in cell polarity, shape and movement. Many of these processes require tight control of the size and shape of these cytoskeletal structures. A key question in cell biology is how these structures maintain a particular size and shape despite the rapid turnover of their components. In this thesis I show that the emerging mechanisms by which cells control and regulate the size of filamentous cytoskeletal structures can be classified using key parameters related to their assembly and disassembly kinetics. First, I examine quantitative models based on these specific molecular mechanisms of length control and make experimentally testable predictions that can be used to distinguish different mechanisms of length-control. Second, I study the length control of actin cables in budding yeast cells. Inspired by recent experimental observations in cells, I propose a novel antenna mechanism for cable length control which involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. My results provide testable predictions of the antenna mechanism of actin-cable length control. Next I consider the question of how different sized structures can co-exist in the same cytoplasm while making use of the same building blocks. Using theory, I discover limitations imposed by physics on the finite monomer pool as a mechanism of size control and conclude that additional length control mechanisms are required if a cell is to maintain multiple structures. While the primary focus of this thesis is on cytoskeletal structures, the broader principles and mechanisms discussed herein will apply to a range of

  14. Cell cytoskeletal conformation under reversible thermal control

    NASA Astrophysics Data System (ADS)

    Chang, Ting-Ya; Yang, Chung-Yao; Liao, Kai-Wei; Andrew Yeh, J.; Cheng, Chao-Min

    2013-12-01

    In order to assess the role of cytoskeletal structure in modulating cell surface topography during cell transformation, we investigated cytoskeletal organization of Madin-Darby canine kidney (MDCK) epithelial cells at different thermal gradients. Specifically, we examined actin polymerization as a function of temperature in a controlled thermal environment. After applying an increase in temperature of 5 °C, we observed fewer actin filaments in the network, as these molecular polymers depolymerized. Partial stress fibers of MDCK cells could be rearranged, but some of them were disrupted irreversibly after a second thermal treatment, and MDCK cells underwent apoptosis at higher temperatures as well.

  15. Kidney keratins: cytoskeletal stress responders with biomarker potential.

    PubMed

    Snider, Natasha T

    2016-04-01

    Keratins are cytoskeletal filamentous proteins that support the structural integrity of epithelial cells. Deficiency of the major simple epithelial keratins K8, K18, and K19 increases susceptibility to hepatobiliary injury, but keratin function in kidney injury has not been addressed. Djudjaj et al. examined renal keratins in health and disease, in both mice and humans. Their findings lay the foundation for pursuing keratins as markers and regulators of renal tubular epithelial injury. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  16. Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

    PubMed Central

    Menon, Shalini; Gupton, Stephanie L.

    2016-01-01

    Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development. PMID:26940519

  17. Measurement of Force-Dependent Release Rates of Cytoskeletal Motors.

    PubMed

    Can, Sinan; Yildiz, Ahmet

    2017-01-01

    Optical tweezers permit measuring motor-filament rupture forces with piconewton sensitivity. For deeper structural and mechanistic understanding of motors, different structural constraints can be induced by pulling motor proteins at various positions and manipulating the direction of the exerted force. Here, we present an optical-trapping approach to investigate the effect of the magnitude and direction of tension applied to the linker element of cytoskeletal motors on motor-filament interactions. Using this approach, force-dependent microtubule release rates of monomeric kinesins can be directly measured by pulling on kinesin's "neck linker" with a constant force.

  18. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  19. Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization.

    PubMed

    Miki, H; Takenawa, T

    1998-02-04

    Verprolin is a yeast protein whose inactivation leads to a cytoskeletal defect characterized by the abnormal organization of actin filaments. Recently, two mammalian proteins previously shown to regulate the actin cytoskeleton, Wiskott-Aldrich Syndrome Protein (WASP) and its homolog expressed in neurons (N-WASP), were found to possess short peptide motifs homologous to one part of verprolin. However, the physiological function of the homologous regions (verprolin-homology domain, VPH domain) remains unknown. Here we report the importance of the VPH domain as the direct actin binding region. In the case of N-WASP, the VPH domain co-acts with the cofilinhomologous region to sever actin filaments in vitro. Furthermore, the VPH domain is indispensable for the reorganization of the actin cytoskeleton by N-WASP downstream of tyrosine kinases in living cells. All data demonstrate that the VPH domain plays critical roles in the regulation of the actin cytoskeleton.

  20. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  1. Altered cytoskeletal organization characterized lethal but not surviving Brtl+/− mice: insight on phenotypic variability in osteogenesis imperfecta

    PubMed Central

    Bianchi, Laura; Gagliardi, Assunta; Maruelli, Silvia; Besio, Roberta; Landi, Claudia; Gioia, Roberta; Kozloff, Kenneth M.; Khoury, Basma M.; Coucke, Paul J.; Symoens, Sofie; Marini, Joan C.; Rossi, Antonio; Bini, Luca; Forlino, Antonella

    2015-01-01

    Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl+/− to investigate the molecular basis of OI phenotypic variability. Brtl+/− resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl+/− mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl+/− lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment. PMID:26264579

  2. CONSERVED ROLES FOR CYTOSKELETAL COMPONENTS IN DETERMINING LATERALITY

    PubMed Central

    McDowell, Gary S.; Lemire, Joan M.; Paré, Jean-Francois; Cammarata, Garrett; Lowery, Laura Anne; Levin, Michael

    2016-01-01

    SUMMARY Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently “rescued” by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking

  3. Length-Dependent Modulation of Cytoskeletal Remodeling and Mechanical Energetics in Airway Smooth Muscle

    PubMed Central

    Kim, Hak Rim; Liu, Katrina; Roberts, Thomas J.; Hai, Chi-Ming

    2011-01-01

    Actin cytoskeletal remodeling is an important mechanism of airway smooth muscle (ASM) contraction. We tested the hypothesis that mechanical strain modulates the cholinergic receptor–mediated cytoskeletal recruitment of actin-binding and integrin-binding proteins in intact airway smooth muscle, thereby regulating the mechanical energetics of airway smooth muscle. We found that the carbachol-stimulated cytoskeletal recruitment of actin-related protein-3 (Arp3), metavinculin, and talin were up-regulated at short muscle lengths and down-regulated at long muscle lengths, suggesting that the actin cytoskeleton–integrin complex becomes enriched in cross-linked and branched actin filaments in shortened ASM. The mechanical energy output/input ratio during sinusoidal length oscillation was dependent on muscle length, oscillatory amplitude, and cholinergic activation. The enhancing effect of cholinergic stimulation on mechanical energy output/input ratio at short and long muscle lengths may be explained by the length-dependent modulation of cytoskeletal recruitment and crossbridge cycling, respectively. We postulate that ASM functions as a hybrid biomaterial, capable of switching between operating as a cytoskeleton-based mechanical energy store at short muscle lengths to operating as an actomyosin-powered mechanical energy generator at long muscle lengths. This postulate predicts that targeting the signaling molecules involved in cytoskeletal recruitment may provide a novel approach to dilating collapsed airways in obstructive airway disease. PMID:20705939

  4. Length-dependent modulation of cytoskeletal remodeling and mechanical energetics in airway smooth muscle.

    PubMed

    Kim, Hak Rim; Liu, Katrina; Roberts, Thomas J; Hai, Chi-Ming

    2011-06-01

    Actin cytoskeletal remodeling is an important mechanism of airway smooth muscle (ASM) contraction. We tested the hypothesis that mechanical strain modulates the cholinergic receptor-mediated cytoskeletal recruitment of actin-binding and integrin-binding proteins in intact airway smooth muscle, thereby regulating the mechanical energetics of airway smooth muscle. We found that the carbachol-stimulated cytoskeletal recruitment of actin-related protein-3 (Arp3), metavinculin, and talin were up-regulated at short muscle lengths and down-regulated at long muscle lengths, suggesting that the actin cytoskeleton--integrin complex becomes enriched in cross-linked and branched actin filaments in shortened ASM. The mechanical energy output/input ratio during sinusoidal length oscillation was dependent on muscle length, oscillatory amplitude, and cholinergic activation. The enhancing effect of cholinergic stimulation on mechanical energy output/input ratio at short and long muscle lengths may be explained by the length-dependent modulation of cytoskeletal recruitment and crossbridge cycling, respectively. We postulate that ASM functions as a hybrid biomaterial, capable of switching between operating as a cytoskeleton-based mechanical energy store at short muscle lengths to operating as an actomyosin-powered mechanical energy generator at long muscle lengths. This postulate predicts that targeting the signaling molecules involved in cytoskeletal recruitment may provide a novel approach to dilating collapsed airways in obstructive airway disease.

  5. Exploring the roles of integrin binding and cytoskeletal reorganization during mesenchymal stem cell mechanotransduction in soft and stiff hydrogels subjected to dynamic compression.

    PubMed

    Steward, Andrew J; Wagner, Diane R; Kelly, Daniel J

    2014-10-01

    The objective of this study was to explore how the response of mesenchymal stem cells (MSCs) to dynamic compression (DC) depends on their pericellular environment and the development of their cytoskeleton. MSCs were first seeded into 3% agarose hydrogels, stimulated with the chondrogenic growth factor TGF-β3 and exposed to DC (~10% strain at 1Hz) for 1h on either day 7, 14, or 21 of culture. At each time point, the actin, vimentin and tubulin networks of the MSCs were assessed using confocal microscopy. Similar to previous results, MSCs displayed a temporal response to DC; however, no dramatic changes in gross cytoskeletal organization were observed with time in culture. Vinculin (a membrane-cytoskeletal protein in focal adhesions) staining appeared more intense with time in culture. We next aimed to explore how changes to the pericellular environment, independent of the duration of exposure to TGF-β3, would influence the response of MSCs to DC. To this end, MSCs were encapsulated into either 'soft' or 'stiff' agarose hydrogels that are known to differentially support pericellular matrix (PCM) development. The application of DC led to greater relative increases in the expression of chondrogenic marker genes in the stiffer hydrogels, where the MSCs were found to have a more well developed PCM. These increases in gene expression were not observed following the addition of RGDS, an integrin blocker, suggesting that integrin binding plays a role in determining the response of MSCs to DC. Microtubule organization in MSCs was found to adapt in response to DC, but this effect was not integrin mediated, as this cytoskeletal reorganization was also observed in the presence of RGDS. In conclusion, although the PCM, integrin binding, and cytoskeletal reorganization are all involved in mechanotransduction of DC, none of these factors in isolation was able to completely explain the temporal mechanosensitivity of MSCs to dynamic compression.

  6. Modeling Cytoskeletal Active Matter Systems

    NASA Astrophysics Data System (ADS)

    Blackwell, Robert

    Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many important cellular processes. One of the most important microtubule-motor protein assemblies is the mitotic spindle, a self-organized active liquid-crystalline structure that forms during cell division and that ultimately separates chromosomes into two daughter cells. Although the spindle has been intensively studied for decades, the physical principles that govern its self-organization and function remain mysterious. To evolve a better understanding of spindle formation, structure, and dynamics, I investigate course-grained models of active liquid-crystalline networks composed of microtubules, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of active crosslinks, modeled as hookean springs that can adsorb to microtubules and and translocate at finite velocity along the microtubule axis. This model is investigated using a combination of brownian dynamics and kinetic monte carlo simulation. I have further refined this model to simulate spindle formation and kinetochore capture in the fission yeast S. pombe. I then make predictions for experimentally realizable perturbations in motor protein presence and function in S. pombe.

  7. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

    PubMed Central

    Denker, Sheryl P.; Barber, Diane L.

    2002-01-01

    Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration. PMID:12486114

  8. Rho-kinase mediated cytoskeletal stiffness in skinned smooth muscle

    PubMed Central

    Lan, Bo; Wang, Lu; Zhang, Jenny; Pascoe, Chris D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Solomon, Dennis; Paré, Peter D.; Deng, Linhong

    2013-01-01

    The structurally dynamic cytoskeleton is important in many cell functions. Large gaps still exist in our knowledge regarding what regulates cytoskeletal dynamics and what underlies the structural plasticity. Because Rho-kinase is an upstream regulator of signaling events leading to phosphorylation of many cytoskeletal proteins in many cell types, we have chosen this kinase as the focus of the present study. In detergent skinned tracheal smooth muscle preparations, we quantified the proteins eluted from the muscle cells over time and monitored the muscle's ability to respond to acetylcholine (ACh) stimulation to produce force and stiffness. In a partially skinned preparation not able to generate active force but could still stiffen upon ACh stimulation, we found that the ACh-induced stiffness was independent of calcium and myosin light chain phosphorylation. This indicates that the myosin light chain-dependent actively cycling crossbridges are not likely the source of the stiffness. The results also indicate that Rho-kinase is central to the ACh-induced stiffness, because inhibition of the kinase by H1152 (1 μM) abolished the stiffening. Furthermore, the rate of relaxation of calcium-induced stiffness in the skinned preparation was faster than that of ACh-induced stiffness, with or without calcium, suggesting that different signaling pathways lead to different means of maintenance of stiffness in the skinned preparation. PMID:24072407

  9. Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress.

    PubMed

    Fidalgo, Miguel; Guerrero, Ana; Fraile, María; Iglesias, Cristina; Pombo, Celia M; Zalvide, Juan

    2012-03-30

    While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.

  10. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  11. Changes in gene expression, protein content and morphology of chondrocytes cultured on a 3D Random Positioning Machine and 2D rotating clinostat

    NASA Astrophysics Data System (ADS)

    Aleshcheva, Ganna; Hauslage, Jens; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela; Sahana, Jayashree

    Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  12. Proteomic and Microscopic Strategies towards the Analysis of the Cytoskeletal Networks in Major Neuropsychiatric Disorders

    PubMed Central

    Coumans, Joëlle V. F.; Palanisamy, Suresh K. A.; McFarlane, Jim; Moens, Pierre D. J.

    2016-01-01

    Mental health disorders have become worldwide health priorities. It is estimated that in the next 20 years they will account for a 16 trillion United State dollars (US$) loss. Up to now, the underlying pathophysiology of psychiatric disorders remains elusive. Altered cytoskeleton proteins expression that may influence the assembly, organization and maintenance of cytoskeletal integrity has been reported in major depressive disorders, schizophrenia and to some extent bipolar disorders. The use of quantitative proteomics, dynamic microscopy and super-resolution microscopy to investigate disease-specific protein signatures holds great promise to improve our understanding of these disorders. In this review, we present the currently available quantitative proteomic approaches use in neurology, gel-based, stable isotope-labelling and label-free methodologies and evaluate their strengths and limitations. We also reported on enrichment/subfractionation methods that target the cytoskeleton associated proteins and discuss the need of alternative methods for further characterization of the neurocytoskeletal proteome. Finally, we present live cell imaging approaches and emerging dynamic microscopy technology that will provide the tools necessary to investigate protein interactions and their dynamics in the whole cells. While these areas of research are still in their infancy, they offer huge potential towards the understanding of the neuronal network stability and its modification across neuropsychiatric disorders. PMID:27104521

  13. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  14. Mitochondrial and cytoskeletal alterations are involved in the pathogenesis of hydronephrosis in ICR/Mlac-hydro mice.

    PubMed

    Isarangkul, Duangnate; Wiyakrutta, Suthep; Kengkoom, Kanchana; Reamtong, Onrapak; Ampawong, Sumate

    2015-01-01

    The pathogenesis of congenital hydronephrosis in laboratory animals has been studied for many years, yet little is known about the underlying mechanism of this disease. In this study, we investigated a MS-based comparative proteomics approach to characterize the differently expressed proteins between kidney tissue samples of ICR/Mlac-hydro and wild-type mice. Interestingly, proteomic results exhibited several mitochondrial protein alterations especially the up-regulation of 60 kDa heat shock protein (Hsp60), stress-70 protein (GRP75) dysfunction, and down-regulation of voltage-dependent anion-selective channel protein 1 (VDAC-1). The results demonstrated that mitochondrial alteration may lead to inadequate energy-supply to maintain normal water reabsorption from the renal tubule, causing hydronephrosis. Moreover, the alteration of cytoskeleton proteins in the renal tubule, in particular the up-regulation of tubulin beta-4B chain (Tb4B) and N-myc downstream-regulated gene 1 protein (Ndr-1) may also be related due to their fundamental roles in maintaining cell morphology and tissue stability. In addition, cytoskeletal alterations may consequence to the reduction of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), cytoplasmic enzyme, which modulates the capacity of structural proteins. Our findings highlight a number of target proteins that may play a crucial role in congenital hydronephrosis and emphasize that the disorder of mitochondria and cytoskeleton proteins may be involved.

  15. Mitochondrial and cytoskeletal alterations are involved in the pathogenesis of hydronephrosis in ICR/Mlac-hydro mice

    PubMed Central

    Isarangkul, Duangnate; Wiyakrutta, Suthep; Kengkoom, Kanchana; Reamtong, Onrapak; Ampawong, Sumate

    2015-01-01

    The pathogenesis of congenital hydronephrosis in laboratory animals has been studied for many years, yet little is known about the underlying mechanism of this disease. In this study, we investigated a MS-based comparative proteomics approach to characterize the differently expressed proteins between kidney tissue samples of ICR/Mlac-hydro and wild-type mice. Interestingly, proteomic results exhibited several mitochondrial protein alterations especially the up-regulation of 60 kDa heat shock protein (Hsp60), stress-70 protein (GRP75) dysfunction, and down-regulation of voltage-dependent anion-selective channel protein 1 (VDAC-1). The results demonstrated that mitochondrial alteration may lead to inadequate energy-supply to maintain normal water reabsorption from the renal tubule, causing hydronephrosis. Moreover, the alteration of cytoskeleton proteins in the renal tubule, in particular the up-regulation of tubulin beta-4B chain (Tb4B) and N-myc downstream-regulated gene 1 protein (Ndr-1) may also be related due to their fundamental roles in maintaining cell morphology and tissue stability. In addition, cytoskeletal alterations may consequence to the reduction of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), cytoplasmic enzyme, which modulates the capacity of structural proteins. Our findings highlight a number of target proteins that may play a crucial role in congenital hydronephrosis and emphasize that the disorder of mitochondria and cytoskeleton proteins may be involved. PMID:26309577

  16. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  17. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  18. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  19. Strain engineering for improved expression of recombinant proteins in bacteria

    PubMed Central

    2011-01-01

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins. PMID:21569582

  20. Cytoskeletal Tension inhibits Hippo signaling through an Ajuba-Warts complex

    PubMed Central

    Sun, Gongping; Pan, Yuanwang; Irvine, Kenneth D.

    2014-01-01

    Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. We report that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein Jub, a negative regulator of Warts within the Hippo pathway. We further show that Jub associates with α-catenin, and that its localization to adherens junctions and association with α-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. Our observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth. PMID:24995985

  1. Cytoskeletal elements in the bacterium Mycoplasma pneumoniae

    NASA Astrophysics Data System (ADS)

    Hegermann, Jan; Herrmann, Richard; Mayer, Frank

    2002-09-01

    Mycoplasma pneumoniae is a pathogenic eubacterium lacking a cell wall. Three decades ago, a "rod", an intracellular cytoskeletal structure, was discovered that was assumed to define and stabilize the elongated cell shape. Later, by treatment with detergent, a "Triton shell" (i.e. a fraction of detergent-insoluble cell material) could be obtained, believed to contain additional cytoskeletal elements. Now, by application of a modified Triton X-100 treatment, we are able to demonstrate that M. pneumoniae possesses a cytoskeleton consisting of a blade-like rod and a peripheral lining located close to the inner face of the cytoplasmic membrane, exhibiting features of a highly regular network. Attached "stalks" may support the cytoplasmic membrane. The rod was connected to the cell periphery by "spokes" and showed a defined ultrastructure. Its proximal end was found to be attached to a wheel-like complex. Fibrils extended from the proximal end of the rod into the cytoplasm.

  2. The cytoskeletal arrangements necessary to neurogenesis

    PubMed Central

    Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

    2016-01-01

    During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation. PMID:26760504

  3. The cytoskeletal arrangements necessary to neurogenesis.

    PubMed

    Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

    2016-04-12

    During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation.

  4. Effects of GnRH on Neurite Outgrowth, Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos.

    PubMed

    Quintanar, J Luis; Calderón-Vallejo, Denisse; Hernández-Jasso, Irma

    2016-10-01

    It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.

  5. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  6. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  7. Cell Forces and Cytoskeletal Order Parameters

    NASA Astrophysics Data System (ADS)

    Discher, Dennis

    2012-02-01

    Nematic, Smectic and Isotropic Order parameters have found wide-spread use in characterizing all manner of soft matter systems, but have not yet been applied to characterize and understand the structures within living cells, particularly cytoskeletal structures. Several examples will be used to illustrate the utility of such analyses, ranging from experiments on stem cells attached to or in various elastic matrices to embryonic heart tissue and simulations of membrane cytoskeletons under all manner of stressing. Recently developed theory will be shown to apply in general with account of cell contractility, matrix elasticity and dimensionality as well as cell shape and a newly defined ``cytoskeletal polarizability.'' The latter property of cells is likely different between different cell types due to different amounts of key cytoskeletal components with some types of stem cells being more polarizable than others. Evidence of coupling to the nucleus as a viscoelastic inclusion will also be presented. [4pt] References: (1) P. Dalhaimer, D.E. Discher, T. Lubensky. Crosslinked actin networks exhibit liquid crystal elastomer behavior, including soft-mode elasticity. Nature Physics 3: 354-360 (2007). (2) A. Zemel, F.Rehfeldt, A.E.X. Brown, D.E. Discher, and S.A. Safran. Optimal matrix rigidity in the self-polarization of stem cells. Nature Physics 6: 468 - 473 (2010).

  8. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics

    PubMed Central

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-01-01

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  9. Systems Genetics Implicates Cytoskeletal Genes in Oocyte Control of Cloned Embryo Quality

    PubMed Central

    Cheng, Yong; Gaughan, John; Midic, Uros; Han, Zhiming; Liang, Cheng-Guang; Patel, Bela G.; Latham, Keith E.

    2013-01-01

    Cloning by somatic cell nuclear transfer is an important technology, but remains limited due to poor rates of success. Identifying genes supporting clone development would enhance our understanding of basic embryology, improve applications of the technology, support greater understanding of establishing pluripotent stem cells, and provide new insight into clinically important determinants of oocyte quality. For the first time, a systems genetics approach was taken to discover genes contributing to the ability of an oocyte to support early cloned embryo development. This identified a primary locus on mouse chromosome 17 and potential loci on chromosomes 1 and 4. A combination of oocyte transcriptome profiling data, expression correlation analysis, and functional and network analyses yielded a short list of likely candidate genes in two categories. The major category—including two genes with the strongest genetic associations with the traits (Epb4.1l3 and Dlgap1)—encodes proteins associated with the subcortical cytoskeleton and other cytoskeletal elements such as the spindle. The second category encodes chromatin and transcription regulators (Runx1t1, Smchd1, and Chd7). Smchd1 promotes X chromosome inactivation, whereas Chd7 regulates expression of pluripotency genes. Runx1t1 has not been associated with these processes, but acts as a transcriptional repressor. The finding that cytoskeleton-associated proteins may be key determinants of early clone development highlights potential roles for cytoplasmic components of the oocyte in supporting nuclear reprogramming. The transcriptional regulators identified may contribute to the overall process as downstream effectors. PMID:23307892

  10. HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span.

    PubMed

    Baird, Nathan A; Douglas, Peter M; Simic, Milos S; Grant, Ana R; Moresco, James J; Wolff, Suzanne C; Yates, John R; Manning, Gerard; Dillin, Andrew

    2014-10-17

    The conserved heat shock transcription factor-1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

  11. Cytoskeletal pinning controls phase separation in multicomponent lipid membranes.

    PubMed

    Arumugam, Senthil; Petrov, Eugene P; Schwille, Petra

    2015-03-10

    We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Visualization of Cytoskeletal Elements by the Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Berdyyeva, Tamara; Woodworth, Craig; Sokolov, Igor

    2004-03-01

    We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquids visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total volume of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3 dimensional technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.

  13. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

    PubMed Central

    Ray, Poulomi; Chapman, Susan C.

    2015-01-01

    Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

  14. Cdk6 contributes to cytoskeletal stability in erythroid cells.

    PubMed

    Uras, Iris Z; Scheicher, Ruth M; Kollmann, Karoline; Glösmann, Martin; Prchal-Murphy, Michaela; Tigan, Anca S; Fux, Daniela A; Altamura, Sandro; Neves, Joana; Muckenthaler, Martina; Bennett, Keiryn L; Kubicek, Stefan; Hinds, Philip W; von Lindern, Marieke; Sexl, Veronika

    2017-03-02

    Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in the cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeletal organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We show further that Cdk6 regulates the transcription of a panel of genes involved in actin (de-) polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

  15. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

    PubMed

    Ray, Poulomi; Chapman, Susan C

    2015-01-01

    Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

  16. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells

    PubMed Central

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-01-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  17. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

    PubMed

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

  18. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  19. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization.

    PubMed

    Guo, Zhenhua; Stephenson, Robert; Qiu, Jiazhang; Zheng, Shijun; Luo, Zhao-Qing

    2014-03-01

    Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. Differential expression of microtubule associated protein MAP-2 in developing cochleovestibular neurons and its modulation by neurotrophin-3.

    PubMed

    San José, I; Vázquez, E; García-Atares, N; Huerta, J J; Vega, J A; Represa, J

    1997-06-01

    Microtubule associated proteins (MAPs) are essential cytoskeletal proteins in developing neurons. The present study was undertaken to analyze the expression of MAP2 and its isoforms (a,b,c) during the embryonal and early post-hatching development of chicken cochleovestibular ganglion (CVG) neurons. Moreover, we have investigated MAP2 expression in primary cultures of CVG neurons, and whether it is regulated by neurotrophin-3 (NT3). The expression of MAP2 immunoreactivity (IR) was studied using both Western blot and immunohistochemistry on tissue sections and primary cultures. In vivo MAP2c was expressed from incubation day 4 (E4) to E10, and MAP2b was found in all embryonal stages studied and at post-hatching day 10 (P10), whereas MAP2a was restricted to the post-hatching periods. The cellular localization of IR was in the neuronal perikarya and their peripheral processes (dendrites) but not in axons. Primary cultures matched the in vivo pattern of MAP2 expression, and IR was localized in neuronal cell bodies and the initial segment of the neuronal processes. Exogenous NT3 regulated the expression of MAP2 isoforms in a dose dependent manner. At the survival dose of 0.5 ng/ml NT3, the main MAP2 expression was MAP2c. Conversely, at the neuritogenic dose of 5 ng/ml NT3 increased MAP2b and MAP2a expression, but not MAP2c. The present results demonstrate that MAP2 isoforms are developmentally regulated, thus suggesting that each isoform is specifically involved in CVG neuron maturation. Furthermore, we provide evidence of MAP2 regulation in culture by the neurotrophic factor NT3.

  1. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  2. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  3. Differential protein expression using proteomics from a crustacean brine shrimp (Artemia sinica) under CO2-driven seawater acidification.

    PubMed

    Chang, Xue-Jiao; Zheng, Chao-Qun; Wang, Yu-Wei; Meng, Chuang; Xie, Xiao-Lu; Liu, Hai-Peng

    2016-11-01

    Gradually increasing atmospheric CO2 partial pressure (pCO2) has caused an imbalance in carbonate chemistry and resulted in decreased seawater pH in marine ecosystems, termed seawater acidification. Anthropogenic seawater acidification is postulated to affect the physiology of many marine calcifying organisms. To understand the possible effects of seawater acidification on the proteomic responses of a marine crustacean brine shrimp (Artemia sinica) three groups of cysts were hatched and further raised in seawater at different pH levels (8.2 as control and 7.8 and 7.6 as acidification stress levels according to the predicted levels at the end of this century and next century, respectively) for 1, 7 and 14 days followed by examination of the protein expression changes via two-dimensional gel electrophoresis. Searches of protein databases revealed that 67 differential protein spots were altered due to lower pH level (7.6 and 7.8) stress in comparison to control groups (pH 8.2) by mass spectrometry. Generally, these differentially expressed proteins included the following: 1) metabolic process-related proteins involved in glycolysis and glucogenesis, nucleotide/amino acid/fatty acid metabolism, protein biosynthesis, DNA replication and apoptosis; 2) stress response-related proteins, such as peroxiredoxin, thioredoxin peroxidase, 70-kDa heat shock protein, Na/K ATPase, and ubiquinol-cytochrome c reductase; 3) immune defence-related proteins, such as prophenoloxidase and ferritin; 4) cytoskeletal-related proteins, such as myosin light chain, TCP1 subunit 2, tropomyosin and tubulin alpha chain; and 5) signal transduction-related proteins, such as phospholipase C-like protein, 14-3-3 zeta, translationally controlled tumour protein and RNA binding motif protein. Taken together, these data support the idea that CO2-driven seawater acidification may affect protein expression in the crustacean A. sinica and possibly also in other species that feed on brine shrimp in the

  4. The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

    PubMed Central

    Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

    2015-01-01

    Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

  5. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  6. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization

    PubMed Central

    Hummel, Thomas; Leifker, Karin; Klämbt, Christian

    2000-01-01

    In Drosophila, the correct formation of the segmental commissures depends on neuron–glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2–SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding. PMID:10766742

  7. Quantitative proteomics analysis of varicose veins: identification of a set of differentially expressed proteins related to ATP generation and utilization.

    PubMed

    Kuo, Chao-Jen; Liang, Shih-Shin; Hsi, Edward; Chiou, Shyh-Horng; Lin, Sin-Daw

    2013-11-01

    Although morphological and anatomical studies indicate that varicose veins are characterized by venous wall weakening and subendothelial fibrosis, the exact underlying biochemical mechanism of their development remains unknown. Additionally, no quantitative proteomic study of venous proteins leading to decreased contractility of varicose veins has been reported to date. Therefore, to elucidate the molecular mechanism of altered vascular contractility, this study performed shotgun proteomic analysis to obtain protein expression profiles in patients with varicose veins. Stable isotope dimethyl labeling coupled with nanoLC-MS/MS revealed downregulation in 12 polypeptides, including myosin light chain kinase, creatine kinase B-type, ATP synthase, phosphoglycerate kinase, and pyruvate kinase. However, analyses of protein species associated with cytoskeletal assembly or with cellular morphology showed no clear up- or down-regulation. These results indicate that defects in ATP generation and utilization may account for the dysfunction of vascular smooth muscle following formation of varicose veins. Collectively, the severity of varicose veins depends on the regulatory roles of various protein factors in the metabolic coordination of physiological functions. This pilot study improves understanding of the pathogenesis of varicose veins and lays the foundation for further validation and clinical translation of biomarkers for targeted therapies in treating this disease. Copyright © 2013. Published by Elsevier B.V.

  8. Abnormal cytoskeletal assembly in platelets from uremic patients.

    PubMed Central

    Escolar, G.; Díaz-Ricart, M.; Cases, A.; Castillo, R.; Ordinas, A.; White, J. G.

    1993-01-01

    The mechanisms involved in the hemostatic abnormality of uremic patients remain obscure. We have explored the response of normal and uremic platelets to surface activation at the ultrastructural level and analyzed changes in the composition of proteins associated with normal and uremic platelet cytoskeletons after stimulation with thrombin (0.01 and 0.1 U/ml). Cytoskeletons were obtained by extraction with Triton X-100, processed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of cytoskeletal proteins analyzed by densitometry. Under static conditions, uremic platelets spread with difficulty on formvar-coated grids. The percentage of platelets that spread fully on this polymer surface was statistically reduced compared with that of control platelets (11 +/- 1.4 vs. 21 +/- 1.6; P < 0.05). An impairment of cytoskeletal organization was observed in resting uremic platelets but abnormalities were more evident after thrombin activation. The incorporation of actin into the cytoskeletons of thrombin-stimulated uremic platelets was significantly reduced with respect to controls (6 +/- 3% vs. 29 +/- 5%; P < 0.01 after 0.01 U/ml and 28 +/- 9% vs. 59 +/- 10%; P < 0.05 after 0.1 U/ml). Decreased associations of actin-binding protein (P < 0.01), alpha-actinin (P < 0.05), and tropomyosin (P < 0.05) with the cytoskeletons of uremic platelets were also noted. No difference was observed for the incorporation of myosin into the cytoskeletons of activated uremic platelets. These results suggest functional and biochemical alterations of the platelet cytoskeleton in uremia, which may contribute to the impairment of platelet function observed in uremic patients. Images Figure 2 Figure 3 Figure 4 PMID:8362980

  9. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  10. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  11. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  12. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  13. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  14. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  15. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  16. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  17. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  18. Engineering cells to improve protein expression.

    PubMed

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Expression of heat shock protein genes in insect stress responses

    USDA-ARS?s Scientific Manuscript database

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  20. PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

    PubMed

    Mansour, Mariam; Nievergall, Eva; Gegenbauer, Kristina; Llerena, Carmen; Atapattu, Lakmali; Hallé, Maxime; Tremblay, Michel L; Janes, Peter W; Lackmann, Martin

    2016-01-15

    Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling. © 2016. Published by The Company of Biologists Ltd.

  1. Emerging technology of in situ cell free expression protein microarrays.

    PubMed

    Nand, Amita; Gautam, Anju; Pérez, Javier Batista; Merino, Alejandro; Zhu, Jinsong

    2012-02-01

    Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.

  2. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  3. Tristetraprolin functions in cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Liu, Xiaohui; Li, Xiaoyan; Ma, Rujun; Xiong, Bo; Sun, Shao-Chen; Liu, Honglin; Gu, Ling

    2016-01-01

    Tristetraprolin (TTP), a member of TIS11 family containing CCCH tandem zinc finger, is one of the best characterized RNA-binding proteins. However, to date, the role of TTP in mammalian oocytes remains completely unknown. In the present study, we report the altered maturational progression and cytokinesis, upon specific knockdown of TTP in mouse oocytes. Furthermore, by confocal scanning, we observe the failure to form cortical actin cap during meiosis of TTP-depleted oocytes. Loss of TTP in oocytes also results in disruption of meiotic spindle morphology and chromosome alignment. In support of these findings, incidence of aneuploidy is accordingly increased when TTP is abated in oocytes. Our results suggest that TTP as a novel cytoskeletal regulator is required for spindle morphology/chromosome alignment and actin polymerization in oocytes. PMID:27458159

  4. HSF-1 mediated cytoskeletal integrity determines thermotolerance and lifespan

    PubMed Central

    Baird, Nathan A.; Douglas, Peter M.; Simic, Milos S.; Grant, Ana R.; Moresco, James J.; Wolff, Suzanne C.; Yates, John R.; Manning, Gerard; Dillin, Andrew

    2015-01-01

    The conserved transcription factor HSF-1 is essential to cellular stress resistance and organismal lifespan determination. The canonical function of HSF-1 is to regulate a network of molecular chaperones that maintain protein homeostasis during extrinsic environmental stresses or intrinsic age related deterioration. In the metazoan C. elegans, we engineered a modified HSF-1 strain that increases stress resistance and longevity without enhancing chaperone induction. This HSF-1 dependent health assurance acts through the regulation of pat-10. Upon heat stress pat-10 upregulation maintains a functional actin cytoskeleton and endocytic network. Loss of pat-10 causes a collapse of organismal health and failure of stress resistance. Furthermore, overexpression of pat-10 is sufficient to increase both thermotolerance and longevity by mechanisms that affect actin stability. Our findings indicate that in addition to chaperone induction, HSF-1 plays a prominent role in cytoskeletal integrity to ensure proper cellular function during times of stress and aging. PMID:25324391

  5. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  6. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  7. Immunohistochemical Studies of Cytoskeletal and Extracellular Matrix Components in Dogfish Scyliorhinus canicula L. Notochordal Cells.

    PubMed

    Restović, Ivana; Vukojević, Katarina; Paladin, Antonela; Saraga-Babić, Mirna; Bočina, Ivana

    2015-10-01

    Immunofluorescence and immunohistochemical techniques were used to define the distribution of cytoskeletal (cytokeratin 8, vimentin) and extracellular matrix components (collagen type I, collagen type II, hyaluronic acid, and aggrecan) and bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) in the notochord of the lesser spotted dogfish Scyliorhinus canicula L. Immunolocalization of hyaluronic acid was observed in the notochord, vertebral centrum, and neural and hemal arches, while positive labeling to aggrecan was observed in the ossified centrum, notochord, and the perichondrium of the hyaline cartilage. Type I collagen was observed in the mineralized cartilage of the vertebral bodies, the notochord, the fibrocartilage of intervertebral disc, and the perichondrium. A positive labeling to type II collagen was observed in the inner part of the cartilaginous vertebral centrum and the notochord, as well as in the neural arch and muscle tissue, but there was no appreciable labeling of the hyaline cartilage. The presence of both BMP4 and BMP7 was seen in the mineralized vertebral centrum, notochordal cells, and neural arch. The notochordal cells expressed both cytokeratin 8 and vimentin, but predominantly vimentin. Hyaluronic acid, collagen type I, and collagen type II expression confirmed the presence of a mixture of notochordal and fibrocartilaginous tissue in the intervertebral disc, while BMPs confirmed the presence of an ossification in the cartilaginous skeleton of the spotted dogfish. © 2015 Wiley Periodicals, Inc.

  8. Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16

    PubMed Central

    Maruthappu, Thiviyani; Chikh, Anissa; Fell, Benjamin; Delaney, Paul J.; Brooke, Matthew A.; Levet, Clemence; Moncada-Pazos, Angela; Ishida-Yamamoto, Akemi; Blaydon, Diana; Waseem, Ahmad; Leigh, Irene M.; Freeman, Matthew; Kelsell, David P.

    2017-01-01

    Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2−/− mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this ‘stress' keratin is regulated. PMID:28128203

  9. Tools for Co-expressing Multiple Proteins in Mammalian Cells

    PubMed Central

    Assur, Zahra; Hendrickson, Wayne A.; Mancia, Filippo

    2013-01-01

    Summary Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multi-protein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate over-expressing lines, and elevated costs. Co-expression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here we present an efficient fluorescence marking procedure for establishing stable cell lines that over-express two proteins in co-ordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more then two components. PMID:21987254

  10. Centrosomal Localization of the Psoriasis Candidate Gene Product, CCHCR1, Supports a Role in Cytoskeletal Organization

    PubMed Central

    Tervaniemi, Mari H.; Siitonen, H. Annika; Söderhäll, Cilla; Minhas, Gurinder; Vuola, Jyrki; Tiala, Inkeri; Sormunen, Raija; Samuelsson, Lena; Suomela, Sari; Kere, Juha; Elomaa, Outi

    2012-01-01

    CCHCR1 (Coiled-Coil α-Helical Rod protein 1), within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene with the psoriasis associated risk allele CCHCR1*WWCC. Although its expression pattern in psoriatic skin differs from healthy skin and its overexpression influences cell proliferation in transgenic mice, its role as a psoriasis effector gene has remained unsettled. The 5′-region of the gene contains a SNP (rs3130453) that controls a 5′-extended open reading frame and thus the translation of alternative isoforms. We have now compared the function of two CCHCR1 isoforms: the novel longer isoform 1 and the previously studied isoform 3. In samples of Finnish and Swedish families, the allele generating only isoform 3 shows association with psoriasis (P<10−7). Both isoforms localize at the centrosome, a cell organelle playing a role in cell division. In stably transfected cells the isoform 3 affects cell proliferation and with the CCHCR1*WWCC allele, also apoptosis. Furthermore, cells overexpressing CCHCR1 show isoform- and haplotype-specific influences in the cell size and shape and alterations in the organization and expression of the cytoskeletal proteins actin, vimentin, and cytokeratins. The isoform 1 with the non-risk allele induces the expression of keratin 17, a hallmark for psoriasis; the silencing of CCHCR1 reduces its expression in HEK293 cells. CCHCR1 also regulates EGF-induced STAT3 activation in an isoform-specific manner: the tyrosine phosphorylation of STAT3 is disturbed in isoform 3-transfected cells. The centrosomal localization of CCHCR1 provides a connection to the abnormal cell proliferation and offers a link to possible cellular pathways altered in psoriasis. PMID:23189171

  11. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  12. A Bpag1 isoform involved in cytoskeletal organization surrounding the nucleus

    SciTech Connect

    Young, Kevin G.; Pinheiro, Bruno; Kothary, Rashmi . E-mail: rkothary@ohri.ca

    2006-01-15

    Bpag1/dystonin proteins are giant cytoskeletal interacting proteins postulated to cross-link cytoskeletal filaments and thereby maintain cellular integrity. Loss of function of non-epithelial Bpag1 isoforms results in neuromuscular dysfunction and early postnatal death in mice. Multiple Bpag1 transcripts have been described, including those encoding protein isoforms that vary at the N-terminal end. Here, we have analyzed the subcellular localizations and cytoskeletal interactions of two isoforms, termed Bpag1a1 and Bpag1a2. We demonstrate that novel sequence at the 5' end of the Bpag1a2 transcript codes for an N-terminal transmembrane domain and targets the protein to the perinuclear region of the cell. Furthermore, we show that the endogenous Bpag1a2 protein is also present in the perinuclear region in myoblast cells. Differences in Bpag1a1 and Bpag1a2 with respect to the extent of their interactions with microtubules and microfilaments are also described, with Bpag1a2 fusion protein serving largely to associate with microfilaments surrounding the nucleus and Golgi apparatus. The overall structure and subcellular localizations of Bpag1a2 indicate possible functions in nuclear envelope structuring, nuclear tethering, and organization of membranous structures surrounding the nucleus.

  13. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  14. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  15. Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs.

    PubMed

    Unachukwu, Uchenna; Trischler, Jordis; Goldklang, Monica; Xiao, Rui; D'Armiento, Jeanine

    2017-06-01

    The present study tested the hypothesis that maternal smoke exposure results in fetal lung growth retardation due to dysregulation in various signaling pathways, including the Wnt (wingless-related integration site)/β-catenin pathway. Pregnant female C57BL/6J mice were exposed to cigarette smoke (100-150 mg/m(3)) or room air, and offspring were humanely killed on 12.5, 14.5, 16.5, and 18.5 d post coitum (dpc). We assessed lung stereology with Cavalieri estimation; apoptosis with proliferating cell nuclear antigen, TUNEL, and caspase assays; and gene expression with quantitative PCR (qPCR) and RNA sequencing on lung epithelium and mesenchyme retrieved by laser capture microdissection. Results demonstrated a significant decrease in body weight and lung volume of smoke-exposed embryos. At 16.5 dpc, the reduction in lung volume was due to loss of lung mesenchymal tissue correlating with a decrease in cell proliferation (n = 10; air: 61.65% vs. smoke: 44.21%, P < 0.05). RNA sequence analysis demonstrated an alteration in the Wnt pathway, and qPCR confirmed an increased expression of secreted frizzled-related protein 1 (sFRP-1) [n = 12; relative quantification (RQ) 1 vs. 2.33, P < 0.05] and down-regulation of Cyclin D1 (n = 7; RQ 1 vs. 0.61, P < 0.05) in mesenchymal tissue. Furthermore, genome expression studies revealed a smoke-induced up-regulation of Rho-GTPase-dependent actin cytoskeletal signaling that can lead to loss of tissue integrity.-Unachukwu, U., Trischler, J., Goldklang, M., Xiao, R., D'Armiento, J. Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs. © FASEB.

  16. Proteomic Analysis of an Immortalized Mouse Pancreatic Stellate Cell Line Identifies Differentially-Expressed Proteins in Activated vs. Non-Proliferating Cell States

    PubMed Central

    Paulo, Joao A.; Urrutia, Raul; Banks, Peter A.; Conwell, Darwin L.; Steen, Hanno

    2011-01-01

    Pancreatic stellate cells (PaSC) are mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of activated to quiescent PaSC may have a significant influence in the development of chronic pancreatitis. We aim to compare differentially expressed proteins from an immortalized cell line of mouse PaSC in the activated and serum-starved cell states using mass spectrometry-based proteomics. PaSC cultured in media supplemented with fetal bovine serum (FBS) proliferate in the activated state, while serum starvation promotes the cellular transition to a “pseudo-quiescent” state. Using these two cell states, we performed a comparative mass spectrometry (GeLC-MS/MS) proteomic analysis. We identified over 2000 non-redundant proteins in PaSC. Qualitative and label-free quantitative analysis revealed several hundred proteins that were differentially abundant between the cell states. Proteins that were more abundant in activated PaSC included cytoskeletal proteins and ribosomal proteins, while those more abundant in pseudo-quiescent PaSC included proteins involved in protein degradation-related pathways (lysosome, ubiquitin-mediated proteolysis, and the proteasome). Investigation of the role of PaSC in the pathogenesis of chronic pancreatitis using the mass spectrometry-based proteomics strategy described herein will lead to further insights into the molecular mechanisms associated with the disease. PMID:21838295

  17. Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

    PubMed Central

    Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

    2014-01-01

    Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

  18. Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading.

    PubMed

    Eto, M; Wong, L; Yazawa, M; Brautigan, D L

    2000-07-01

    Cell migration and cytokinesis require reorganization of the cytoskeleton, involving phosphorylation and dephosphorylation of proteins such as myosin II and moesin. Myosin and moesin bind directly to a regulatory subunit of myosin/moesin phosphatase (MMP) that contains a protein type-1 phosphatase (PP1) catalytic subunit. Here we examined the role of MMP in cytoskeletal dynamics using a phosphorylation-dependent inhibitor protein specific for MMP, called CPI-17. Fibroblasts do not express CPI-17, making them a null background to study effects of expression. Wild type CPI-17 in rat embryo fibroblasts caused (1) abnormal accumulation of cortical F-actin fibers, distinct from the stress fibers induced by expression of active RhoA; (2) progressive contraction of cell area, leaving behind filamentous extensions that stained for F-actin and moesin, but not myosin; and (3) significantly retarded spreading of fibroblasts on fibronectin with elevated myosin II light chain phosphorylation. A phosphorylation site mutant CPI-17(T38A) and inhibitor-2 (Inh2), another PP1-specific inhibitor protein, served as controls and did not elicit these same responses when expressed at the same level as CPI-17. Inhibition of myosin light chain kinase by ML-9 prevented the abnormal accumulation of cortical microfilaments by CPI-17, but did not reverse shrinkage in area, whereas kinase inhibitors HA1077 and H7 prevented CPI-17-induced changes in microfilament distribution and cell contraction. These results highlight the physiological importance of myosin/moesin phosphatase regulation to dynamic remodeling of the cytoskeleton.

  19. Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells.

    PubMed

    Wenzel, Uwe; Herzog, Angelika; Kuntz, Sabine; Daniel, Hannelore

    2004-07-01

    A high dietary intake of plant foods is thought to contribute to the prevention of colorectal cancers in humans and flavonoids as part of such a diet are considered to contribute to those protective effects. Quercetin is a major dietary flavonoid consumed with a diet rich in onions, tea, and apples. We used HT-29 human colon cancer cells and investigated the effects of quercetin on proliferation, apoptosis, and differentiation as processes shown to be disregulated during cancer development. To identify the cellular targets of quercetin action, two-dimensional gel electrophoresis was performed and proteins altered in expression level after quercetin exposure of cells were identified by mass spectrometry of peptide fragments generated by tryptic digestion. Quercetin inhibited the proliferation of HT-29 cells with an IC(50)-value of 81.2 +/- 6.6 microM. Cell differentiation based on surface expression of alkaline phosphatase was enhanced 4-fold and the activity of the pro-apoptotic effector caspase-3 increased 3-fold. Those effects were associated with the regulation of heat-shock proteins and annexins shown to both play a crucial role in the process of apoptosis. Cytoskeletal caspase substrates were found as regulated as well and various proteins involved in intermediary metabolism and in gene regulation showed altered steady-state expression levels upon quercetin treatment of cells. In conclusion, quercetin alters the levels of a variety of proteins involved in growth, differentiation, and apoptosis of colon cancer cells. Their identification as molecular targets of quercetin may explain the anti-cancer activities of this flavonoid.

  20. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  1. Exocyst complex protein expression in the human placenta.

    PubMed

    Gonzalez, I M; Ackerman, W E; Vandre, D D; Robinson, J M

    2014-07-01

    Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Improving membrane protein expression by optimizing integration efficiency.

    PubMed

    Niesen, Michiel J M; Marshall, Stephen S; Miller, Thomas F; Clemons, William M

    2017-09-16

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effect of double mutations, on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  4. Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

    PubMed

    Alfei, L; Medolago-Albani, L; Zezze, G M; Stefanelli, A

    1992-01-01

    A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.

  5. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  6. Rapid Label-Free Identification of Estrogen-Induced Differential Protein Expression In Vivo from Mouse Brain and Uterine Tissue

    PubMed Central

    Prokai, Laszlo; Stevens, Stanley M.; Rauniyar, Navin; Nguyen, Vien

    2009-01-01

    Protein abundance profiling from tissue using liquid chromatograph—tandem mass spectrometry-based ‘shotgun’ proteomics and label-free relative quantitation was evaluated for the investigation of estrogen-regulated protein expression in the mouse brain and uterus. Sample preparation involved a 30-min protein extraction in 8 M aqueous urea solution, followed by disulphide reduction, thiol alkylation and trypsin digestion of the extracted proteins, and was performed on 3–4 mg of tissue in order to evaluate the suitability of this methodology to expedite the survey of cellular pathways that are affected in vivo by an experimental therapeutic intervention in an animal model. The label-free proteomic approach (spectral counting) was suitable to identify even subtle changes in cortical protein levels and revealed significant estrogen-induced upregulation of ATP synthase (both α- and β-isoforms), aspartate aminotransferase 2 and mitochondrial malate dehydrogenase without any prior subcellular fractionation of the tissue or the use of multidimensional chromatographic separation. The methodology was also suitable to observe various up- and downregulated proteins in the uterine tissue of ovariectomized mice upon treatment with 17β-estradiol. In addition to confirming a very significant decrease in the abundance of glutathione S-transferase recognized as a marker of estrogen’s impact, our studies have also revealed potential new protein markers such as desmin and lumican that are critical components of cytoskeletal arrangement and, hence, regulation of their abundance could contribute to major morphological changes in the uterus occurring upon estrogenic stimulation. PMID:19545149

  7. Precortical phase of Alzheimer’s disease (AD)-related tau cytoskeletal pathology

    PubMed Central

    Stratmann, Katharina; Heinsen, Helmut; Korf, Horst-Werner; Del Turco, Domenico; Ghebremedhin, Estifanos; Seidel, Kay; Bouzrou, Mohamed; Grinberg, Lea T.; Bohl, Jürgen; Wharton, Stephen B; den Dunnen, Wilfred; Rüb, Udo

    2015-01-01

    Alzheimer’s disease (AD) represents the most frequent progressive neuropsychiatric disorder worldwide leading to dementia and accounts for 60 to 70% of demented individuals. In view of the early appearance of neuronal deposits of the hyperphosphorylated cytoskeletal protein tau in the transentorhinal and entorhinal regions of the allocortex (i.e. in Braak and Braak AD stage I in the evolution of the AD-related cortical tau cytoskeletal pathology) it has been believed for a long time that these allocortical regions represent the first brain targets of the AD-related tau cytoskeletal pathology. However, recent pathoanatomical studies suggested that the subcortical brain nuclei that send efferent projections to the transentorhinal and entorhinal regions may also comprise AD-related cytoskeletal changes already at very early Braak and Braak AD stages. In order to corroborate these initial results we systematically investigated the presence and extent of the AD-related cytoskeletal pathology in serial thick tissue sections through all the subcortical nuclei known to send efferent projections to these vulnerable allocortical regions of three individuals with Braak and Braak AD stage 0 and fourteen individuals with Braak and Braak AD stage I by means of immunostainings with the anti-tau antibody AT8. These investigations revealed consistent AT8 immunoreactive neuronal tau cytoskeletal pathology in a subset of these subcortical nuclei (i.e. medial septal nucleus, nuclei of the vertical and horizontal limbs of the diagonal band of Broca, basal nucleus of Meynert; claustrum; hypothalamic ventromedial, tuberomamillary and supramamillary nuclei, perifornical region and lateral area; thalamic central medial, laterodorsal, subparafascicular, and central lateral nuclei, medial pulvinar and limitans-suprageniculate complex; peripeduncular nucleus, dopaminergic substantia nigra and ventral tegmental area, periaqueductal gray, midbrain and pontine dorsal raphe nuclei, locus

  8. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  9. Rectal 1% Tenofovir Gel Use Associates with Altered Epidermal Protein Expression

    PubMed Central

    Romas, Laura; Birse, Kenzie; Mayer, Kenneth H.; Abou, Max; Westmacott, Garrett; Giguere, Rebecca; Febo, Irma; Cranston, Ross D.; Carballo-Diéguez, Alex; McGowan, Ian

    2016-01-01

    Abstract Rectal use of a 1% tenofovir (TFV) gel is currently being evaluated for HIV prevention. While careful assessment of mucosal safety of candidate microbicides is a primary concern, tools to assess mucosal toxicity are limited. Mass spectrometry-based proteomics is a sensitive and high-throughput technique that can provide in-depth information on inflammation processes in biological systems. In this study, we utilized a proteomics approach to characterize mucosal responses in study participants involved in a phase 1 clinical trial of a rectal TFV-based gel. Project Gel was a phase 1 randomized (1:1), double-blind, multisite, placebo-controlled trial in which 24 participants received rectal TFV or a universal placebo [hydroxyethyl cellulose (HEC)] over a course of 8 daily doses. Rectal mucosal swabs were collected after 0, 1, and 8 doses and were analyzed by label-free tandem mass spectrometry. Differential protein expression was evaluated using a combination of paired (time-effects) and unpaired (across study arm) t-tests, and multivariate [least absolute shrinkage and selection operator (LASSO)] modeling. Within the TFV arm, 7% (17/249, p < .05) and 10% (25/249, p < .05) of total proteins changed after 1 and 8 daily applications of TFV gel, respectively, compared to 3% (7/249, p < .05) and 6% (16/249, p < .05) in the HEC arm. Biofunctional analysis associated TFV use with a decrease in epidermal barrier proteins (adj. p = 1.21 × 10−10). Multivariate modeling identified 13 proteins that confidently separated TFV gel users (100% calibration and 96% cross-validation accuracy), including the epithelial integrity factors (FLMNB, CRNN, CALM), serpins (SPB13, SPB5), and cytoskeletal proteins (VILI, VIME, WRD1). This study suggested that daily rectal applications of a 1% TFV gel may be associated with mucosal proteome changes involving epidermal development. Further assessment of more extended use of TFV-gel is recommended to validate

  10. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  11. Molecular characterization and different expression patterns of the muscle ankyrin repeat protein (MARP) family during porcine skeletal muscle development in vitro and in vivo.

    PubMed

    Wang, Linjie; Lei, Minggang; Xiong, Yuanzhu

    2011-04-01

    CARP, ANKRD2, and DARP belong to the ankyrin repeat protein (MARP) family and play a critical role in the integration of cytoskeletal architecture, stress response, and transcriptional regulation. In this study, we cloned the cDNA and promoter sequences of porcine ankyrin repeat protein (MARP) gene family. RT-PCR analysis revealed that porcine CARP gene was predominantly expressed in heart. ANKRD2 was widely expressed in many tissues, a high expression level was observed in the skeletal muscle and heart. DARP gene was expressed specifically in skeletal muscle and heart. Moreover, the expression of CARP and ANKRD2 was significantly different in porcine skeletal muscle among different developmental stages and between the two breeds. Expression analysis in porcine satellite cells showed that CARP and ANKRD2 were induced in differentiated porcine satellite cells, suggesting a role of them in myogenic differentiation. This result suggests that the MARP gene family may be important genes for skeletal muscle growth and provides useful information for further studies on their roles in porcine skeletal muscle.

  12. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  13. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  15. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  16. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  17. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  18. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  19. Expression and Purification of Mini G Proteins from Escherichia coli.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

  20. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  1. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  2. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  3. Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts.

    PubMed

    Pezzetti, Furio; Carinci, Francesco; Palmieri, Annalisa; Vizzotto, Laura; Moscheni, Claudia; Vertemati, Maurizio; Calastrini, Carla; Pellati, Agnese; Stabellini, Giordano

    2009-01-01

    Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts. Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis. In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated. Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.

  4. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  5. Modulation of expression of genes encoding nuclear proteins following exposure to JANUS neutrons or {gamma}-rays

    SciTech Connect

    Woloschak, G.E.; Chang-Liu, Chin-Mei

    1994-05-01

    Previous work has shown that exposure of cells to ionizing radiations causes modulation of a variety of genes, including those encoding c-fos, interleukin-1, tumor necrosis factor, and cytoskeletal elements. The experiments reported herein were designed to examine the effects of either JANUS neutron or {gamma}-ray exposure on expression of genes encoding nucleus-associated proteins (H4-histone, c-jun, c-myc, Rb, and p53). Cycling Syrian hamster embryo cells were irradiated with varying doses and dose rates of either JANUS fission-spectrum neutrons or {gamma}-rays; after incubation of the cell cultures for 1 h following radiation exposure, mRNA was harvested and analyzed by Northern blot. Results revealed induction of transcripts for c-jun, H4-histone, and (to a lesser extent) Rb following {gamma}-ray but not following neutron exposure. Expression of p53 and c-myc genes was unaffected by radiation exposure. Radiations at different doses and dose rates were compared for each of the genes studied.

  6. Survival motor neuron protein deficiency impairs myotube formation by altering myogenic gene expression and focal adhesion dynamics.

    PubMed

    Bricceno, Katherine V; Martinez, Tara; Leikina, Evgenia; Duguez, Stephanie; Partridge, Terence A; Chernomordik, Leonid V; Fischbeck, Kenneth H; Sumner, Charlotte J; Burnett, Barrington G

    2014-09-15

    While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility. Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  7. Ethanol-Induced Changes in the Expression of Proteins Related to Neurotransmission and Metabolism in Different Regions of the Rat Brain

    PubMed Central

    Zahr, Natalie M.; Bell, Richard L.; Ringham, Heather N.; Sullivan, Edith V.; Witzmann, Frank A.; Pfefferbaum, Adolf

    2011-01-01

    Despite extensive description of the damaging effects of chronic alcohol exposure on brain structure, mechanistic explanations for the observed changes are just emerging. To investigate regional brain changes in protein expression levels following chronic ethanol treatment, one rat per sibling pair of male Wistar rats was exposed to intermittent (14 hr/day) vaporized ethanol, the other to air for 26 weeks. At the end of 24 weeks of vapor exposure, the ethanol group had blood ethanol levels averaging 450 mg %, had not experienced a protracted (>16 hr) withdrawal from ethanol, and revealed only mild evidence of hepatic steatosis. Extracted brains were micro-dissected to isolate the prefrontal cortex (PFC), dorsal striatum (STR), corpus callosum genu (CCg), CC body (CCb), anterior vermis (AV), and anterior dorsal lateral cerebellum (ADLC) for protein analysis with two-dimensional gel electrophoresis. Expression levels for 54 protein spots were significantly different between the ethanol- and air- treated groups. Of these 54 proteins, tandem mass spectroscopy successfully identified 39 unique proteins, the levels of which were modified by ethanol treatment: 13 in the PFC, 7 in the STR, 2 in the CCg, 7 in the CCb, 7 in the AV, and 5 in the ADLC. The functions of the proteins altered by chronic ethanol exposure were predominately associated with neurotransmitter systems in the PFC and cell metabolism in the STR. Stress response proteins were elevated only in the PFC, AV, and ADLC perhaps supporting a role for frontocerebellar circuitry disruption in alcoholism. Of the remaining proteins, some had functions associated with cytoskeletal physiology (e.g., in the CCb) and others with transcription/translation (e.g., in the ADLC). Considered collectively, all but 4 of the 39 proteins identified in the present study have been previously identified in ethanol gene- and/or protein- expression studies lending support for their role in ethanol-related brain alterations. PMID

  8. Ethanol-induced changes in the expression of proteins related to neurotransmission and metabolism in different regions of the rat brain.

    PubMed

    Zahr, Natalie M; Bell, Richard L; Ringham, Heather N; Sullivan, Edith V; Witzmann, Frank A; Pfefferbaum, Adolf

    2011-09-01

    Despite extensive description of the damaging effects of chronic alcohol exposure on brain structure, mechanistic explanations for the observed changes are just emerging. To investigate regional brain changes in protein expression levels following chronic ethanol treatment, one rat per sibling pair of male Wistar rats was exposed to intermittent (14 h/day) vaporized ethanol, the other to air for 26 weeks. At the end of 24 weeks of vapor exposure, the ethanol group had blood ethanol levels averaging 450 mg%, had not experienced a protracted (> 16 h) withdrawal from ethanol, and revealed only mild evidence of hepatic steatosis. Extracted brains were micro-dissected to isolate the prefrontal cortex (PFC), dorsal striatum (STR), corpus callosum genu (CCg), CC body (CCb), anterior vermis (AV), and anterior dorsal lateral cerebellum (ADLC) for protein analysis with two-dimensional gel electrophoresis. Expression levels for 54 protein spots were significantly different between the ethanol- and air-treated groups. Of these 54 proteins, tandem mass spectroscopy successfully identified 39 unique proteins, the levels of which were modified by ethanol treatment: 13 in the PFC, 7 in the STR, 2 in the CCg, 7 in the CCb, 7 in the AV, and 5 in the ADLC. The functions of the proteins altered by chronic ethanol exposure were predominantly associated with neurotransmitter systems in the PFC and cell metabolism in the STR. Stress response proteins were elevated only in the PFC, AV, and ADLC perhaps supporting a role for frontocerebellar circuitry disruption in alcoholism. Of the remaining proteins, some had functions associated with cytoskeletal physiology (e.g., in the CCb) and others with transcription/translation (e.g., in the ADLC). Considered collectively, all but 4 of the 39 proteins identified in the present study have been previously identified in ethanol gene- and/or protein-expression studies lending support for their role in ethanol-related brain alterations.

  9. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  10. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  11. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  12. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  13. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ

  14. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  15. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  16. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  17. Non-redundant role of Shc in Erk activation by cytoskeletal reorganization.

    PubMed

    Faisal, Amir; Kleiner, Sandra; Nagamine, Yoshikuni

    2004-01-30

    We have shown previously that cytoskeletal reorganization (CSR) induced by pharmacological reagents such as colchicine or cytochalasins can up-regulate the urokinase-type plasminogen activator (uPA) gene via the Ras/Erk signaling pathway. In this present study using the small interfering RNA technique, we have found that ShcA adapter proteins play a rather active role in CSR-induced Erk activation, contrary to their mostly redundant role in other signaling pathways, e.g. growth factor-induced Erk activation, where Grb2 can bind directly to the receptor tyrosine kinase and activate Erk in the absence of ShcA. ShcA knockdown abolished CSR-induced activation of both Erk and the uPA promoter. Expression of small interfering RNA-escaping silent mutants of p52 or p46 but not p66 ShcA isoform efficiently rescued CSR-induced Erk activation. Moreover, we have shown that phosphorylation of either Tyr-239/Tyr-240 or Tyr-313 in p52(ShcA) can mediate CSR-induced Erk activation equally well. In a quest for molecules upstream of ShcA in this signaling, we found that CSR-induced ShcA tyrosine phosphorylation, its association with Grb2, Erk activation, and uPA gene expression were all dependent on Rho kinase, p38 mitogen-activated protein kinase, and Src. In summary, we have found a novel, non-redundant role for ShcA in contrast to its redundant role in many other signaling pathways.

  18. The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

    PubMed Central

    Malliri, Angeliki; Symons, Marc; Hennigan, Robert F.; Hurlstone, Adam F.L.; Lamb, Richard F.; Wheeler, Tricia; Ozanne, Bradford W.

    1998-01-01

    Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion. PMID:9817764

  19. Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

    PubMed Central

    Wilson, Carlos; González-Billault, Christian

    2015-01-01

    A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons. PMID:26483635

  20. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia.

    PubMed

    Ihnatko, Robert; Edén, Ulla; Lagali, Neil; Dellby, Anette; Fagerholm, Per

    2013-12-06

    Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. © 2013 Elsevier B.V. All rights reserved.

  1. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  2. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  3. Expression and purification of GST-FHL2 fusion protein.

    PubMed

    Yu, H; Ma, Q; Lin, J; Sun, Y F; Zheng, F

    2013-12-06

    Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

  4. Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

    PubMed

    Dias, Rosane Borges; Valverde, Ludmila de Faro; Sales, Caroline Brandi Schlaepfer; Guimarães, Vanessa Sousa Nazaré; Cabral, Márcia Grillo; de Aquino Xavier, Flávia Caló; Dos Santos, Jean Nunes; Ramos, Eduardo Antônio Gonçalves; Gurgel Rocha, Clarissa Araújo

    2016-09-01

    The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.

  5. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  6. Regulation of cytoskeletal organization and junctional remodeling by the atypical cadherin Fat

    PubMed Central

    Marcinkevicius, Emily; Zallen, Jennifer A.

    2013-01-01

    The atypical cadherin Fat is a conserved regulator of planar cell polarity, but the mechanisms by which Fat controls cell shape and tissue structure are not well understood. Here, we show that Fat is required for the planar polarized organization of actin denticle precursors, adherens junction proteins and microtubules in the epidermis of the late Drosophila embryo. In wild-type embryos, spatially regulated cell-shape changes and rearrangements organize cells into highly aligned columns. Junctional remodeling is suppressed at dorsal and ventral cell boundaries, where adherens junction proteins accumulate. By contrast, adherens junction proteins fail to accumulate to the wild-type extent and all cell boundaries are equally engaged in junctional remodeling in fat mutants. The effects of loss of Fat on cell shape and junctional localization, but not its role in denticle organization, are recapitulated by mutations in Expanded, an upstream regulator of the conserved Hippo pathway, and mutations in Hippo and Warts, two kinases in the Hippo kinase cascade. However, the cell shape and planar polarity defects in fat mutants are not suppressed by removing the transcriptional co-activator Yorkie, suggesting that these roles of Fat are independent of Yorkie-mediated transcription. The effects of Fat on cell shape, junctional remodeling and microtubule localization are recapitulated by expression of activated Notch. These results demonstrate that cell shape, junctional localization and cytoskeletal planar polarity in the Drosophila embryo are regulated by a common signal provided by the atypical cadherin Fat and suggest that Fat influences tissue organization through its role in polarized junctional remodeling. PMID:23250217

  7. Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

    PubMed

    Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

    2010-11-01

    At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the β2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/β2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel β subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.

  8. Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants1[w

    PubMed Central

    Mathur, Jaideep; Mathur, Neeta; Hülskamp, Martin

    2002-01-01

    Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect. PMID:11891258

  9. Moesin is activated in cardiomyocytes in experimental autoimmune myocarditis and mediates cytoskeletal reorganization with protrusion formation.

    PubMed

    Miyawaki, Akimitsu; Mitsuhara, Yusuke; Orimoto, Aya; Nakayasu, Yusuke; Tsunoda, Shin-Ichi; Obana, Masanori; Maeda, Makiko; Nakayama, Hiroyuki; Yoshioka, Yasuo; Tsutsumi, Yasuo; Fujio, Yasushi

    2016-08-01

    Acute myocarditis is a self-limiting disease. Most patients with myocarditis recover without cardiac dysfunction in spite of limited capacity of myocardial regeneration. Therefore, to address intrinsic reparative machinery of inflamed hearts, we investigated the cellular dynamics of cardiomyocytes in response to inflammation using experimental autoimmune myocarditis (EAM) model. EAM was induced by immunization of BALB/c mice with α-myosin heavy chain peptides twice. The inflammatory reaction was evoked with myocardial damage with the peak at 3 wk after the first immunization (EAM3w). Morphological and functional restoration started from EAM3w, when active protrusion formation, a critical process of myocardial healing, was observed in cardiomyocytes. Shotgun proteomics revealed that cytoskeletal proteins were preferentially increased in cardiomyocytes at EAM3w, compared with preimmunized (EAM0w) hearts, and that moesin was the most remarkably upregulated among them. Immunoblot analyses demonstrated that the expression of both total and phosphorylated moesin was upregulated in isolated cardiomyocytes from EAM3w hearts. Immunofluorescence staining showed that moesin was localized at cardiomyocyte protrusions at EAM3w. Adenoviral vectors expressing wild-type, constitutively active and inactive form of moesin (wtMoesin, caMoesin, and iaMoesin, respectively) were transfected in neonatal rat cardiomyocytes. The overexpression of wtMoesin and caMoesin resulted in protrusion formation, while not iaMoesin. Finally, we found that cardiomyocyte protrusions were accompanied by cell-cell contact formation. The expression of moesin was upregulated in cardiomyocytes under inflammation, inducing protrusion formation in a phosphorylati