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Sample records for microfilaments

  1. Method and apparatus for testing microfilaments

    DOEpatents

    Schleitweiler, Patrick M.; Merten, Jr., Charles W.

    1995-08-01

    A method and apparatus are disclosed for testing tensile strength of microfilaments. Fibers as small as 0.001 inch in diameter and 0.04 inches in length have been tested, although the method and apparatus of the invention are capable of testing fibers of smaller diameter and length. The invention utilizes a method wherein one or both ends of a microfilament is gripped using resin which is softened sufficiently to accept an end of the microfilament and then allowed to harden. The invention also employs the use of a translation stage capable of controlled three-dimensional movement suited to facilitating gripping of the microfilament.

  2. Method and apparatus for testing microfilaments

    DOEpatents

    Schleitweiler, P.M.; Merten, C.W. Jr.

    1995-08-01

    A method and apparatus are disclosed for testing tensile strength of microfilaments. Fibers as small as 0.001 inch in diameter and 0.04 inches in length have been tested, although the method and apparatus of the invention are capable of testing fibers of smaller diameter and length. The invention utilizes a method wherein one or both ends of a microfilament is gripped using resin which is softened sufficiently to accept an end of the microfilament and then allowed to harden. The invention also employs the use of a translation stage capable of controlled three-dimensional movement suited to facilitating gripping of the microfilament. 2 figs.

  3. Dynamic aspects of microfilament-membrane attachments.

    PubMed

    Jockusch, B M; Wiegand, C; Temm-Grove, C J; Nikolai, G

    1993-01-01

    Microfilament-membrane attachment sites are complex structures that are essential for tissue differentiation in animals. In this article, we focus on the assembly and dynamics of such contact sites as seen in two cell types differentiating in cultures of the embryonic chicken heart, cardiocytes and fibroblasts. Concentrating on the cytoplasmic domain, we refer to previous biochemical, light, and electron microscopic studies on the structure and dynamics of these regions and supplement them with our own recent data. Although many details are still to be elucidated, we would like to propose the following model. Actin, alpha-actinin and vinculin are the major structural components of all microfilament-membrane contacts. Various subtypes of junctions are characterised by additional structural components or by specific isoforms. Temporal regulation of contact sites is linked to assembly and disassembly of microfilaments and might be controlled by special regulatory proteins. Finally, the cytoplasmic domains of junctional complexes may serve as structural matrices for the positioning of proteins involved in signal transduction pathways.

  4. Nomofungin: a new microfilament disrupting agent.

    PubMed

    Ratnayake, A S; Yoshida, W Y; Mooberry, S L; Hemscheidt, T K

    2001-12-28

    A new alkaloid, nomofungin, has been isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa L. The structure of nomofungin was determined by application of spectroscopic methods. The absolute stereochemistry of nomofungin was assigned by using the exciton chirality method. Nomofungin disrupts microfilaments in cultured mammalian cells and is moderately cytotoxic with minimum inhibitory concentrations (MICs) of 2 and 4.5 microg/mL against LoVo and KB cells, respectively. The ring system of nomofungin is unprecedented.

  5. Microfilament distribution in protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Walker, L. M.; Sack, F. D.

    1995-01-01

    Microfilaments were visualized in dark-grown protonemata of the moss Ceratodon to assess their possible role in tip growth and gravitropism. The relative effectiveness of rhodamine phalloidin (with or without m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)) and of immunofluorescence (using the C4 antibody) was evaluated for actin localization in the same cell type. Using immunofluorescence, microfilaments were primarily in an axial orientation within the apical cell. However, a more complex network of microfilaments was observed using rhodamine phalloidin after MBS pretreatment, especially when viewed by confocal laser scanning microscopy. This method revealed a rich three dimensional network of fine microfilaments throughout the apical cell, including the extreme apex. Although there were numerous internal microfilaments, peripheral microfilaments were more abundant. No major redistribution of microfilaments was detected after gravistimulation. The combination of MBS, rhodamine phalloidin, and confocal laser scanning microscopy preserves and reveals microfilaments remarkably well and documents perhaps the most extensive F-actin network visualized to date in any tip-growing cell.

  6. Microfilament distribution in protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Walker, L. M.; Sack, F. D.

    1995-01-01

    Microfilaments were visualized in dark-grown protonemata of the moss Ceratodon to assess their possible role in tip growth and gravitropism. The relative effectiveness of rhodamine phalloidin (with or without m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)) and of immunofluorescence (using the C4 antibody) was evaluated for actin localization in the same cell type. Using immunofluorescence, microfilaments were primarily in an axial orientation within the apical cell. However, a more complex network of microfilaments was observed using rhodamine phalloidin after MBS pretreatment, especially when viewed by confocal laser scanning microscopy. This method revealed a rich three dimensional network of fine microfilaments throughout the apical cell, including the extreme apex. Although there were numerous internal microfilaments, peripheral microfilaments were more abundant. No major redistribution of microfilaments was detected after gravistimulation. The combination of MBS, rhodamine phalloidin, and confocal laser scanning microscopy preserves and reveals microfilaments remarkably well and documents perhaps the most extensive F-actin network visualized to date in any tip-growing cell.

  7. Helical Microfilaments with Alternating Imprinted Intrinsic Curvatures.

    PubMed

    Silva, Pedro Emanuel Santos; Godinho, Maria Helena

    2017-03-01

    There has been an intense research for developing techniques that can produce filaments with helical shapes, given the widespread of potential applications. In this work, how helices with different curvatures can be precisely imprinted in microfilaments is shown. It is also shown that using this technique, it is possible to produce, in a single fiber, helices with different curvatures. This striking and innovative behavior is observed when one side of the stretched filaments is irradiated with UV light, modifying the mechanical properties at surface. Upon release, the regions with higher curvature start to curl first, while regions with lower intrinsic curvature remain stretched until start to curl later. The results presented here can be important to understand why structures adopt a helical shape in general, which can be of interest in nanotechnology, biomolecular science, or even to understand why plant filaments curl. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A Microfilament-eruption Mechanism for Solar Spicules

    NASA Astrophysics Data System (ADS)

    Sterling, Alphonse C.; Moore, Ronald L.

    2016-09-01

    Recent investigations indicate that solar coronal jets result from eruptions of small-scale chromospheric filaments, called minifilaments; that is, the jets are produced by scaled-down versions of typical-sized filament eruptions. We consider whether solar spicules might in turn be scaled-down versions of coronal jets, being driven by eruptions of microfilaments. Assuming a microfilament's size is about a spicule's width (∼300 km), the estimated occurrence number plotted against the estimated size of erupting filaments, minifilaments, and microfilaments approximately follows a power-law distribution (based on counts of coronal mass ejections, coronal jets, and spicules), suggesting that many or most spicules could result from microfilament eruptions. Observed spicule-base Ca ii brightenings plausibly result from such microfilament eruptions. By analogy with coronal jets, microfilament eruptions might produce spicules with many of their observed characteristics, including smooth rise profiles, twisting motions, and EUV counterparts. The postulated microfilament eruptions are presumably eruptions of twisted-core micro-magnetic bipoles that are ∼1.″0 wide. These explosive bipoles might be built and destabilized by merging and cancelation of approximately a few to 100 G magnetic-flux elements of size ≲ 0\\buildrel{\\prime\\prime}\\over{.} 5{--}1\\buildrel{\\prime\\prime}\\over{.} 0. If, however, spicules are relatively more numerous than indicated by our extrapolated distribution, then only a fraction of spicules might result from this proposed mechanism.

  9. Statoliths pull on microfilaments: experiments under microgravity.

    PubMed

    Buchen, B; Braun, M; Hejnowicz, Z; Sievers, A

    1993-01-01

    Previous videomicroscopy of Chara rhizoids during parabolic flights of rockets showed that the weightless statoliths moved basipetally. A hypothesis was offered that the removal of gravity force disturbed the initial balance between this force and the basipetally acting forces generated in a dynamic interaction of statoliths with microfilaments (MFs). The prediction of this hypothesis that the statoliths would not be displaced basipetally during the microgravity phase (MG-phase) after disorganizing the MFs was tested by videomicroscopy of a rhizoid treated with cytochalasin D (CD) immediately before the flight. The prediction was fully supported by the flight experiment. Additionally, by chemical fixation of many rhizoids at the end of the MG-phase it was shown that all rhizoids treated with CD before the flight had statoliths at the same location. i.e., sedimented an the apical cell wall, while all untreated rhizoids had statoliths considerably displaced basipetally from their normal position. Thus, a dynamical interaction involving shearing forces between MFs and statoliths appears highly probable.

  10. Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization.

    PubMed Central

    Staiger, CJ; Cande, WZ

    1991-01-01

    Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis. PMID:12324607

  11. Epithelial microfilament regulators show regional distribution in mouse conjunctiva

    PubMed Central

    Zhu, Hong-Yuan; Riau, A.K.

    2010-01-01

    Purpose The conjunctival epithelium is a continuous sheet of cells with regional characteristics that appear to be similar. This study was designed to investigate the distribution and levels of expression of a subset of microfilament regulators in the forniceal, palpebral, and bulbar conjunctival epithelia. Methods Balb/C mice were used. The localizations of paxillin, focal adhesion kinase, vinculin, talin1, cofilin, profilin, gelsolin, integrin β1, and integrin α6 were studied with the use of cross-sectional immunofluorescent staining. For a detailed cellular analysis, positioning and ablation with the laser microbeam (PALM) Combi System was used to obtain forniceal, bulbar, and palpebral conjunctival epithelia for expression comparison with the use of western blot analysis and quantitative real-time polymerase chain reaction. Results Immunostaining showed that focal adhesion kinase, cofilin, profilin, gelsolin, talin1, and vinculin were expressed in all layers of the forniceal, palpebral, and bulbar conjunctival epithelia. Paxillin, integrin β1, and α6 was found to be located in the basal cell layer in all three of these areas. Quantitative real-time polymerase chain reaction showed that the transcript levels of these microfilament regulators in the forniceal conjunctivae were higher than those levels found in the bulbar and palpebral conjunctivae. Western blot analysis confirmed the differential expression levels of these microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae. Conclusions Differences in the levels of microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae suggest different modes of interaction with their microenvironment and within cell layers. PMID:21151337

  12. Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

    NASA Astrophysics Data System (ADS)

    Luo, Mingzhi; Yang, Zhouqi; Li, Jingbao; Xu, Huiyun; Li, Shengsheng; Zhang, Wei; Qian, Airong; Shang, Peng

    2013-06-01

    We have explored the role of Ca2+ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca2+]i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca2+]i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca2+]i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca2+ influx through stretch-activated channels, then results in microfilament disruption.

  13. Actin Microfilament Organization in the Transition Zone of Arabidopsis-ABD2-GFP Roots Under Clinorotation

    NASA Astrophysics Data System (ADS)

    Shevchenko, Galina

    2012-12-01

    Seedlings of Arabidopsis thaliana-ABD2-GFP were grown under slow clinorotation (2 rpm) and treated with actin and tubulin disrupting drugs in order to characterize the role of actin microfilaments in cell growth and gravisensing. Changes in microfilament organization and cell parameters have shown that the transition root zone (TZ) is rather sensitive to microfilament disruption in control plants. It is assumed that under clinorotation, organization of actin cytoskeleton in the TZ is coordinated in a different way than in the control. Organization of microfilaments depends upon organization of microtubules and clinorotation does not influence this interrelation significantly.

  14. Selective, pulsed CVD of platinum on microfilament gas sensors

    SciTech Connect

    Manginell, R.P.; Smith, J.H.; Ricco, A.J.; Moreno, D.J.; Hughes, R.C.; Huber, R.J.; Senturia, S.D.

    1996-05-01

    A post-processing, selective micro-chemical vapor deposition (``micro-CVD``) technology for the deposition of catalytic films on surface-micromachined, nitride-passivated polysilicon filaments has been investigated. Atmospheric pressure deposition of Pt on microfilaments was accomplished by thermal decomposition of Pt acetylacetonate; deposition occurs selectively only on those filaments which are electrically heated. Catalyst morphology, characterized by SEM, can be controlled by altering deposition time, filament temperature, and through the use of pulsed heating of the filament during deposition. Morphology plays an important role in determining the sensitivity of these devices when used as combustible gas sensors.

  15. Trypanosoma cruzi infection induces microfilament depletion in human placenta syncytiotrophoblast.

    PubMed

    Sartori, M J; Pons, P; Mezzano, L; Lin, S; de Fabro, S P

    2003-08-01

    Congenital Chagas disease, endemic in Latin America, is associated with premature labour, miscarriage, and placentitis. Metacyclic trypomastigotes adhere to specific receptors on the outer membrane of host cells as a prelude to intracellular invasion, causing calcium ion mobilization, rearrangement of host cell microfilaments, recruitment of lysosomes and parasite internalization. The actin cytoskeleton plays an important role in many cellular processes including the parasite invasion into mammalian cells. In order to observe if placental cytoskeleton is altered in the process of parasite invasion into placental villi, actin microfilaments were studied. Using immunohistochemical techniques, it was observed that the presence of actin in the syncytiotrophoblast was intense throughout the brush border in control placentae belonging to non-chagasic women. But after culture with the trypomastigote, this labelling disappeared, indicating that the parasite induced disassembly of the cortical actin cytoskeleton when the placenta was infected. As a control, placentae from chagasic women were studied, and no actin was found. The same results were obtained by the electron microscope. We confirmed that cortical actin rearrangements may be an early step in the Trypanosoma cruzi invasion mechanism into placental cells, in order to allow lysosomes access to the plasma membrane, and formation of the parasitophorous vacuole. The recruitment of lysosomes occurs directly beneath the invasion site, and this process is required for parasite internalization.

  16. Time-sequential observation of spindle and phragmoplast orientation in BY-2 cells with altered cortical actin microfilament patterning.

    PubMed

    Kojo, Kei H; Yasuhara, Hiroki; Hasezawa, Seiichiro

    2014-01-01

    Precise division plane determination is essential for plant development. At metaphase, a dense actin microfilament meshwork appears on both sides of the cell center, forming a characteristic cortical actin microfilament twin peak pattern in BY-2 cells. We previously reported a strong correlation between altered cortical actin microfilament patterning and an oblique mitotic spindle orientation, implying that these actin microfilament twin peaks play a role in the regulation of mitotic spindle orientation. In the present study, time-sequential observation was used to reveal the progression from oblique phragmoplast to oblique cell plate orientation in cells with altered cortical actin microfilament patterning. In contrast to cells with normal actin microfilament twin peaks, oblique phragmoplast reorientation was rarely observed in cells with altered cortical actin microfilament patterning. These results support the important roles of cortical actin microfilament patterning in division plane orientation.

  17. Actin microfilaments in presumptive statocytes of root caps and coleoptiles

    NASA Technical Reports Server (NTRS)

    White, R. G.; Sack, F. D.

    1990-01-01

    Rhodamine-phalloidin was used to determine the distribution of actin microfilament bundles (mfb) in cells thought to be the site of gravity perception (statocytes) in coleoptiles and root caps of Zea mays and Hordeum vulgare. In coleoptile cells, amyloplasts were usually observed in close proximity to thick mfb, which often appeared to divide into finer mfb adjacent to individual amyloplasts. The nucleus in these cells was surrounded by an extensive network of mfb, which were connected to thicker transvacuolar mfb. Columella cells of the root cap contained an extensive reticulum of fine mfb throughout the protoplast, but lacked the much thicker mfb seen in coleoptile cells. The distribution and extent of mfb observed in fixed cells correlates with patterns of streaming and amyloplast movement seen in living cells. A possible role for actin mfb in the perception of gravity is discussed.

  18. Microfilament Depolymerization Is a Pre-requisite for Stem Cell Formation During In vitro Shoot Regeneration in Arabidopsis

    PubMed Central

    Tang, Li Ping; Li, Xiao Ming; Dong, Yu Xiu; Zhang, Xian Sheng; Su, Ying Hua

    2017-01-01

    De novo shoot regeneration is widely used in fundamental studies and agricultural applications. Actin microfilaments are involved in many aspects of plant cell division, cell morphogenesis and cell signal transduction. However, the function of actin microfilaments during de novo shoot regeneration is poorly understood. Here, we investigated the organization of actin microfilaments during this process and found that stem cell formation was associated with microfilament depolymerization. Furthermore, inhibition of microfilament depolymerization by phalloidin treatment or downregulation of actin depolymerizing factors (ADFs) restrained stem cell initiation and shoot regeneration. Inhibition of ADF expression affected the architecture of microfilaments during stem cell formation, and the polar transport and distribution of auxin were also disrupted. Together, our results demonstrate that organization of the microfilament cytoskeleton play important roles in stem cell formation and shoot meristem induction during shoot regeneration. PMID:28261231

  19. The nitrate reductase inhibitor, tungsten, disrupts actin microfilaments in Zea mays L.

    PubMed

    Adamakis, Ioannis-Dimosthenis S; Panteris, Emmanuel; Eleftheriou, Eleftherios P

    2014-05-01

    Tungsten is a widely used inhibitor of nitrate reductase, applied to diminish the nitric oxide levels in plants. It was recently shown that tungsten also has heavy metal attributes. Since information about the toxic effects of tungsten on actin is limited, and considering that actin microfilaments are involved in the entry of tungsten inside plant cells, the effects of tungsten on them were studied in Zea mays seedlings. Treatments with sodium tungstate for 3, 6, 12 or 24 h were performed on intact seedlings and seedlings with truncated roots. Afterwards, actin microfilaments in meristematic root and leaf tissues were stained with fluorescent phalloidin, and the specimens were examined by confocal laser scanning microscopy. While the actin microfilament network was well organized in untreated seedlings, in tungstate-treated ones it was disrupted in a time-dependent manner. In protodermal root cells, the effects of tungsten were stronger as cortical microfilaments were almost completely depolymerized and the intracellular ones appeared highly bundled. Fluorescence intensity measurements confirmed the above results. In the meristematic leaf tissue of intact seedlings, no depolymerization of actin microfilaments was noticed. However, when root tips were severed prior to tungstate application, both cortical and endoplasmic actin networks of leaf cells were disrupted and bundled after 24 h of treatment. The differential response of root and leaf tissues to tungsten toxicity may be due to differential penetration and absorption, while the effects on actin microfilaments could not be attributed to the nitric oxide depletion by tungsten.

  20. Statolith positioning by microfilaments in Chara rhizoids and protonemata

    NASA Astrophysics Data System (ADS)

    Hodick, Dieter; Buchen, Brigitte; Sievers, Andreas

    The rhizoids of the green alga Chara are tip-growing cells with a precise positive gravitropism. In rhizoids growing downwards the statoliths never sediment upon the cell wall at the very tip but keep a minimal distance of approximately 10 μm from the cell vertex. It has been argued that this position is attained by a force acting upon the statoliths in the basal direction and that this force is generated by an interaction between actin microfilaments and myosin on the statolith membrane. This hypothesis received experimental support from (1) effects of the actin-attacking drug cytochalasin, (2) experiments under microgravity conditions, and (3) clinostat experiments. Using video-microscopy it is now shown that this basipetal force also acts on statoliths during sedimentation. As a result, many statoliths in Chara rhizoids do not simply fall along the plumb line while sedimenting during gravistimulation, but move basipetally. This statolith movement is compared to the ones occurring in the unicellular Chara protonemata during gravistimulation. Dark-grown protonemata morphologically closely resemble the rhizoids but respond negatively gravitropic. In contrast to the rhizoids a gravistimulation of the protonemata induces a transport of statoliths towards the tip. This transport is mainly along the cell axis and not parallel to the gravity vector. It is stressed that the sedimentation of statoliths in Chara rhizoids and protonemata as well as in gravity sensing cells in mosses and higher plants is accompanied by statolith movements based on interactions with the cytoskeleton. In tip-growing cells these movements direct the statoliths to a definite region of the cell where they can sediment and elicit a gravitropic curvature. In the statocytes of higher plants the interactions of the statoliths with the cytoskeleton probably do not serve primarily to move the statoliths but to transduce mechanical stresses from the sedimenting statoliths to the plasma membrane.

  1. Confocal microscopic analysis of the microfilament configurations from human vitrification-thawed oocytes matured in vitro.

    PubMed

    Ci, Qianqian; Li, Mei; Zhang, Yingchun; Ma, Shuiying; Gao, Qin; Shi, Yuhua

    2014-01-01

    The alteration in microfilaments of human oocytes by vitrification has not been understood well. To evaluate the effect of vitrification on the microfilaments of human in vitro matured oocytes and the time needed for the repair. Human in vitro matured oocytes were divided into the control group (Group 1) and the vitrified group. The vitrified oocytes were further divided into four sub-groups that were cultured after thawing for 1 h, 2 h, 3 h and 4 h, respectively (Groups 2, 3, 4 and 5). The survival rate of oocytes was 87.4% after vitrification and thawing. The percentage of oocytes with a normal configuration of microfilaments in Group 2 and Group 3 was significantly lower than that of the fresh control group (Group 1), whereas the percentage of oocytes with normal microfilament configuration in Group 4 and Group 5 was comparable to the control group. Vitrification alters microfilament structure of oocytes, which takes at least 3h after thawing for the repair and recovery.

  2. Interrelation between microtubules and microfilaments in the elongation zone of Arabidopsis root under clinorotation

    NASA Astrophysics Data System (ADS)

    Shevchenko, G. V.; Kalinina, Ya. M.; Kordyum, E. L.

    Arabidopsis thaliana plants stably transformed with a MAP4-GFP construct were grown under slow clinorotation to characterize the role of tubulin microtubules in cell growth and gravisensing in the distal elongation zone (DEZ) of the root. Plants were treated with actin and tubulin disrupting drugs to investigate the changes in microtubule distribution and orientation between clinostat grown and control plants. It has been shown that organization of microtubules in the meristem and DEZ of Arabidopsis seedling root is interrelated with the organization of microfilaments. Cooperation between cytoskeleton elements is dependent upon the type of cell differential growth and aimed to provide the stabilization of cell growth on earth. In contrast, under clinorotation the cross-talk between microtubules and microfilaments was less pronounced. This phenomenon suggests the existence of complex mechanism for regulation of microtubules and microfilaments which is probably, unmasked in simulated microgravity.

  3. The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

    PubMed Central

    Nair, Sreelaja; Marlow, Florence; Abrams, Elliott; Kapp, Lee; Mullins, Mary C.; Pelegri, Francisco

    2013-01-01

    Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation. PMID:23637620

  4. Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

    PubMed

    Hesketh, J E; Horne, Z; Campbell, G P

    1991-02-01

    Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

  5. The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division[W][OA

    PubMed Central

    Li, Yanhua; Shen, Yuan; Cai, Chao; Zhong, Chenchun; Zhu, Lei; Yuan, Ming; Ren, Haiyun

    2010-01-01

    Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division. PMID:20709814

  6. Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte.

    PubMed

    Wessel, Gary M; Conner, Sean D; Berg, Linnea

    2002-09-01

    Cortical granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 micro m in diameter (Lytechinus variegatus), many of the cortical granules translocate upwards of 60 micro m to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and cortical granule translocation are inseparable. We also learned that cortical granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though cortical granules do not use them. However, just after GVBD, cortical granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the cortical granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of cortical granules to the egg cortex.

  7. Cytoplasmic streaming emerges naturally from hydrodynamic self-organisation of a microfilament suspension

    NASA Astrophysics Data System (ADS)

    Woodhouse, Francis; Goldstein, Raymond

    2013-03-01

    Cytoplasmic streaming is the ubiquitous phenomenon of deliberate, active circulation of the entire liquid contents of a plant or animal cell by the walking of motor proteins on polymer filament tracks. Its manifestation in the plant kingdom is particularly striking, where many cells exhibit highly organised patterns of flow. How these regimented flow templates develop is biologically unclear, but there is growing experimental evidence to support hydrodynamically-mediated self-organisation of the underlying microfilament tracks. Using the spirally-streaming giant internodal cells of the characean algae Chara and Nitella as our prototype, we model the developing sub-cortical streaming cytoplasm as a continuum microfilament suspension subject to hydrodynamic and geometric forcing. We show that our model successfully reproduces emergent streaming behaviour by evolving from a totally disordered initial state into a steady characean ``conveyor belt'' configuration as a consequence of the cell geometry, and discuss applicability to other classes of steadily streaming plant cells.

  8. Depolymerization of macrophage microfilaments prevents induction and inhibits activity of nitric oxide synthase.

    PubMed

    Fernandes, P D; Araujo, H M; Riveros-Moreno, V; Assreuy, J

    1996-12-01

    We have investigated the relationship between peritoneal murine macrophage cytoskeleton and nitric oxide (NO) synthase (NOS). Activation of the cells with lipopolysaccharide plus interferon-gamma (LI) induced iNOS, detected by nitrite or by labeled L-citrulline production and by a specific antibody against macrophage iNOS. Addition of cytochalasin B (a microfilament-depolymerizing agent) caused a dose-dependent inhibition in NO production by macrophages, whereas colchicine (a microtubule depolymerizing agent) inhibited it only by 20% and not dose-dependently. Addition of cytochalasin B together with LI abolished nitrite and L-citrulline accumulation as well as the amount of iNOS antigen in activated macrophage. Moreover, addition of cytochalasin B 6 or 12 h after stimulus, also decreased the nitrite and L-citrulline production by macrophages although iNOS antigen content by Western blot was the same in the presence or in the absence of cytochalasin B added 12 h after activation. Since cytochalasin B failed to inhibit iNOS activity directly, its inhibitory effects on NO production by macrophages is likely to be indirect, through microfilament network in central regions of cells, but not in filaments seen at pseudopodia or edging processes. Our findings demonstrate that disruption of microfilaments but not of microtubules prevents the iNOS induction process and inhibits its enzymatic activity in activated macrophages.

  9. Cloned pigmented retinal epiehtlium. The role of microfilaments in the differentiation of cell shape

    PubMed Central

    1979-01-01

    3-wk-old clones of pigmented epithelial cells from chick retina can be divided into four zones on the basis of cellular morphology and pigmentation. These zones appear to represent different stages in the re-expression of differentiation: those cells with essentially no differentiated characteristics are at the outer edge and those with the greatest number are at the center. Cells of the colony exhibit three different types of movement when analyzed by time-lapse cinephotomicrography: focal contractions, extension and retraction of apical protrusions, and undulations of the lateral membranes. All the cells of the colony contain microfilaments, 4--7 nm in Diam, which are primarily arranged as apical and basal webs. In addition, less well defined filamentous networks are found in the apical protrusions and lateral interdigitations. When colonies are treated with 10 micrograms/ml of the drug cytochalasin B (CCB), the apical microfilament arrays are disrupted and movement stops. Both phenomena are reversible upon removal of the drug. During the process of redifferentiation, the cells change their shape from squamous to cuboidal, and the greatest change is found where the colony exhibits the greatest number of focal contractions. The evidence suggests that the apical microfilament arrays are directly responsible for the observed movements, particularly the focal contractions, and that focal contractions contribute to the development of the differentiated cellular shape. Possible roles for the other movements are discussed. PMID:572829

  10. Mitosis in Oedogonium: spindle microfilaments and the origin of the kinetochore fiber.

    PubMed

    Schibler, M J; Pickett-Heaps, J D

    1980-10-01

    New ultrastructural observations of mitosis in the closed spindle of Oedogonium cardiacum have been made using cells fixed with glutaraldehyde and tannic acid. Fine filaments 5 to 8 nm in diameter are attached to kinetochores from prophase through anaphase. Some are free in the early division nucleus while others emanate from forming kinetochores at prophase when few if any microtubules (MTs) are inside the nucleus. During prometaphase, MTs invade the nucleus from the poles and appear to interact with the microfilaments. Early in prometaphase, numerous MTs are laterally associated with kinetochores, and the kinetochore fiber is often formed first at one kinetochore of a pair. During metaphase and anaphase, the microfilaments are interspersed among the MTs of these kinetochore fibers. There also is an ill-defined matrix concentrated in the kinetochore fiber, and MTs are often coated irregularly with osmiophilic material. Live mitotic cells of Oedogonium were studied using time lapse cinematography, and we correlate these observations with the above results. We conclude that these microfilaments may constitute one structural component of the traction apparatus that moves chromosomes during metakinesis and anaphase, and that at least some (and possibly many) of the MTs of the kinetochore fiber are derived from those entering the nucleus at prometaphase.

  11. Permeable guidance channels containing microfilament scaffolds enhance axon growth and maturation.

    PubMed

    Cai, Jie; Peng, Xuejun; Nelson, Kevin D; Eberhart, Robert; Smith, George M

    2005-11-01

    Successful peripheral nerve regeneration is still limited in artificial conduits, especially for long lesion gaps. In this study, porous poly(L-lactide-co-DL-lactide, 75:25) (PLA) conduits were manufactured with 16 poly(L-lactide) (PLLA) microfilaments aligned inside the lumen. Fourteen and 18 mm lesion gaps were created in a rat sciatic nerve lesion model. To evaluate the combined effect of permeable PLA conduits and microfilament bundles on axon growth, four types of implants were tested for each lesion gap: PLA conduits with 16 filaments; PLA conduits without filaments; silicone conduits with 16 filaments; and silicone conduits without filaments. Ten weeks following implantation, regeneration within the distal nerve was compared between corresponding groups. Antibodies against the markers S100, calcitonin gene related peptide (CGRP), RMDO95, and P0 were used to identify Schwann cells, unmyelinated axons, myelinated axons, and myelin, respectively. Results demonstrated that the filament scaffold enhanced tissue cable formation and Schwann cell migration in all groups. The filament scaffold enhanced axonal regeneration toward the distal stump, especially across long lesion gaps, but significance was only achieved with PLA conduits. When compared to corresponding silicone conduits, permeable PLA conduits enhanced myelinated axon regeneration across both lesion gaps and achieved significance only in combination with filament scaffolds. Myelin staining indicated PLA conduits supported axon myelination with better myelin quantity and quality when compared to silicone conduits.

  12. Microfilament Orientation Constrains Vesicle Flow and Spatial Distribution in Growing Pollen Tubes

    PubMed Central

    Kroeger, Jens H.; Daher, Firas Bou; Grant, Martin; Geitmann, Anja

    2009-01-01

    Abstract The dynamics of cellular organelles reveals important information about their functioning. The spatio-temporal movement patterns of vesicles in growing pollen tubes are controlled by the actin cytoskeleton. Vesicle flow is crucial for morphogenesis in these cells as it ensures targeted delivery of cell wall polysaccharides. Remarkably, the target region does not contain much filamentous actin. We model the vesicular trafficking in this area using as boundary conditions the expanding cell wall and the actin array forming the apical actin fringe. The shape of the fringe was obtained by imposing a steady state and constant polymerization rate of the actin filaments. Letting vesicle flux into and out of the apical region be determined by the orientation of the actin microfilaments and by exocytosis was sufficient to generate a flux that corresponds in magnitude and orientation to that observed experimentally. This model explains how the cytoplasmic streaming pattern in the apical region of the pollen tube can be generated without the presence of actin microfilaments. PMID:19804712

  13. Microfilament-organizing centers in areas of cell contact: cytoskeletal interactions during cell attachment and locomotion

    PubMed Central

    Geiger, B.; Avnur, Z.; Rinnerthaler, G.; Hinssen, H.; Small, V. J.

    1984-01-01

    In this article we discuss three aspects of cell contact formation: (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane- and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion. We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts: some proteins are specifically associated with focal contacts (vinculin and talin); alpha-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and alpha-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction. Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions. PMID:6430912

  14. [Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo].

    PubMed

    Foucault, G; Raymond, M N; Coffe, G; Pudles, J

    1984-01-01

    Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X-100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.

  15. Tributyltin induces disruption of microfilament in HL7702 cells via MAPK-mediated hyperphosphorylation of VASP.

    PubMed

    Tu, Wei-Wei; Ji, Lin-Dan; Qian, Hai-Xia; Zhou, Mi; Zhao, Jin-Shun; Xu, Jin

    2016-11-01

    Tributyltin (TBT) has been widely used for various industrial purposes, and it has toxic effects on multiple organs and tissues. Previous studies have found that TBT could induce cytoskeletal disruption, especially of the actin filaments. However, the underlying mechanisms remain unclear. The aim of the present study was to determine whether TBT could induce microfilament disruption using HL7702 cells and then to assess for the total levels of various microfilament-associated proteins; finally, the involvement of the MAPK pathway was investigated. The results showed that after TBT treatment, F-actin began to depolymerize and lost its characteristic filamentous structure. The protein levels of Ezrin and Cofilin remained unchanged, the actin-related protein (ARP) 2/3 levels decreased slightly, and the vasodilator-stimulated phosphoprotein (VASP) decreased dramatically. However, the phosphorylation levels of VASP increased 2.5-fold, and the ratio of phosphorylated-VASP/unphosphorylated-VASP increased 31-fold. The mitogen-activated protein kinases (MAPKs) ERK and JNK were discovered to be activated. Inhibition of ERK and JNK not only largely diminished the TBT-induced hyperphosphorylation of VASP but also recovered the cellular morphology and rescued the cells from death. In summary, this study demonstrates that TBT-induced disruption of actin filaments is caused by the hyperphosphorylation of VASP through MAPK pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1530-1538, 2016. © 2015 Wiley Periodicals, Inc.

  16. Evolution of laser microfilaments in the wake of a femtosecond driving pulse

    NASA Astrophysics Data System (ADS)

    Romanov, D. A.; Levis, R. J.

    2013-06-01

    A theoretical model for subnanosecond evolution of a nonequilibrium, inhomogeneous free-electron gas in a laser filament or microfilament wake channel is presented. The spatial distributions of electron density and temperature calculated in axially symmetric geometry as a function of time reveal dynamics on the picosecond time scale that is principally driven by a combination of thermal conduction in the electron gas and impact ionization of residual neutral atoms. At high laser intensity, the electron density evolves into a widened distribution with a sharp edge while the temperature distribution evolves to a central peak surrounded by a wide plateau. At low laser intensity, little change is seen in the electron density while the temperature again evolves to a wide plateau. The calculations show that the homogeneous electron-density approximation becomes progressively invalid at higher laser intensity. Pump-probe experiments addressing Fraunhofer diffraction patterns, four-wave mixing, and dynamic Rabi sidebands are proposed for experimental verification of the results.

  17. [Alterations of microtubule and microfilament expression in spinal cord of carbon disulfide intoxicated rats].

    PubMed

    Pan, Guang-bing; Song, Fu-yong; Zhao, Xiu-lan; Yu, Li-hua; Zhou, Gui-zhen; Xie, Ke-qin

    2007-03-01

    To investigate whether the alterations of microtubule and microfilament expression are responsible for the neurotoxicity of carbon disulfide. Wistar rats were administered with carbon disulfide by gavage at a dosage of 300 or 500 mg/kg for continuous 12 weeks (five times per week). Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet and a corresponding supernatant fraction. Then, the contents of alpha-tubulin, beta-tubulin, and beta-actin in both fractions were determined by immunoblotting. In the meantime, their mRNA levels in spinal cords were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR). In the supernatant fraction, the contents of beta-tubulin and beta-actin in both treated groups increased significantly (P < 0.01) the content of beta-tubulin increased by 141% and 158% respectively, and the content of beta-actin increased by 19% and 32% respectively. In the pellet fraction, the content of beta-tubulin in both groups increased by 107%(P < 0.01) and 118%(P < 0.01) respectively, and the others keep unaffected. In the meantime, the levels of of mRNA expression of beta-tubulin and beta-actin gene were elevated consistently in CS(2)-treated groups (P < 0.01) the levels of mRNA expression of beta-tubulin increased by 207% and 212% respectively, and the levels of mRNA expression of beta-actin increased by 94% and 91% respectively. Carbon disulfide intoxication results in alternations of microtubule and microfilament expression, and the alternations might be related to its neurotoxicity.

  18. Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13762 rat mammary ascites tumor cells

    SciTech Connect

    Vanderpuye, L.A.; Carraway, C.A.C.; Carraway, K.L. )

    1988-10-01

    Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. Phalloidin shift analyses on velocity sedimentation gradients of Triton X-100 extracts of ({sup 3}H)-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Since the Triton-insoluble membrane residue is enriched in actin-containing transmembrane complex, which contains a different glycoprotein, the authors suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

  19. Microfilament regulatory protein MENA increases activity of RhoA and promotes metastasis of hepatocellular carcinoma.

    PubMed

    Lin, Ling; Yang, Xiao-Mei; Li, Jun; Zhang, Yan-Li; Qin, Wenxin; Zhang, Zhi-Gang

    2014-09-10

    Mammalian enabled (MENA), usually known as a direct regulator of microfilament polymerization and bundling, promotes metastasis in various cancers. Here we focus on the role of MENA in hepatocellular carcinoma (HCC) metastasis and the relevant mechanism from the view of RhoA activity regulation. By HCC tissue microarray analysis, we found that MENA expression was positively associated with satellite lesions (P<0.01) and vascular invasion (P<0.01). Cases with membrane reinforcement of MENA staining in HCC tissues had significantly higher rates of early recurrence in the intermediate MENA expression group. Knockdown of MENA significantly suppressed HCC cell migration and invasion in vitro, as well as their intrahepatic and distant metastasis in vivo. Knockdown of MENA also decreased filopodia and stress fibers in SMMC-7721 cells. Furthermore, a decrease of RhoA activity was detected by a pull-down assay in SMMC-7721-shMENA cells. The ROCK inhibitor, Y-27632, suppressed migration of both MENA knockdown SMMC-7721 cells and control cells, but diminished their difference. Thus, our findings suggest that MENA promotes HCC cell motility by activating RhoA.

  20. The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Calarco, Patricia G.

    2005-04-01

    Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

  1. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots.

    PubMed

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-07-01

    The plant cytoskeleton plays a crucial role in the cells' growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton.

  2. Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization

    PubMed Central

    Woodhouse, Francis G.; Goldstein, Raymond E.

    2013-01-01

    Many cells exhibit large-scale active circulation of their entire fluid contents, a process termed cytoplasmic streaming. This phenomenon is particularly prevalent in plant cells, often presenting strikingly regimented flow patterns. The driving mechanism in such cells is known: myosin-coated organelles entrain cytoplasm as they process along actin filament bundles fixed at the periphery. Still unknown, however, is the developmental process that constructs the well-ordered actin configurations required for coherent cell-scale flow. Previous experimental works on streaming regeneration in cells of Characean algae, whose longitudinal flow is perhaps the most regimented of all, hint at an autonomous process of microfilament self-organization driving the formation of streaming patterns during morphogenesis. Working from first principles, we propose a robust model of streaming emergence that combines motor dynamics with both microscopic and macroscopic hydrodynamics to explain how several independent processes, each ineffectual on its own, can reinforce to ultimately develop the patterns of streaming observed in the Characeae and other streaming species. PMID:23940314

  3. Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization.

    PubMed

    Woodhouse, Francis G; Goldstein, Raymond E

    2013-08-27

    Many cells exhibit large-scale active circulation of their entire fluid contents, a process termed cytoplasmic streaming. This phenomenon is particularly prevalent in plant cells, often presenting strikingly regimented flow patterns. The driving mechanism in such cells is known: myosin-coated organelles entrain cytoplasm as they process along actin filament bundles fixed at the periphery. Still unknown, however, is the developmental process that constructs the well-ordered actin configurations required for coherent cell-scale flow. Previous experimental works on streaming regeneration in cells of Characean algae, whose longitudinal flow is perhaps the most regimented of all, hint at an autonomous process of microfilament self-organization driving the formation of streaming patterns during morphogenesis. Working from first principles, we propose a robust model of streaming emergence that combines motor dynamics with both microscopic and macroscopic hydrodynamics to explain how several independent processes, each ineffectual on its own, can reinforce to ultimately develop the patterns of streaming observed in the Characeae and other streaming species.

  4. Two classes of actin microfilaments are associated with the inner cytoskeleton of axons

    PubMed Central

    1988-01-01

    The distribution and length of actin microfilaments (MF) was determined in axoplasm extruded from the giant axons of the squid (Loligo pealeii). Extruded axoplasm that was separated from the axonal cortex contains approximately 92% of the total axonal actin, and 60% of this actin is polymerized (Morris, J., and R. Lasek. 1984. J. Cell Biol. 98:2064-2076). Localization of MF with rhodamine-phalloidin indicated that the MF were organized in fine columns oriented longitudinally within the axoplasm. In the electron microscope, MF were surrounded by a dense matrix and they were associated with the microtubule domains of the axoplasm. The surrounding matrix tended to obscure the MF which may explain why MF have rarely been recognized before in the inner regions of the axon. The axoplasmic MF are relatively short (number average length of 0.55 micron). Length measurements of MF prepared either in the presence or absence of the actin-filament stabilizing drug phalloidin indicate that axoplasm contains two populations of MF: stable MF (number average length of 0.79 micron) and metastable MF (number average length of 0.41 micron). Although individual axonal MF are much shorter than axonal microtubules, the combined length of the total MF is twice that of the total microtubules. Apparently, these numerous short MF have an important structural role in the architecture of the inner axonal cytoskeleton. PMID:3417765

  5. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

    PubMed Central

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-01-01

    The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

  6. Effect of thiol-oxidation of glutathione with diamide on corneal endothelial function, junctional complexes, and microfilaments.

    PubMed

    Edelhauser, H F; Van Horn, D L; Miller, P; Pederson, H J

    1976-03-01

    Intracellular-reduced glutathione (GSH) was removed by thiol-oxidation with diamide during in vitro perfusion of the corneal endothelium. By 15 min the normal mosaic-like pattern of the endothelial cells was disrupted by serpentine-like lines of cell separation at the cell juntions. After 45 min of perfusion, infividual clusters of cells formed cup-shaped islands. The resultant exposure of Descemet's membrane to the perfusion solution resulted in corneal swelling. Transmission electron microscopy revealed that the endothelial cells separated at the apical junctions and that the microfilaments in the apical cytoplasm of cells formed dense bands, whereas the other subcellular organelles were normal in appearance. The change in cellular shape may be due to loss of cellular adhesion which results in the condensation of the microfilaments or contraction of the microfilaments. The addition of glucose to the perfusate prevented the diamide effect, and the diamide effect could be reversed upon removal and perfusion of a glutathione bicarbonate Ringer's solution. These results suggest that the ratio of reduced to oxidized glutathione in the endothelial cells plays a role in the maintenance of the endothelial cell barrier function.

  7. Effect of thiol-oxidation of glutathione with diamide on corneal endothelial function, junctional complexes, and microfilaments

    PubMed Central

    1976-01-01

    Intracellular-reduced glutathione (GSH) was removed by thiol-oxidation with diamide during in vitro perfusion of the corneal endothelium. By 15 min the normal mosaic-like pattern of the endothelial cells was disrupted by serpentine-like lines of cell separation at the cell juntions. After 45 min of perfusion, infividual clusters of cells formed cup-shaped islands. The resultant exposure of Descemet's membrane to the perfusion solution resulted in corneal swelling. Transmission electron microscopy revealed that the endothelial cells separated at the apical junctions and that the microfilaments in the apical cytoplasm of cells formed dense bands, whereas the other subcellular organelles were normal in appearance. The change in cellular shape may be due to loss of cellular adhesion which results in the condensation of the microfilaments or contraction of the microfilaments. The addition of glucose to the perfusate prevented the diamide effect, and the diamide effect could be reversed upon removal and perfusion of a glutathione bicarbonate Ringer's solution. These results suggest that the ratio of reduced to oxidized glutathione in the endothelial cells plays a role in the maintenance of the endothelial cell barrier function. PMID:1035910

  8. The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

    PubMed

    Collings, David A; Harper, John D I; Vaughn, Kevin C

    2003-12-01

    We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

  9. Ezrin: a regulator of actin microfilaments in cell junctions of the rat testis

    PubMed Central

    Gungor-Ordueri, N Ece; Celik-Ozenci, Ciler; Cheng, C Yan

    2015-01-01

    Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis. PMID:25652626

  10. Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components.

    PubMed

    Head, Brian P; Patel, Hemal H; Roth, David M; Murray, Fiona; Swaney, James S; Niesman, Ingrid R; Farquhar, Marilyn G; Insel, Paul A

    2006-09-08

    Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.

  11. Microtubules and Microfilaments in Fixed and Permeabilized Cells are Selectively Decorated by Nerve Growth Factor

    NASA Astrophysics Data System (ADS)

    Nasi, S.; Cirillo, D.; Naldini, L.; Marchisio, P. C.; Calissano, P.

    1982-02-01

    A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PC12 cells) and nontarget (3T3, BKH 2I, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form of stress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies. Binding of 125I-labeled NGF to PC12 cells was not competitively inhibited by a 100-fold excess of several positively charged proteins but it was markedly decreased in the presence of DNase I. 125I-Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range of concentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.

  12. Transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized IPEC-J2 cells.

    PubMed

    Zhao, Shanshan; Gao, Junkai; Zhu, Liqi; Yang, Qian

    2014-11-04

    Viral infection converts the normal constitution of a cell to optimise viral entry, replication, and virion production. These conversions contain alterations or disruptions of the tight and adherens junctions between cells as part of their pathogenesis, and reorganise cellular microfilaments that initiate, sustain and spread the viral infections and so on. Using porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV) and a model of normal intestinal epithelial cells (IPEC-J2), we researched the interaction between tight and adherens junctions and microfilaments of IPEC-J2 cells with these viruses. In our work, the results showed that IPEC-J2 cells were susceptible to TGEV and PEDV infection. And TGEV could impair the barrier integrity of IPEC-J2 cells at early stages of infection through down-regulating some proteins of tight and adherens junctions, while PEDV cloud cause a slight of damage in the integrity of epithelial barrier. In addition, they also could affect the microfilaments remodelling of IPEC-J2 cells, and the drug-interfered microfilaments could inhibit viral replication and release. Furthermore, PEDV+TGEV co-infection was more aggravating to damage of tight junctions and remodelling of microfilaments than their single infection. Finally, the PEDV and TGEV infection affected the MAPK pathway, and inhibition of MAPK pathway regulated the changes of tight junctions and microfilaments of cells. These studies provide a new insight from the perspective of the epithelial barrier and microfilaments into the pathogenesis of PEDV and TGEV. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability.

    PubMed Central

    Ji, Jun-Yuan; Haghnia, Marjan; Trusty, Cory; Goldstein, Lawrence S B; Schubiger, Gerold

    2002-01-01

    Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage. PMID:12454065

  14. Enrichment of distinct microfilament-associated and GTP-binding-proteins in membrane/microvilli fractions from lymphoid cells

    PubMed Central

    Hao, Jian-Jiang; Wang, Guanghui; Pisitkun, Trairak; Patino-Lopez, Genaro; Nagashima, Kunio; Knepper, Mark A.; Shen, Rong-Fong; Shaw, Stephen

    2008-01-01

    Summary Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shear-based procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by LC/MS/MS analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G. Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament- and microtubule-related proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells. Lymphocyte microvilli initiate adhesion to endothelium during movement from blood into tissue. Using LC/MS/MS and label

  15. Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

    PubMed Central

    Higaki, Takumi; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro

    2008-01-01

    Background Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown. Results Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2) fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER) revealed that MFs were involved in

  16. Centrifugation causes adaptation of microfilaments: studies on the transport of statoliths in gravity sensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1993-01-01

    The actin cytoskeleton is involved in the positioning of statoliths in tip growing Chara rhizoids. The balance between the acropetally acting gravity force and the basipetally acting net outcome of cytoskeletal force results in the dynamically stable position of the statoliths 10-30 micrometers above the cell tip. A change of the direction and/or the amount of one of these forces in a vertically growing rhizoid results in a dislocation of statoliths. Centrifugation was used as a tool to study the characteristics of the interaction between statoliths and microfilaments (MFs). Acropetal and basipetal accelerations up to 6.5 g were applied with the newly constructed slow-rotating-centrifuge-microscope (NIZEMI). Higher accelerations were applied by means of a conventional centrifuge, namely acropetally 10-200 g and basipetally 10-70 g. During acropetal accelerations (1.4-6g), statoliths were displaced to a new stable position nearer to the cell vertex (12-6.5 micrometers distance to the apical cell wall, respectively), but they did not sediment on the apical cell wall. The original position of the statoliths was reestablished within 30 s after centrifugation. Sedimentation of statoliths and reduction of the growth rates of the rhizoids were observed during acropetal accelerations higher than 50 g. When not only the amount but also the direction of the acceleration were changed in comparison to the natural condition, i.e., during basipetal accelerations (1.0-6.5 g), statoliths were displaced into the subapical zone (up to 90 micrometers distance to the apical cell wall); after 15-20 min the retransport of statoliths to the apex against the direction of acceleration started. Finally, the natural position in the tip was reestablished against the direction of continuous centrifugation. Retransport was observed during accelerations up to 70 g. Under the 1 g condition that followed the retransported statoliths showed an up to 5-fold increase in sedimentation time onto the

  17. A stochastic thermostat algorithm for coarse-grained thermomechanical modeling of large-scale soft matters: Theory and application to microfilaments

    SciTech Connect

    Li, Tong; Gu, YuanTong

    2014-04-15

    As all-atom molecular dynamics method is limited by its enormous computational cost, various coarse-grained strategies have been developed to extend the length scale of soft matters in the modeling of mechanical behaviors. However, the classical thermostat algorithm in highly coarse-grained molecular dynamics method would underestimate the thermodynamic behaviors of soft matters (e.g. microfilaments in cells), which can weaken the ability of materials to overcome local energy traps in granular modeling. Based on all-atom molecular dynamics modeling of microfilament fragments (G-actin clusters), a new stochastic thermostat algorithm is developed to retain the representation of thermodynamic properties of microfilaments at extra coarse-grained level. The accuracy of this stochastic thermostat algorithm is validated by all-atom MD simulation. This new stochastic thermostat algorithm provides an efficient way to investigate the thermomechanical properties of large-scale soft matters.

  18. Effects of Phorbol Esters and Lipopolysaccharide on Endothelial Cell Microfilaments: Laser Scanning Confocal Microscopy and Quantitative Morphometry of Dose Dependent Changes

    DTIC Science & Technology

    1988-11-29

    the same ECmo (1-5 nM) for both biochemical and morphological processes. -PDB was less potent in inducing the disruption of microfilament structure...but the agent was less potent than PMA, with an ECmo of about 80 nM. The agent 4a-phorbol, which is not a tumor-promoter, had no effect on the

  19. A novel anti-microfilament antibody, anti-135 kD, is associated with Raynaud's disease, undifferentiated connective tissue disease and systemic autoimmune diseases.

    PubMed

    Girard, D; Senécal, J L

    1996-01-01

    We report herein the characterization of a human IgG antibody reactive with a nonmuscle 135 kD microfilament-associated protein, anti-135 kD. Using nonmuscle epithelial PtK2 cells as substrate in indirect immunofluorescence, we identified a distinctive pattern of reactivity with microfilaments in sera from 12 of 165 (7.3%) patients investigated for systemic autoimmune diseases and in only 2 of 171 (1.2%) normal and rheumatic disease controls (P < 0.006, 95% Cl 1.46 to 30.1). An association between anti-135 kD and Raynaud's phenomenon (n = 12/14, 85.7%) with or without an associated systemic autoimmune disease was noted. The anti-135 kD specificity was established by several criteria. (1) The fluorescence was periodically distributed along microfilaments and concentrated at focal adhesions for all sera (n = 14). (2) On immunoblots, the 14 sera reacted with a PtK2 polypeptide of 135 kD. (3) IgG purified by blot-affinity from the 135 kD band (alpha-135) reproduced the fluorescent pattern of the original sera while IgG purified from other bands did not. (4) Double immunofluorescence with alpha-135 and anti-alpha-actinin mAb indicated absence of antibody fluorescence at ruffling membranes where a-actinin was distributed. (5) IgG subclass analysis of anti-135 kD revealed that 12 (85.7%) sera are of IgG3 isotype and 2 (14.3%) are of IgG1 isotype while the light chain expression was kappa restricted. This is the first report of an antibody to a 135 kD microfilament protein. Anti-135 kD expand the repertoire of anti-microfilament and anticytoskeletal antibodies in human sera.

  20. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells.

    PubMed

    Chardin, P; Boquet, P; Madaule, P; Popoff, M R; Rubin, E J; Gill, D M

    1989-04-01

    Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

  1. ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis

    PubMed Central

    Lin, Deshu; Dhonukshe, Pankaj; Zhang, Xingxing; Friml, Jiri; Scheres, Ben; Fu, Ying; Yang, Zhenbiao

    2012-01-01

    Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis. PMID:22509133

  2. MicroFilament Analyzer, an image analysis tool for quantifying fibrillar orientation, reveals changes in microtubule organization during gravitropism.

    PubMed

    Jacques, Eveline; Buytaert, Jan; Wells, Darren M; Lewandowski, Michal; Bennett, Malcolm J; Dirckx, Joris; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-06-01

    Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high-throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi-stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  3. Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments

    PubMed Central

    Zimmermann, Pascale; Tomatis, Daniela; Rosas, Marcela; Grootjans, Johan; Leenaerts, Iris; Degeest, Gisèle; Reekmans, Gunter; Coomans, Christien; David, Guido

    2001-01-01

    Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, β-catenin, and α-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes. PMID:11179419

  4. VLN2 Regulates Plant Architecture by Affecting Microfilament Dynamics and Polar Auxin Transport in Rice[OPEN

    PubMed Central

    Wu, Shengyang; Xie, Yurong; Guo, Xiuping; Sheng, Peike; Wang, Juan; Wu, Chuanyin; Wang, Haiyang; Wan, Jianmin

    2015-01-01

    As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport. PMID:26486445

  5. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells

    PubMed Central

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L. Andrew; Mineyuki, Yoshinobu

    2016-01-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. PMID:27053663

  6. The force induced by organelles' weight in the microfilament is in the range of 0.1-1 pN

    NASA Astrophysics Data System (ADS)

    Yang, Chun; Wei, Dong; Zhuang, Feng Y.

    It has been well documented that a microgravity environment can bring about many changes in cell metabolism. Can mammalian cells feel the gravity directly? At present, arguments surrounding the problem are difficult to be answered through experiments. However, using finite element simulation to estimate the force exerted on the microfilament meshwork model, we demonstrated a possible way through which gravity acts on the cytoskeleton system. This system, which includes microfilaments, microtubules, and intermediate filaments, is responsible for the retention of cell shape and plays a role in many aspects related to cell proliferation and function. Many organelles, such as ribosomes and nucleus, are deposited, hinged, or attached on the cytoskeleton system. The weight of organelles can deform the cytoskeleton system and can induce force in it. Simulation results showed that the force induced by organelles' weight in the microfilament is in the range of 0.1-1 pN. The magnitude of the force is near the single Van der Waals bond force between the proteins, which is large enough to influence the hinge motion of proteins.

  7. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

    PubMed

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L Andrew; Mineyuki, Yoshinobu

    2016-06-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. © 2016 Takeuchi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments.

    PubMed

    Angel, Carlos A; Lutz, Lindy; Yang, Xiaohua; Rodriguez, Andres; Adair, Adam; Zhang, Yu; Leisner, Scott M; Nelson, Richard S; Schoelz, James E

    2013-09-01

    The gene VI product, protein 6 (P6), of Cauliflower mosaic virus (CaMV) assembles into large, amorphous inclusion bodies (IBs) that are considered sites for viral protein synthesis and viral genome replication and encapsidation. P6 IBs align with microfilaments and require them for intracellular trafficking, a result implying that P6 IBs function to move virus complexes or virions within the cell to support virus physiology. Through a yeast two-hybrid screen we determined that CHUP1, a plant protein allowing chloroplast transport through an interaction with chloroplast and microfilament, interacts with P6. The interaction between CHUP1 and P6 was confirmed through colocalization in vivo and co-immunoprecipitation assays. A truncated CHUP1 fused with enhanced cyan fluorescent protein, unable to transport chloroplasts, inhibited intracellular movement of P6-Venus inclusions. Silencing of CHUP1 in N. edwardsonii impaired the ability of CaMV to infect plants. The findings suggest that CHUP1 supports CaMV infection through an interaction with P6.

  9. Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape

    PubMed Central

    1988-01-01

    Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA- modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture. PMID:3276711

  10. 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.

  11. Contraction-mediated pinocytosis of RGD-peptide by dermal fibroblasts: inhibition of matrix attachment blocks contraction and disrupts microfilament organisation.

    PubMed

    Sethi, K K; Mudera, V; Sutterlin, R; Baschong, W; Brown, R A

    2002-08-01

    Force generation in collagen and matrix contraction are basic functions of fibroblasts and important elements of tissue repair. Cell-matrix attachment is critical to this contraction, involving RGD-binding integrins. We have investigated how this process operates, in terms of force generation (in the Culture Force Monitor) and cytoskeletal structure, using a synthetic RGD-decapeptide. The RGD-peptide blocked force generation over the first 6 h, followed by near complete recovery by 20 h. However, dose response was complex indicating multiple processes were operating. Analysis of cytoskeletal structure after treatment with RGD-peptide indicated major disruption with condensed aggregates of actin and microtubular fragmentation. Fluorescent labeling and tracking of the RGD-peptide demonstrated intracellular uptake into discrete cytoplasmic aggregates. Critically, these RGD-peptide pools co-localised with the condensed actin microfilament aggregates. It is concluded that RGD-peptide uptake was by a form of contraction-mediated pinocytosis, resulting from mechanical tension applied to the untethered RGD-peptide-integrin, as contractile microfilament were assembled. These findings emphasize the importance of sound mechanical attachment of ligand-occupied integrins (e.g., to extracellular matrix) for normal cytoskeletal function. Conversely, this aspect of unrestrained cytoskeletal contraction may have important pathogenic and therapeutic applications. Copyright 2002 Wiley-Liss, Inc.

  12. Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats.

    PubMed

    Chen, Haiqi; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2016-05-01

    Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis.

  13. The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules1[W][OA

    PubMed Central

    Harries, Phillip A.; Palanichelvam, Karuppaiah; Yu, Weichang; Schoelz, James E.; Nelson, Richard S.

    2009-01-01

    The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV. PMID:19028879

  14. Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats

    PubMed Central

    Chen, Haiqi; Mruk, Dolores D.; Lee, Will M.

    2016-01-01

    Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis. PMID:26990065

  15. A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

    NASA Astrophysics Data System (ADS)

    AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

    2015-01-01

    Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such

  16. Microfilament-mediated surface change in starfish oocytes in response to 1-methyladenine: implications for identifying the pathway and receptor sites for maturation-inducing hormones

    PubMed Central

    1981-01-01

    Oocytes of the starfish Pisaster ochraceus exhibit an early response to 1-methyladenine (the maturation-inducing hormone), which is described for the first time. In this response approximately 6,500 spikelike surface projections, much larger than microvilli, emerge transiently from oocytes stripped of their follicle cells and then treated with the hormone in vitro. Each spike contains a prominent bundle of microfilaments, possibly composed of actin. The distribution of spikes when follicle cells are only partially removed and the morphological details of the normal junctional association between follicle cells and oocytes suggest that 1-methyladenine-sensitive sites (receptor sites) can be identified with the approximately 6,500 postjunctional specializations that are part of the oocyte surface. This finding in turn is employed to construct a set of hypotheses concerning the route that 1-methyladenine normally takes from the follicle cells to an oocyte during stimulation of maturation; it is postulated that, for each oocyte, 1-methyladenine is transported along approximately 6,500 thin follicle-cell processes, it is transmitted across the junctional gaps of an equivalent number of junctions between follicle cells and an oocyte, and then interacts with the postjunctional sites where 1- methyladenine receptors are thought to be clustered. Comparative aspects of this mode of intercellular communication are discussed. PMID:6270153

  17. Tau dephosphorylation and microfilaments disruption are upstream events of the anti-proliferative effects of DADS in SH-SY5Y cells

    PubMed Central

    Aquilano, Katia; Vigilanza, Paola; Filomeni, Giuseppe; Rotilio, Giuseppe; Ciriolo, Maria R

    2010-01-01

    Abstract Garlic organosulphur compounds have been successfully used as redox anti-proliferative agents. In this work, we dissect the effects of diallyl disulphide (DADS) focusing on the events upstream of cell cycle arrest and apoptosis induced in neuroblastoma SH-SY5Y cells. We demonstrate that DADS is able to cause early morphological changes, cytoskeleton oxidation, microfilaments reduction and depolymerization of microtubules. These events are attenuated in cells stably overexpressing the antioxidant enzyme SOD1, suggesting that superoxide plays a crucial role in destabilizing cytoskeleton. Moreover, we evidence that the main microtubules-associated protein Tau undergoes PP1-mediated dephosphorylation as demonstrated by treatment with okadaic acid as well as by immunoreaction with anti-Tau-1 antibody, which specifically recognizes its dephosphorylated forms. Tau dephosphorylation is inhibited by the two-electron reductants NAC and GSH ester but not by SOD1. The inability of DADS to induce apoptosis in neuroblastoma-differentiated cells gives emphasis to the anti-proliferative activity of DADS, which can be regarded as a promising potent anti-neuroblastoma drug by virtue of its widespread cytoskeleton disrupting action on proliferating cells. PMID:19040422

  18. Actin microfilaments participate in the regulation of the COL1A1 promoter activity in ROS17/2.8 cells under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Dai, Zhongquan; Li, Yinghui; Ding, Bai; Zhang, Xiaoyou; Tan, Yingjun; Wan, Yumin

    2006-01-01

    IntroductionMicrogravity is thought to decrease osteoblastic activity and induce osteoporosis during spaceflight, but the mechanisms, particularly the attendant changes in gene expression, are not well understood. It is suspected that the cytoskeletal system is involved in the manifold changes of cell shape, function, and signaling under microgravity conditions. MethodsWe constructed cell lines stably transfected with pJI36EGFP and pJI23EGFP, which contained a 3.6 and a 2.3 kb fragment, respectively, of the α1(I) collagen gene (COL1A1) promoter fused with the enhanced green fluorescence protein (EGFP) reporter gene. We then developed a semi-quantitative analysis of EGFP fluorescence intensity to evaluate the effects of clinorotation and/or cytochalasin B on the activity of the COL1A1 promoter. Simultaneously, we assessed the collagen type I protein content versus total protein content in clinorotated or control osteoblasts, using immunocytochemistry and the Bradford method, respectively. ResultsThe fluorescence intensity analysis revealed that the expression of COL1A1-EGFP increased in GFP-ROS cells clinorotated for 24 or 48 h, as compared with stationary control cultures. We observed a similar trend in collagen type I content, as assessed by immunocytochemistry. We found that the osteoblast microfilaments tended to disassemble and show a reduction in stress fibers under space flight and clinorotation. Treatment with cytochalasin B in normal gravity resulted in a dose-dependent increase of EGFP fluorescence intensity, indicating that disruption of the actin system was associated with increased activity of the COL1A1 promoter. ConclusionOur study demonstrates that disrupting the actin cytoskeleton by treatment with cytochalasin B and real or simulated microgravity conditions led to altered COL1A1 promoter activity. Together, these results suggest that actin may participate in the regulation of the COL1A1 promoter activity under microgravity conditions.

  19. Trapping and wiggling: elastohydrodynamics of driven microfilaments.

    PubMed Central

    Wiggins, C H; Riveline, D; Ott, A; Goldstein, R E

    1998-01-01

    We present an analysis of the planar motion of single semiflexible filaments subject to viscous drag or point forcing. These are the relevant forces in dynamic experiments designed to measure biopolymer bending moduli. By analogy with the "Stokes problems" in hydrodynamics (motion of a viscous fluid induced by that of a wall bounding the fluid), we consider the motion of a polymer, one end of which is moved in an impulsive or oscillatory way. Analytical solutions for the time-dependent shapes of such moving polymers are obtained within an analysis applicable to small-amplitude deformations. In the case of oscillatory driving, particular attention is paid to a characteristic length determined by the frequency of oscillation, the polymer persistence length, and the viscous drag coefficient. Experiments on actin filaments manipulated with optical traps confirm the scaling law predicted by the analysis and provide a new technique for measuring the elastic bending modulus. Exploiting this model, we also present a reanalysis of several published experiments on microtubules. PMID:9533717

  20. Trapping and wiggling: elastohydrodynamics of driven microfilaments.

    PubMed

    Wiggins, C H; Riveline, D; Ott, A; Goldstein, R E

    1998-02-01

    We present an analysis of the planar motion of single semiflexible filaments subject to viscous drag or point forcing. These are the relevant forces in dynamic experiments designed to measure biopolymer bending moduli. By analogy with the "Stokes problems" in hydrodynamics (motion of a viscous fluid induced by that of a wall bounding the fluid), we consider the motion of a polymer, one end of which is moved in an impulsive or oscillatory way. Analytical solutions for the time-dependent shapes of such moving polymers are obtained within an analysis applicable to small-amplitude deformations. In the case of oscillatory driving, particular attention is paid to a characteristic length determined by the frequency of oscillation, the polymer persistence length, and the viscous drag coefficient. Experiments on actin filaments manipulated with optical traps confirm the scaling law predicted by the analysis and provide a new technique for measuring the elastic bending modulus. Exploiting this model, we also present a reanalysis of several published experiments on microtubules.

  1. Microfilament-coordinated adhesion dynamics drives single cell migration and shapes whole tissues

    PubMed Central

    Aguilar-Cuenca, Rocio; Llorente-Gonzalez, Clara; Vicente, Carlos; Vicente-Manzanares, Miguel

    2017-01-01

    Cell adhesion to the substratum and/or other cells is a crucial step of cell migration. While essential in the case of solitary migrating cells (for example, immune cells), it becomes particularly important in collective cell migration, in which cells maintain contact with their neighbors while moving directionally. Adhesive coordination is paramount in physiological contexts (for example, during organogenesis) but also in pathology (for example, tumor metastasis). In this review, we address the need for a coordinated regulation of cell-cell and cell-matrix adhesions during collective cell migration. We emphasize the role of the actin cytoskeleton as an intracellular integrator of cadherin- and integrin-based adhesions and the emerging role of mechanics in the maintenance, reinforcement, and turnover of adhesive contacts. Recent advances in understanding the mechanical regulation of several components of cadherin and integrin adhesions allow us to revisit the adhesive clutch hypothesis that controls the degree of adhesive engagement during protrusion. Finally, we provide a brief overview of the major impact of these discoveries when using more physiological three-dimensional models of single and collective cell migration. PMID:28299195

  2. Processive motions of MreB micro-filaments coordinate cell wall growth

    NASA Astrophysics Data System (ADS)

    Garner, Ethan

    2012-02-01

    Rod-shaped bacteria elongate by the action of cell-wall synthesis complexes linked to underlying dynamic MreB filaments, but how these proteins function to allow continued elongation as a rod remains unknown. To understand how the movement of these filaments relates to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-resolution particle tracking in Bacillus subtilis. We found that both MreB and the elongation machinery move in linear paths across the cell, moving at similar rates (˜20nm / second) and angles to the cell body, suggesting they function as single complexes. These proteins move circumferentially around the cell, principally perpendicular to its length. We find that the motions of these complexes are independent, as they can pause and reverse,and also as nearby complexes move independently in both directions across one surface of the cell. Inhibition of cell wall synthesis with antibiotics or depletions in the cell wall synthesis machinery blocked MreB movement, suggesting that the cell wall synthetic machinery is the motor in this system. We propose that bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that span the plasma membrane and insert radial hoops of new peptidoglycan during their transit.

  3. Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites.

    PubMed

    Olshina, Maya A; Wong, Wilson; Baum, Jake

    2012-05-01

    Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.

  4. The role of host microfilaments and microtubules during opsonin-independent interactions of Cryptococcus neoformans with mammalian lung cells.

    PubMed

    Choo, K K; Chong, P P; Ho, A S H; Yong, P V C

    2015-12-01

    The purpose of this investigation was to characterise the interactions of Cryptococcus neoformans with mammalian host alveolar epithelial cells and alveolar macrophages, with emphasis on the roles of the cryptococcal capsule and the host cell cytoskeletons. The adherence and internalisation of C. neoformans into mammalian lung cells and the roles of host cell cytoskeletons in host-pathogen interactions were studied using in vitro models coupled with a differential fluorescence assay, fluorescence staining, immunofluorescence and drug inhibition of actin and microtubule polymerisation. Under conditions devoid of opsonin and macrophage activation, C. neoformans has a high affinity towards MH-S alveolar macrophages, yet associated poorly to A549 alveolar epithelial cells. Acapsular C. neoformans adhered to and internalised into the mammalian cells more effectively compared to encapsulated cryptococci. Acapsular C. neoformans induced prominent actin reorganisation at the host-pathogen interface in MH-S alveolar macrophages, but minimally affected actin reorganisation in A549 alveolar epithelial cells. Acapsular C. neoformans also induced localisation of microtubules to internalised cryptococci in MH-S cells. Drug inhibition of actin and microtubule polymerisation both reduced the association of acapsular C. neoformans to alveolar macrophages. The current study visualises and confirms the interactions of C. neoformans with mammalian alveolar cells during the establishment of infection in the lungs. The acapsular form of C. neoformans effectively adhered to and internalised into alveolar macrophages by inducing localised actin reorganisation, relying on the host's actin and microtubule activities.

  5. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

    PubMed Central

    1993-01-01

    Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm. PMID:8381123

  6. The control of cellular shape and motility. Mg2+ and tropomyosin regulate the formation and the dissociation of microfilament bundles.

    PubMed Central

    Grazi, E; Cuneo, P; Cataldi, A

    1992-01-01

    At pH 7.14 and 37 degrees C, in 7.2% (w/v) poly(ethylene glycol) 6000, tropomyosin-regulated actin filaments are converted into filament bundles by increasing the free Mg2+ concentration to 1.7-2.0 mM. When free Mg2+ concentration is decreased below 1.7 mM, bundles dissociate back into tropomyosin-regulated actin filaments. Pure actin filaments are insensitive to this mechanism of control and are found as filament bundles in all the range of free Mg2+ concentrations tested (1.37-2.2 mM). The mechanism of regulation described above is likely to operate in the cell, where the concentration of free Mg2+ is linked to the energy charge of the adenine nucleotide system. Images Fig. 3. PMID:1471985

  7. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    PubMed

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  8. Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy

    PubMed Central

    1992-01-01

    The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two- thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F- actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F- actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A). PMID:1360014

  9. Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.

    PubMed Central

    Jung, E; Fucini, P; Stewart, M; Noegel, A A; Schleicher, M

    1996-01-01

    Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. Images PMID:8635456

  10. Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

    PubMed

    Manuel-Cabrera, Carlos Alberto; Vallejo-Cardona, Alba Adriana; Padilla-Camberos, Eduardo; Hernández-Gutiérrez, Rodolfo; Herrera-Rodríguez, Sara Elisa; Gutiérrez-Ortega, Abel

    2016-11-29

    Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

  11. [Development and differentiation of the rat epididymis. II: histochemical aspects of the peritubular zone].

    PubMed

    Francavilla, F; Santiemma, V; Francavilla, S; Forcella, G; Moscardelli, S; Properzi, G

    1979-07-15

    Development of the contractile peritubular structures of rat testis, from birth to full maturation, was investigated by histochemical evaluation of alkaline phosphatase activity (A.P.A.) at 0, 16, 22 e 35 days of age. A.P.A. appeared at 6 days, age of appearance also of the cytoplasmic microfilaments of peritubular cells. The correlation between cytoplasmic microfilaments and A.P.A. was confirmed by the pattern of A.P.A. positivity at 6 degree day, when the inner peritubular cells layer, in which the microfilaments are present, are more positive than the outer layer in which the microfilaments are not yet present. These findings give further support to the belief that the cytoplasmic microfilaments of peritubular cells are contractile structures.

  12. Differing requirements for actin and myosin by plant viruses for sustained intercellular movement.

    PubMed

    Harries, Phillip A; Park, Jong-Won; Sasaki, Nobumitsu; Ballard, Kimberly D; Maule, Andrew J; Nelson, Richard S

    2009-10-13

    The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.

  13. New Clue Found to Growth Factor Action.

    ERIC Educational Resources Information Center

    Hoffman, Michelle

    1991-01-01

    Discussed is the discovery which may help to explain epidermal growth factor effects on the cell skeleton. The role of a protein called profilin in the regulation of the microfilament system is described. (CW)

  14. New Clue Found to Growth Factor Action.

    ERIC Educational Resources Information Center

    Hoffman, Michelle

    1991-01-01

    Discussed is the discovery which may help to explain epidermal growth factor effects on the cell skeleton. The role of a protein called profilin in the regulation of the microfilament system is described. (CW)

  15. Secretory mechanism of fibroin, a silk protein, in the posterior silk gland cells of Bombyx mori.

    PubMed

    Sasaki, S; Nakagaki, I

    1980-01-01

    There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intracellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.

  16. Formins: Actin nucleators that regulate cytoskeletal dynamics during spermatogenesis

    PubMed Central

    Li, Nan; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris KC; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

    2015-01-01

    Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis. PMID:26413414

  17. Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

    PubMed Central

    1987-01-01

    We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm- mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells. PMID:3680376

  18. Formins: Actin nucleators that regulate cytoskeletal dynamics during spermatogenesis.

    PubMed

    Li, Nan; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris Kc; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

    2015-01-01

    Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis.

  19. Actin cytoskeleton rearrangements in Arabidopsis roots under stress and during gravitropic response

    NASA Astrophysics Data System (ADS)

    Pozhvanov, Gregory; Medvedev, Sergei; Suslov, Dmitry; Demidchik, Vadim

    Among environmental factors, gravity vector is the only one which is constant in direction and accompanies the whole plant ontogenesis. That said, gravity vector can be considered as an essential factor for correct development of plants. Gravitropism is a plant growth response against changing its position relative to the gravity vector. It is well estableshed that gravitropism is directed by auxin redistribution across the gravistimulated organ. In addition to auxin, actin cytoskeleton was shown to be involved in gravitropism at different stages: gravity perception, signal transduction and gravitropic bending formation. However, the relationship between IAA and actin is still under discussion. In this work we studied rearrangements of actin cytoskeleton during root gravitropic response. Actin microfilaments were visualized in vivo in GFP-fABD2 transgenic Arabidopsis plants, and their angle distribution was acquired from MicroFilament Analyzer software. The curvature of actin microfilaments in root elongation zone was shown to be increased within 30-60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. In particular, the fraction of transversally oriented microfilaments (i.e. parallel to the gravity vector) increased 3-5 times. Under 10 min of sub-lethal salt stress impact, actin microfilament orientations widened from an initial axial orientation to a set of peaks at 15(°) , 45(°) and 90(°) . We conclude that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed. Having common stress-related features, gravity-induced actin cytoskeleton rearrangement is slower but results in higher number of g-vector-parallel microfilaments when compared to salt stress-induced rearrangement. Also, differences in gravistimulated root

  20. Induction of Plant Curvature by Magnetophoresis and Cytoskeletal Changes during Root Graviresponse

    NASA Technical Reports Server (NTRS)

    Hasenstein, Karl H.; Kuznetsov, Oleg A.; Blancaflor, Eilson B.

    1996-01-01

    High gradient magnetic fields (HGMF) induce curvature in roots and shoots. It is considered that this response is likely to be based on the intracellular displacement of bulk starch (amyloplasts) by the ponderomotive force generated by the HGMF. This process is called magnetophoresis. The differential elongation during the curvature along the concave and convex flanks of growing organs may be linked to the microtubular and/or microfilament cytoskeleton. The possible existence of an effect of the HGMF on the cytoskeleton was tested for, but none was found. The application of cytoskeletal stabilizers or depolymerizers showed that neither microtubules, nor microfilaments, are involved in the graviresponse.

  1. Cytochalasin D does not inhibit gravitropism in roots

    NASA Technical Reports Server (NTRS)

    Staves, M. P.; Wayne, R.; Leopold, A. C.

    1997-01-01

    It is generally thought that sedimenting plastids are responsible for gravity sensing in higher plants. We directly tested the model generated by the current statolith hypothesis that the gravity sensing that leads to gravitropism results from an interaction between the plastids and actin microfilaments. We find that the primary roots of rice, corn, and cress undergo normal gravitropism and growth even when exposed to cytochalasin D, a disruptor of actin microfilaments. These results indicate that an interaction between amyloplasts and the actin cytoskeleton is not critical for gravity sensing in higher plants and weaken the current statolith hypothesis.

  2. Influence of marihuana on cellular structures and biochemical activities.

    PubMed

    Tahir, S K; Zimmerman, A M

    1991-11-01

    Cannabinoids are known to affect a number of cellular systems and functions, but the basis for their action is unclear. In this paper we review the current evidence describing cannabinoid effects on various levels of cellular structure and activity and we present our current studies on the influence of delta-9-tetrahydrocannabinol, cannabidiol and cannabinol on one cellular system, the cytoskeleton. The organization of two cytoskeletal structures, microtubules and microfilaments, were examined and the mRNA levels of tubulin and actin, the major protein components of microtubules and microfilaments, respectively, were analysed.

  3. The Mechanosensitive Ca2+ Channel as a Central Regulator of Prostate Tumor Cell Migration and Invasiveness

    DTIC Science & Technology

    2010-01-01

    nM for 2 hours), an agent reported to promote actin polymerization also did not alter MscCa transient gating in LNCaP cells. However, curcumin (60...channels. J Cell Sci 2008; 121: 496-503. 17. Holy J. Curcumin inhibits cell motility and alters microfilaments organization and function in

  4. Inside the Cell: The New Frontier of Medical Science. Series: A New Medical Science for the 21st Century.

    ERIC Educational Resources Information Center

    Pines, Maya

    Provides information on cellular morphology and physiology, including general cell characteristics, the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, microtubules, microfilaments, and membranes. Focuses on membranes which are postulated to play an important role in many aspects of health and disease.…

  5. The terminal web of the duodenal enterocyte.

    PubMed

    Leeson, T S

    1982-06-01

    The terminal web-brush border complex of rodent duodenal enterocytes has been studied by electron microscopy to investigate its structure in relation to currently accepted models of motility in this region. The main adherens zone is composed chiefly of a fine feltwork of 5 to 7 nm filaments, some of which originate in zonulae adherentes. In some cells, this is not a complete layer or sheet. Passing into it from its deep aspect are 10 nm tonofilaments, which also form the basal zone. The filament density in the basal zone is less than that of the adherens zone, and many of the tonofilaments are associated with spot desmosomes. The apical zone contains a loose meshwork of 5 to 7 nm filaments with more filaments lying adjacent to plasmalemmae of the zonula occludens. The core of each microvillus contains a bundle of 17 to 48 microfilaments, 5-7 nm in diameter, apparently attached to the apical plasmalemma and with some slender cross filaments between core filaments and the plasmalemma. In the main, these core bundles of microfilaments pass deeply into and often through the adherens zone of the terminal web where they terminate abruptly. Filaments of the terminal web appear to interconnect microfilaments of adjacent core bundles but without positive evidence of 'splaying' of microfilaments of a core bundle within the adherens zone. These findings are discussed in relation to movement of microvilli.

  6. Effect of the meiotic inhibitor cilostamide on resumption of meiosis and cytoskeletal distribution in buffalo oocytes.

    PubMed

    Li, Qing-Yang; Lou, Juan; Yang, Xiao-Gan; Lu, Yang-Qing; Lu, Sheng-Sheng; Lu, Ke-Huan

    2016-11-01

    Improving the quality of in vitro maturated buffalo oocytes is essential for embryo production. We report here the effects on microtubules and microfilaments in oocytes and embryo development that result from treating buffalo oocytes with the phosphodiesterase 3 (PDE3) inhibitor cilostamide. Addition of 20μM or 50μM cilostamide for 24h during in vitro maturation showed no differences in the percentage of oocytes arrested at the germinal vesicle (GV) stage. When 20μM cilostamide was added to the pre-maturation culture for 6h, 12h or 24h and continued for another 24h without cilostamide, oocytes resumed meiosis, but with significantly lower (P<0.01) maturation rates in the 24h group than that in the other two groups. During oocyte maturation in vitro, no microtubules were detected before GV breakdown (GVBD). After GVBD, microtubules combined with condensed chromatin to form the meiotic metaphase spindle. Microfilaments covered a thick area around the cellular cortex and overlying chromosomes. Cilostamide had no effects on microtubules and microfilaments in metaphase II oocytes, and there were no significant differences in the rates of cleavage, blastocyst formation and number of blastocyst cells between oocytes treated pre-maturation with inhibitor for 6h and those of the control group (P>0.05). In summary, cilostamide reversibly arrested the resumption of meiosis without any adverse impact on the dynamic changes in microtubules and microfilaments in buffalo oocytes and their in vitro developmental capacity.

  7. Actin- and myosin-like filaments in rat brain pericytes.

    PubMed

    Le Beux, Y J; Willemot, J

    1978-04-01

    Heavy meromyosin (HMM) labeling was used to identify the nature of the filaments which form bundles in the cytoplasm of the pericytes in brain tissue. Rat brain tissue pieces were incubated in glycerol solutions at 4 degrees and then transferred into buffer (pH 7.0), (1) without HMM, (2) with HMM, (3) with HMM + 5 mM ATP, and (4) with HMM + 2.5 mM Na+ pyrophosphate. In pericytes from untreated tissue, smooth-surfaced microfilaments, averaging 6 nm in diameter, appear to branch and anastomose and to anchor on the plasma membrane. After exposure to HMM, the number and the density of the microfilaments are strikingly increased. These tightly-packed microfilaments are now heavily coated with exogeneous HMM thus increasing in width to 18-20 mm. They intertwine in closely-woven networks. After incubation in HMM solutions containing ATP or Na+ phosphate, they are no longer coated with thick sidearms. It can thus be concluded that these microfilaments are of actin-like nature. In addition, after incubation in ATP, they are intermingled with, and converge onto the surfaces of, thick, tapered filaments, which we have tentatively identified as of myosin-like nature. Thus, it appears that certain of the major elements necessary for contraction are present in brain pericytes.

  8. The Molecules of the Cell Matrix.

    ERIC Educational Resources Information Center

    Weber, Klaus; Osborn, Mary

    1985-01-01

    Cytoplasmic proteins form a highly structured yet changeable matrix that affects cell shape, division, motion, and transport of vesicles and organelles. Types of microfilaments, research techniques, actin and myosin, tumor cells, and other topics are addressed. Evidence indicates that the cell matrix might have a bearing on metabolism. (DH)

  9. Motility and centrosomal organization during sea urchin and mouse fertilization

    NASA Technical Reports Server (NTRS)

    Schatten, Heide; Schatten, Gerald

    1986-01-01

    It is noted that microfilaments are essential for incorporation of sperm in sea urchins and for pronuclear apposition in mice. The ability of sea urchin sperm to fertilize eggs is lowered by latrunculin, giving evidence that acrosomal microfilaments are of importance to the process of fertilization. Due to the uncertainty regarding the presence of microfilaments in various mammalian sperm, it is interesting that latrunculin does not noticeably affect the ability of mouse sperm to fertilize oocytes. The movements of the sperm and egg nuclei at the time of sea urchin fertilization are dependent on microtubules arranged into a radial monastral array (the sperm aster). In the mouse egg, microtubule activity is also required during pronuclear apposition, but they are arranged by a number of egg cytoplasmic sites. Results of the investigations show that both microtubules and microfilaments are necessary for the successful completion of fertilization in both mice and sea urchins, but at different stages. Also, it is demonstrated that centrosomes are contributed by the sperm in the process of sea urchin fertilization, but in mammals they may be inherited maternally.

  10. Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae).

    PubMed

    Lee, Jung Sick; Lee, Yeon Gyu; Park, Jung Jun; Shin, Yun Kyung

    2012-10-01

    In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  11. Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation.

    PubMed

    Somfai, T; Kikuchi, K; Kaneda, M; Akagi, S; Watanabe, S; Mizutani, E; Haraguchi, S; Dang-Nguyen, T Q; Inaba, Y; Geshi, M; Nagai, T

    2011-10-01

    We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

  12. Low dose radiation-induced endothelial cell retraction.

    PubMed

    Kantak, S S; Diglio, C A; Onoda, J M

    1993-09-01

    We characterized in vitro the effects of gamma-radiation (12.5-100 cGy) on pulmonary microvascular endothelial cell (PMEC) morphology and F-actin organization. Cellular retraction was documented by phase-contrast microscopy and the organization of actin microfilaments was determined by immunofluorescence. Characterization included radiation dose effects, their temporal duration and reversibility of the effects. A dose-dependent relationship between the level of exposure (12.5-100 cGy) and the rate and extent of endothelial retraction was observed. Moreover, analysis of radiation-induced depolymerization of F-actin microfilament stress fibres correlated positively with the changes in PMEC morphology. The depolymerization of the stress fibre bundles was dependent on radiation dose and time. Cells recovered from exposure to reform contact inhibited monolayers > or = 24 h post-irradiation. Concomitantly, the depolymerized microfilaments reorganized to their preirradiated state as microfilament stress fibres arrayed parallel to the boundaries of adjacent contact-inhibited cells. The data presented here are representative of a series of studies designed to characterize low-dose radiation effects on pulmonary microvascular endothelium. Our data suggest that post-irradiation lung injuries (e.g. oedema) may be induced with only a single fraction of therapeutic radiation, and thus microscopic oedema may initiate prior to the lethal effects of radiation on the microvascular endothelium, and much earlier than would be suggested by the time course for clinically-detectable oedema.

  13. Imaging and quantitative methods for studying cytoskeletal rearrangements during root development and gravitropism.

    PubMed

    Jacques, Eveline; Wells, Darren M; Bennett, Malcolm J; Vissenberg, Kris

    2015-01-01

    High-resolution imaging of cytoskeletal structures paves the way for standardized methods to quantify cytoskeletal organization. Here we provide a detailed description of the analysis performed to determine the microtubule patterns in gravistimulated roots, using the recently developed software tool MicroFilament Analyzer.

  14. The Molecules of the Cell Matrix.

    ERIC Educational Resources Information Center

    Weber, Klaus; Osborn, Mary

    1985-01-01

    Cytoplasmic proteins form a highly structured yet changeable matrix that affects cell shape, division, motion, and transport of vesicles and organelles. Types of microfilaments, research techniques, actin and myosin, tumor cells, and other topics are addressed. Evidence indicates that the cell matrix might have a bearing on metabolism. (DH)

  15. New dynamics in an old friend: dynamic tubular vacuoles radiate through the cortical cytoplasm of red onion epidermal cells.

    PubMed

    Wiltshire, Elizabeth J; Collings, David A

    2009-10-01

    The textbook image of the plant vacuole sitting passively in the centre of the cell is not always correct. We observed vacuole dynamics in the epidermal cells of red onion (Allium cepa) bulbs, using confocal microscopy to detect autofluorescence from the pigment anthocyanin. The central vacuole was penetrated by highly mobile transvacuolar strands of cytoplasm, which were also visible in concurrent transmitted light images. Tubular vacuoles also extended from the large central vacuole and radiated through the cortical cytoplasm. These tubules were thin, having a diameter of about 1.5 microm, and were connected to the central vacuole as shown by fluorescence recovery after photobleaching (FRAP) experiments. The tubules were bounded by the tonoplast, as revealed by transient expression of green fluorescent protein (GFP) targeted to the vacuolar membrane and through labeling with the dye MDY-64. Expression of endoplasmic reticulum-targeted GFP demonstrated that the vacuolar tubules were distinct from the cortical endoplasmic reticulum. Movement of the tubular vacuoles depended on actin microfilaments, as microfilament disruption blocked tubule movement and caused their collapse into minivacuoles. The close association of the tubules with GFP-tagged actin microfilaments suggests that the tubules are associated with myosin, and that tubules likely move along microfilaments. Tubular vacuoles do not require anthocyanin for their formation, as tubules were also present in white onion cells that lack anthocyanin. The function of these tubular vacuoles remains unknown, but as they greatly increase the surface area of the tonoplast, they might increase transport rates between the cytoplasm and vacuole.

  16. Aberrant Vimentin DNA Methylation in Stool — EDRN Public Portal

    Cancer.gov

    The VIM gene encodes a member of the intermediate filament family. VIM proteins are class-III intermediate filaments found in various non-epithelial cells, especially mesenchymal cells. These intermediate filaments, along with microtubules and actin microfilaments, make up the cytoskeleton.

  17. [What is your diagnosis? A "fat arm". Onchocerciasis].

    PubMed

    Bousquet, A; Larréché, S; Elhadji Toumane, C; Dupin, M; Avignant, J; Mérens, A; Maccari, F

    2014-01-01

    We report a case of edema of the right forearm with pruriginous papules and eosinophilia in the blood. The patient had lived in a forested area of Cameroon. The clinical, laboratory, and geographical findings led to a diagnosis of onchocerciasis, despite the absence of microfilaments on skin biopsy samples. The patient was successfully treated with ivermectin and doxycycline.

  18. Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation.

    PubMed

    Talbot, N C; Garrett, W M

    2001-09-01

    Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.

  19. Changes in cell migration due to the combined effects of sonodynamic therapy and photodynamic therapy on MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Wang, Haiping; Wang, Pan; Zhang, Kun; Wang, Xiaobing; Liu, Quanhong

    2015-03-01

    Sono-photodynamic therapy is an emerging method with an increasing amount of research having demonstrated its anti-cancer efficacy. However, the impacts of cell migration ability after sono-photodynamic therapy have seldom been reported. In this study, we identified cell migration by wound healing and transwell assays. Significant inability of cell migration was observed in combined groups accompanied by the decline of cell adhesion. Cells in combined treatment groups showed serious microfilament network collapse as well as decreased expression of matrix metalloproteinases-9. These results suggested that sono-photodynamic therapy could inhibit MDA-MB-231 cell migration and that the microfilament and matrix metalloproteinases-9 disorder might be involved.

  20. Soluble lymphocytic mediators

    PubMed Central

    Pick, E.

    1974-01-01

    The effect of a number of drugs on the production of macrophage migration inhibitory factor (MIF) by antigen-stimulated sensitized guinea-pig lymph node cells was studied. The drugs were present during the entire culture period and eliminated from supernatants by dialysis. It was found that MIF secretion is inhibited by exogenous dibutyryl cyclic AMP and by theophylline and chlorphenesin, two agents raising the endogenous level of cyclic AMP. On the other hand, isoproterenol, which stimulates cyclic AMP generation in several tissues, did not block MIF production. The formation of the mediator was also suppressed by the microfilament-affecting drug, cytochalasin B. The microtubular disruptive agents, colchicine and vinblastine sulphate, did not influence MIF production. It is concluded that: (a) endogenous cyclic AMP may act as a regulator of MIF production; (b) the activity of contractile microfilaments is probably required for MIF formation; and (c) microtubules are not involved in the secretory process. PMID:4369184

  1. [Alpha-actinin in the electric organ of Electrophorus electricus, L].

    PubMed

    Dos Santos Mermelstein, C; Moura Neto, V; Chagas, C

    1988-01-01

    We have identified Alpha-actinin from the electric organ of the Electrophorus electricus, L. It was analysed by polyacrylamide gel electrophoresis, and identified by immunoblotting. This protein was also found in a membrane fraction of the electric organ enriched with components of the cytoskeleton. Our results suggest that this protein might play a role either in the organization of the microfilaments or its interactions with the membrane to maintain a polarized electrocyte.

  2. Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method

    PubMed Central

    1984-01-01

    Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system. PMID:6207180

  3. Association of actin with alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

  4. Vasopressin Receptor Signaling and Cycling of Water Channels in Renal Epithelia

    DTIC Science & Technology

    1992-08-22

    August 22, 1992 TYPE OF REPORT: ANNUAL REPORT PREPARED FOR: U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND FORT DETRICK FEDERICK , MARYLAND 21702-5012...fusion events are dependent very likely on the presence of cytosolic microtubules and microfilaments ( Taylor et al., 1973; Hardy and DiBona, 1982; Hays...et al., 1982; Pearl and Taylor , 1983; Mia et al., 1991). Amphibian epithelia also contain an ADH V1 receptor coupled to phosphoinositide metabolism and

  5. Microfilamin - a new protein of the cytoskeleton with molecular weight 53,000

    SciTech Connect

    Fridlyanskaya, I.I.; Galaktionov, K.I.; D'yakonova, M.Yu.; Pinaev, G.P.

    1987-01-01

    The microfilaments in cultured cells participate in many vitally important processes: endocytosis, movement of cells, division, etc. One of the approaches to the study of the proteins of the microfilaments is the use of the hybridoma technique. This method permits the use of cell structures as an immunogen and the investigation of their protein assortment by means of monoclonal antibodies. Using such an approach, the authors were able to identify a protein with molecular weight 53 kilodaltons, not previously described in the microfilaments. BABL/c mice were immunized with an extract of the cytoskeleton of murine cells 3T3. It was produced by treating the cells with 0.5% Triton in a buffer stabilizing the cytoskeleton (10 mM sodium phosphate buffer, pH 6.8, 0.1 M KCl, 1 mMEGTA, 0.1 mM EDTA, 5 mM MgCl/sub 2/, and 2 M sucrose), and then with the same buffer, containing 1 micrograms/ml DNase I, 1mM PMSF, and 20 units/ml trasylol. The extract was clarified by successive centrifugation at 1000, 1200, and 10,000 g. Splenocytes of immune animals were hybridized with murine myeloma SP 2/0-Ag 14 cells and screening of the hybridomas was performed by the method of indirect immunofluorescence on murine cells of the 3T3 line stained with Coomassie blue. Data from autograph after incubation with E9-48 antibodies and /sup 125/I-labeled rabbit anti-mouse immunoglobulins are presented as well. The authors named this new technique microfilamin since it is detected in structures containing microfilaments: stress fibrils, the active edge, surface villi, and others.

  6. Cytoskeleton in Mast Cell Signaling

    PubMed Central

    Dráber, Pavel; Sulimenko, Vadym; Dráberová, Eduarda

    2012-01-01

    Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca2+. Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling. PMID:22654883

  7. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite

    PubMed Central

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-01-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs. PMID:25565321

  8. Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture.

    PubMed

    Ciesielski-Treska, J; Bader, M F; Aunis, D

    1982-03-01

    Microtubules and microfilament patterns in cultured astrocytes were revealed by using indirect immunofluorescent microscopy in conjunction with anti-tubulin immune serum and anti-actin immunoglobulins respectively. In flat epitheloid astroglial cells (either polygonal or elongated) colchicine-sensitive immunofluorescent fibres, which correspond to bundles of microtubules, extend from the perinuclear cytoplasm into the cell periphery by running for long distances through the different focal planes. These patterns of organization differ markedly from the patterns of organization of microfilaments which are arranged in fibres parallel to each other and often oriented along the cell boundary. In response to the combined treatments of serum withdrawal and administration of dBcAMP, flat epitheloid astrocytes adopt a morphology similar to that of the mature astrocytes in situ in the CNS, that is of stellate process-bearing cells. This is prevented or is reverted by the administration of colchicine at the appropriate times. There are strong suggestions indicating that during cell processes formation the microtubular network is reorganized and microtubules assembled into dense bundles which are oriented along the axis of the cell processes. In view of these results, we suggest that, in contrast to microfilaments, microtubules are not determinant for the maintenance of cellular shape in elongated or polygonal flat epitheloid astroglial cells but they are required for both the formation and maintenance of processes in stellate astrocytes.

  9. [Nimotuzumab significantly enhances chemosensitivity of
PC9 human lung adenocarcinoma cells to paclitaxel in vitro].

    PubMed

    Xiao, Yu; Cao, Baoshan; Liang, Li

    2015-02-01

    Nimotuzumab is a humanized IgG1 type monoclonal antibody targeting epidermal growth factor receptor, and can enhance chemosensitivity and radiosensitivity of certain cancers. The aim of this study is to investigate the effects of nimotuzumab on the chemosensitivities of PC9 human lung adenocarcinoma cells to common chemtherapeutic drugs including ciaplatin, gemcitabine, paclitaxel, pemetrexed and vinorelbine, and to elucidate possible mechanisms. PC9 human lung adenocarcinoma cell line was used in the study. Cell proliferation was determined by WST-1 assay and cell apoptosis was detected by TUNEL assay. Cell cycle distribution was analyzed by DNA analysis with FACS. Tublin and microfilaments were observed by immunofluorescence staining. Nimotuzumab significantly enhanced the chemosensitivity of PC9 cells to paclitaxel. Cell proliferation was inhibited significantly (P<0.05) and cell apoptosis rate was higher in nimotuzumab combined with low dose paclitaxel (0.05 μg/mL) group (P=0.013). G2/M arrest was increased significantly by nimotuzumab combined with paclitaxel group (P<0.05). Nimotuzumab caused aggregation of tublin and microfilaments into well organized microtubules. Nimotuzumab enhanced the chemosensitivity of PC9 cell to paclitaxel by enhancing G2/M arrest and aggregation of tublin and microfilaments. Therefore, Nimotuzumab combined with taxane drugs could be a potential effective regimen in non-small cell lung cancer.

  10. Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs.

    PubMed

    Connors, S A; Kanatsu-Shinohara, M; Schultz, R M; Kopf, G S

    1998-08-01

    Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.

  11. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  12. Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

  13. Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1994-04-01

    The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

  14. Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

    PubMed Central

    Witze, Eric S.; Connacher, Mary Katherine; Houel, Stephane; Schwartz, Michael P.; Morphew, Mary K.; Reid, Leah; Sacks, David B.; Anseth, Kristi S.; Ahn, Natalie G.

    2013-01-01

    SUMMARY Wnt5a directs the assembly of “Wnt-receptor-actin-myosin-polarity (WRAMP)” structure, which integrates cell adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies. The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here, we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca2+. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca2+ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction. PMID:24091015

  15. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite.

    PubMed

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-04-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.

  16. Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

  17. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    PubMed

    Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

    2016-11-07

    An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

  18. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  19. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  20. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture.

    PubMed

    Schatten, H; Hedrick, J; Chakrabarti, A

    1998-12-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  1. Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells.

    PubMed

    Sartori, M J; Mezzano, L; Lin, S; Muñoz, S; de Fabro, S P

    2003-09-01

    In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.

  2. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Langer, R.; Ingber, D. E.

    1995-01-01

    This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In

  3. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Langer, R.; Ingber, D. E.

    1995-01-01

    This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In

  4. Occluding junctions and cytoskeletal components in a cultured transporting epithelium

    PubMed Central

    Meza, I; Ibarra, G; Sabanero, M; Martinez-Palomo, A; Cereijido, M

    1980-01-01

    MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect

  5. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    PubMed

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  6. Rational Identification of Enoxacin as a Novel V-ATPase-Directed Osteoclast Inhibitor

    PubMed Central

    Toro, Edgardo J; Ostrov, David A; Wronski, Thomas J; Holliday, L Shannon

    2012-01-01

    Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actin-binding activity has been demonstrated in the B-subunit of yeast. A conserved “profilin-like” domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the “profilin-like” domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the “housekeeping” functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis. PMID:22044158

  7. Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

    PubMed

    Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

    2014-12-01

    The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean

  8. Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation

    NASA Astrophysics Data System (ADS)

    Shevchenko, G. V.; Kordyum, E. L.

    Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.

  9. Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Wong, Chris K. C.; Han, Daishu; Lee, Will M.

    2015-01-01

    During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics. PMID:25901598

  10. Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents.

    PubMed

    Aggeler, J

    1990-01-01

    Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with

  11. Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

    PubMed Central

    Phan, Quynh T.; Eng, David K.; Mostowy, Serge; Park, Hyunsook; Cossart, Pascale; Filler, Scott G.

    2013-01-01

    ABSTRACT Candida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism. PMID:24345743

  12. Regulation of PTHrP expression by cyclic mechanical strain in postnatal growth plate chondrocytes.

    PubMed

    Xu, Tao; Yang, Kaixiang; You, Hongbo; Chen, Anmin; Wang, Jiang; Xu, Kai; Gong, Chen; Shao, Jingfan; Ma, Zhongxi; Guo, Fengjing; Qi, Jun

    2013-10-01

    Mechanical loading has been widely considered to be a crucial regulatory factor for growth plate development, but the exact mechanisms of this regulation are still not completely understood. In the growth plate, parathyroid hormone-related protein (PTHrP) regulates chondrocyte differentiation and longitudinal growth. Cyclic mechanical strain has been demonstrated to influence growth plate chondrocyte differentiation and metabolism, whereas the relationship between cyclic mechanical strain and PTHrP expression is not clear. The objective of this study was to investigate whether short-term cyclic tensile strain regulates PTHrP expression in postnatal growth plate chondrocytes in vitro and to explore whether the organization of cytoskeletal F-actin microfilaments is involved in this process. To this end, we obtained growth plate chondrocytes from 2-week-old Sprague-Dawley rats and sorted prehypertrophic and hypertrophic chondrocytes using immunomagnetic beads coated with anti-CD200 antibody. The sorted chondrocytes were subjected to cyclic tensile strain of varying magnitude and duration at a frequency of 0.5 Hz. We found that cyclic strain regulates PTHrP expression in a magnitude- and time-dependent manner. Incubation of chondrocytes with cytochalasin D, an actin microfilament-disrupting reagent, blocked the induction of PTHrP expression in response to strain. The results suggest that short-term cyclic tensile strain induces PTHrP expression in postnatal growth plate prehypertrophic and hypertrophic chondrocytes and that PTHrP expression by these chondrocytes may subsequently affect growth plate development. The results also support the idea that the organization of cytoskeletal F-actin microfilaments plays an important role in mechanotransduction.

  13. Enoxacin directly inhibits osteoclastogenesis without inducing apoptosis.

    PubMed

    Toro, Edgardo J; Zuo, Jian; Ostrov, David A; Catalfamo, Dana; Bradaschia-Correa, Vivian; Arana-Chavez, Victor; Caridad, Aliana R; Neubert, John K; Wronski, Thomas J; Wallet, Shannon M; Holliday, L Shannon

    2012-05-18

    Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

  14. Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis*

    PubMed Central

    Toro, Edgardo J.; Zuo, Jian; Ostrov, David A.; Catalfamo, Dana; Bradaschia-Correa, Vivian; Arana-Chavez, Victor; Caridad, Aliana R.; Neubert, John K.; Wronski, Thomas J.; Wallet, Shannon M.; Holliday, L. Shannon

    2012-01-01

    Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μm) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the “housekeeping” a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein l-plastin was altered in cells treated with 50 μm enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments. PMID:22474295

  15. Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells.

    PubMed

    Calí, G; Retta, S F; Negri, R; Damiano, I; Gentile, R; Tarone, G; Nitsch, L; Garbi, C

    1998-02-01

    Beta1B is a beta1 integrin splice variant that differs from the ubiquitous beta1A in the terminal portion of the cytosolic tail. The expression of this variant in CHO cells results in reduced fibroblast adhesion and motility (Balzac, E et al., J. Cell Biol. 127, 557-565 (1994)). We have evaluated the phenotypic changes induced by the expression of beta1B in the FRT epithelial cell line. Stable transfectants of FRT cells expressing beta1B or beta1A human integrins were obtained. The transfected integrins associated with the endogenous alpha subunits and were delivered to the plasma membrane. Beta1B expressing cells attached less efficiently and spread less on fibronectin, laminin or type IV collagen coated dishes. A great reduction of fibronectin fibrils associated to the basal membrane of non-confluent beta1B transfected cells was observed. This was paralleled by the disappearance of microfilament bundles and loss of basally located focal adhesions. On the contrary, upon beta1A transfection, a higher amount of fibronectin fibrils, together with microfilament bundles and focal adhesions, was observed. Expression of beta1B did not significantly modify the ability to manifest the polarized phenotype when cells were grown to confluence on filters in two-chamber-systems. Beta1B-transfected cells showed reduced motile properties when embedded as aggregates in type I collagen gels. Moreover, formation of polarized cysts in suspension culture was impaired. The results show that beta1B, by interfering with focal adhesion organization, microfilament and fibronectin assembly, cell spreading and migration, affects some morphogenetic properties of FRT epithelial cells.

  16. Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Sawyer, S. J.; Norvell, S. M.; Ponik, S. M.; Pavalko, F. M.

    2001-01-01

    Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

  17. Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema.

    PubMed

    Onoda, J M; Kantak, S S; Diglio, C A

    1999-01-01

    We determined the effects of low dose radiation (<200 cGy) on the cell-cell integrity of confluent monolayers of pulmonary microvascular endothelial cells (PMEC). We observed dose- and time-dependent reversible radiation induced injuries to PMEC monolayers characterized by retraction (loss of cell-cell contact) mediated by cytoskeletal F-actin reorganization. Radiation induced reorganization of F-actin microfilament stress fibers was observed > or =30 minutes post irradiation and correlated positively with loss of cell-cell integrity. Cells of irradiated monolayers recovered to form contact inhibited monolayers > or =24 hours post irradiation; concomitantly, the depolymerized microfilaments organized to their pre-irradiated state as microfilament stress fibers arrayed parallel to the boundaries of adjacent contact-inhibited cells. Previous studies by other investigators have measured slight but significant increases in mouse lung wet weight >1 day post thoracic or whole body radiation (> or =500 cGy). Little or no data is available concerning time intervals <1 day post irradiation, possibly because of the presumption that edema is mediated, at least in part, by endothelial cell death or irreversible loss of barrier permeability functions which may only arise 1 day post irradiation. However, our in vitro data suggest that loss of endothelial barrier function may occur rapidly and at low dose levels (< or =200 cGy). Therefore, we determined radiation effects on lung wet weight and observed significant increases in wet weight (standardized per dry weight or per mouse weight) in < or =5 hours post thoracic exposure to 50 200 cGy x-radiation. We suggest that a single fraction of radiation even at low dose levels used in radiotherapy, may induce pulmonary edema by a reversible loss of endothelial cell-cell integrity and permeability barrier function.

  18. Actin cytoskeleton mediates BMP2-Smad signaling via calponin 1 in preosteoblast under simulated microgravity.

    PubMed

    Xu, Hongjie; Wu, Feng; Zhang, Hongyu; Yang, Chao; Li, Kai; Wang, Hailong; Yang, Honghui; Liu, Yue; Ding, Bai; Tan, Yingjun; Yuan, Ming; Li, Yinghui; Dai, Zhongquan

    2017-07-01

    Microgravity influences the activity of osteoblast, induces actin microfilament disruption and leads to bone loss during spaceflight. Mechanical stress such as gravity, regulates cell function, response and differentiation through dynamic cytoskeleton changes, but the mechanotransduction mechanism remains to be fully elucidated. Previous, we demonstrated actin microfilament mediated osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity (SMG). Here, we explored a potential molecular and its detailed mechanism of actin cytoskeleton functioning on BMP2-Smad signaling in MC3T3-E1 under SMG. Results showed that the actin microfilament-disrupting agent, cytochalasin B (CB), reduced BMP2-induced activation, translocation of Smad1/5/8 and Runx2 expression. SMG also inhibited BMP2-Smad signaling, which was rescued by actin cytoskeleton stabilizing agent, Jasplakinolide (JAS). Furthermore, we found that siRNA mediated knockdown of calponin 1 (CNN1), an actin binding protein, markedly promoted BMP2-Smad signaling and abolished both inhibition of CB, SMG on BMP2-Smad signaling and the rescue action of JAS. Overexpression of CNN1 inhibited the p-Smad induced by BMP2. Bidirectional Co-IP experiments demonstrated CNN1 could interacted with Smad or p-Smad protein. Furthermore, CB or SMG decreased the phosphorylated CNN1 and increased its interaction with Smad or p-Smad. Combined with the phosphorylation of CNN1 inhibites its actin binding activity, these results indicate that actin cytoskeleton depolymerization inhibites BMP2 signaling via blocking of Smad by dephosphorylated CNN1 in osteoblast cells. Thus, we provide new important insights into the mechanism of mechanotransduction under SMG condition, which probably contribute to bone formation decrease induced by SMG. Copyright © 2017. Published by Elsevier B.V.

  19. Fourier imaging correlation spectroscopy: Technique development and application to colloidal thin films and intracellular mitochondrial transport

    NASA Astrophysics Data System (ADS)

    Knowles, Michelle Kay

    2003-10-01

    Understanding fluid dynamics is fundamentally intriguing and relevant to many areas of applied science, including polymer materials and cellular transport. Many complex fluids are difficult to study using traditional methods, which are limited in sensitivity, dynamic range or spatial information. In this work, a new technique, Fourier Imaging Correlation Spectroscopy (FICS), is developed in order to measure the dynamics of complex fluids over a broad dynamic range with high sensitivity. FICS measures complex fluid structure one length scale at a time and allows for direct calculation of the intermediate scattering function; a function that describes how the system is changing on a given length scale as a function of time. The sensitivity of FICS allows for study of materials with intrinsically low signals, such as thin films. Colloidal thin film measurements provided a proof-of-principle of FICS by comparing the intermediate scattering function calculated from FICS data to results from an established technique, digital video microscopy. FICS is an ideal method for obtaining information about mitochondrial transport within living cells. Mitochondrial dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins. This leads to complex multi-exponential relaxations occurring over a wide range of spatial and temporal scales. The cytoskeleton consists of an interconnected polymer network whose primary components are microfilaments and microtubules. Cytoskeletal filaments work with motor proteins to traffic organelles within the cell. Components of the cytoskeleton were selectively destabilized and the resulting mitochondrial dynamics measured using FICS and digital video microscopy. These studies show that both microfilaments and microtubules are necessary for transport of the mitochondrial reticulum. FICS measurements reveal that microfilaments control short-range (0.8--1.6 mum) dynamics and microtubules are

  20. Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

    PubMed Central

    Peng, ChiehFu Jeff; Wikramanayake, Athula H.

    2013-01-01

    Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved

  1. Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Sawyer, S. J.; Norvell, S. M.; Ponik, S. M.; Pavalko, F. M.

    2001-01-01

    Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

  2. N terminus is essential for tropomyosin functions: N-terminal modification disrupts stress fiber organization and abolishes anti-oncogenic effects of tropomyosin-1.

    PubMed

    Bharadwaj, Shantaram; Hitchcock-DeGregori, Sarah; Thorburn, Andrew; Prasad, G L

    2004-04-02

    Down-regulation of several key actin-binding proteins, such as alpha-actinin, vinculin, gelsolin, and tropomyosins (TMs), is considered to contribute to the disorganized cytoskeleton present in many neoplastic cells. TMs stabilize actin filaments against the gel severing actions of proteins such as cofilin. Among multiple TMs expressed in non-muscle cells, tropomyosin-1 (TM1) isoform induces stress fibers and functions as a suppressor of malignant transformation. However, the molecular mechanisms of TM1-mediated cytoskeletal effects and tumor suppression remain poorly understood. We have hypothesized that the ability of TM1 to stabilize microfilaments is crucial for tumor suppression. In this study, by employing a variant TM1, which contains an N-terminal hemagglutinin epitope tag, we demonstrate that the N terminus is a key determinant of tropomyosin-1 function. Unlike the wild type TM1, the modified protein fails to restore stress fibers and inhibit anchorage-independent growth in transformed cells. Furthermore, the N-terminal modification of TM1 disorganizes the cytoskeleton and delays cytokinesis in normal cells, abolishes binding to F-actin, and disrupts the dimeric associations in vivo. The functionally defective TM1 allows the association of cofilin to stress fibers and disorganizes the microfilaments, whereas wild type TM1 appears to restrict the binding of cofilin to stress fibers. TM1-induced cytoskeletal reorganization appears to be mediated through preventing cofilin interaction with microfilaments. Our studies provide in vivo functional evidence that the N terminus is a critical determinant of TM1 functions, which in turn determines the organization of stress fibers.

  3. Cytoplasmic streaming in Chara rhizoids: studies in a reduced gravitational field during parabolic flights of rockets.

    PubMed

    Buchen, B; Hejnowicz, Z; Braun, M; Sievers, A

    1991-01-01

    In-vivo videomicroscopy of Chara rhizoids under 10(-4)g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.

  4. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    DOEpatents

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  5. Texture sensing of cytoskeletal dynamics in cell migration

    NASA Astrophysics Data System (ADS)

    Das, Satarupa; Lee, Rachel; Hourwitz, Matthew J.; Sun, Xiaoyu; Parent, Carole; Fourkas, John T.; Losert, Wolfgang

    Migrating cells can be directed towards a target by gradients in properties such as chemical concentration or mechanical properties of the surrounding microenvironment. In previous studies we have shown that micro/nanotopographical features on scales comparable to those of natural collagen fibers can guide fast migrating amoeboid cells by aligning actin polymerization waves to such nanostructures. We find that actin microfilaments and microtubules are aligned along the nanoridge topographies, modulating overall cell polarity and directional migration in epithelial cells. This work shows that topographic features on a biologically relevant length scale can modulate migration outcomes by affecting the texture sensing property of the cytoskeleton.

  6. A NEW CRYSTAL-CONTAINING CELL IN HUMAN ADRENAL CORTEX

    PubMed Central

    Magalhães, Maria C.

    1972-01-01

    Electron microscope examination of the adrenal cortex from three male human subjects revealed a special type of cell occurring in periendothelial spaces, in all adrenal cortex zones. It is a clear, spindle-shaped cell the principal cytoplasmic features of which are crystalline inclusions with a structure similar to that of the Reinke crystals of human testicular interstitial cells and an abundance of microfilaments. Enzymatic digestions with pronase, pepsin, and ribonuclease were performed, and no digestion of the crystals was obtained. The crystals had no peroxidase or acid phosphatase activities. This cell appears to be exclusive to human males and it may be related to adrenal androgen secretion. PMID:4347248

  7. Roles of Bifocal, Homer, and F-actin in anchoring Oskar to the posterior cortex of Drosophila oocytes

    PubMed Central

    Babu, Kavita; Cai, Yu; Bahri, Sami; Yang, Xiaohang; Chia, William

    2004-01-01

    Transport, translation, and anchoring of osk mRNA and proteins are essential for posterior patterning of Drosophila embryos. Here we show that Homer and Bifocal act redundantly to promote posterior anchoring of the osk gene products. Disruption of actin microfilaments, which causes delocalization of Bifocal but not Homer from the oocyte cortex, severely disrupts anchoring of osk gene products only when Homer (not Bifocal) is absent. Our data suggest that two processes, one requiring Bifocal and an intact F-actin cytoskeleton and a second requiring Homer but independent of intact F-actin, may act redundantly to mediate posterior anchoring of the osk gene products. PMID:14752008

  8. Effects of inhibitors on 1-methyladenine induced maturation of starfish oocytes

    NASA Astrophysics Data System (ADS)

    Lee, Harold H.; Xu, Quanhan

    1986-12-01

    1-methladenine (1-MA) induces starfish oocytes maturation via surface reaction followed by the appearance of a cytoplasmic maturation factor which in turn induces germinal vesicle breakdown (GVBD) to resume meiosis. Cellular mechanisms involved in GVBD were investigated by microinjection of metabolic inhibitors. Colchicine (Co) inhibited maturation, cytochalasin-B (CB) delayed GVBD and actinomycin-D-(Act-D) and puromycin (Pu) had no effect. It appears that the microtubule and the microfilament systems are associated with the nuclear membrane dissolution during the process of oocyte maturation of starfish.

  9. Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

    PubMed

    Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

    2015-04-01

    Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

  10. Synthesis of antimicrofilament marine macrolides: synthesis and configurational assignment of a C5-C16 degradation fragment of reidispongiolide A.

    PubMed

    Paterson, Ian; Britton, Robert; Ashton, Kate; Knust, Henner; Stafford, Jonathan

    2004-08-17

    Reidispongiolide A is a representative member of the sphinxolide/reidispongiolide group of cytotoxic 26-membered macrolides of marine origin. By interacting with actin in the cell cytoskeleton, the reidispongiolides and sphinxolides are potent microfilament destabilizing agents that represent a promising mechanism of action for developing novel anticancer drugs. An aldol-based synthesis of a library of diastereomers of C(8)-C(16) and C(5)-C(16) fragments and detailed NMR comparison with a reported degradation fragment enabled a configurational assignment for a major part of the reidispongiolide macrocyclic core, thus setting a solid foundation for ongoing synthetic efforts.

  11. Arterioles in the swimming muscles of the leatherjacket Parika scaber (Pisces: Balistidae).

    PubMed

    Davison, W

    1987-06-01

    The leatherjacket (Parika scaber) is a balistiform fish which swims using its dorsal and anal median fins. The muscles controlling these fins are well vascularised, with control of blood flow effected by arterioles. These arterioles are long with a single layer of smooth muscle surrounding the endothelium, although the amount of contractile material is sparse, probably a consequence of the low blood pressure. The endothelial cells contain microfilaments, probably contractile, running along the length of the arteriole. The function of this material is unknown.

  12. Microscopy basics and the study of actin-actin-binding protein interactions.

    PubMed

    Thomasson, Maggie S; Macnaughtan, Megan A

    2013-12-15

    Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Para-hydrogen narrow filament evaporation at low temperature

    NASA Astrophysics Data System (ADS)

    Elizarova, T. G.; Gogolin, A. A.; Montero, S.

    2012-11-01

    Undercooling of liquid para-hydrogen (pH2) below its freezing point at equilibrium (13.8 K) has been shown recently in flowing micro-filaments evaporating in low density background gas [M. Kühnel et al, Phys. Rev. Lett. 106, 245301 (2011)]. An hydrodynamical model accounting for this process is reported here. Analytical expressions for the local temperature T of a filament, averaged over its cross section, are obtained as a function of distance z to the nozzle. Comparison with the experiment is shown. It is shown also that the thermocapillary forces induce a parabolic profile of velocity across the jet.

  14. Regulation of peripheral spindle movement and spindle rotation during mouse oocyte meiosis: new perspectives.

    PubMed

    Ai, Jun-Shu; Wang, Qiang; Yin, Shen; Shi, Li-Hong; Xiong, Bo; OuYang, Ying-Chun; Hou, Yi; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2008-08-01

    Spindle movement, including spindle migration during first meiosis and spindle rotation during second meiosis, is essential for asymmetric divisions in mouse oocytes. Previous studies by others and us have shown that microfilaments are required for both spindle migration and rotation. In the present study, we aimed to further investigate the mechanism controlling spindle movement during mouse oocyte meiosis. By employing drug treatment and immunofluorescence microscopy, we showed that dynamic microtubule assembly was involved in both spindle migration and rotation. Furthermore, we found that the calcium/CaM/CaMKII pathway was important for regulating spindle rotation.

  15. Morphological and biochemical study of cytoskeletal changes in cultured cells after extracellular application of Clostridium novyi alpha-toxin.

    PubMed Central

    Oksche, A; Nakov, R; Habermann, E

    1992-01-01

    Clostridium novyi alpha-toxin caused retraction and rounding of cultured endothelial cells from porcine pulmonary arteries; nevertheless, the endothelial cells firmly adhered to their supports. F-actin stained with fluorescein-labeled phalloidin was condensed around the nucleus, whereas intermediate filaments and microtubules appeared unchanged. The content of F-actin and myosin was decreased, but that of G-actin or vimentin was not. A predominant role of the microfilament system in C. novyi alpha-toxin cytopathic action is suggested. Images PMID:1612767

  16. Interaction of Phalloidin with Actin

    PubMed Central

    Lengsfeld, Anneliese M.; Löw, Irmentraut; Wieland, Theodor; Dancker, Peter; Hasselbach, Wilhelm

    1974-01-01

    Phalloidin, a toxic bicyclic peptide of rapid action from the toadstool, Amanita phalloides, gives rise to polymerization of G-actin to filamentous structures (Ph-actin) in a medium of low ionic strength. Ph-actin closely resembles the microfilaments found in liver membrane fractions (Ph-filaments) after in vivo or in vitro poisoning. Both phalloidin induced filaments are resistant to 0.6 M KI in contrast to F-actin, and become decorated by heavy meromyosin. After preincubation with cytochalasin B significantly fewer actin filaments are observed. Images PMID:4368830

  17. Method for localized deposition of noble metal catalysts with control of morphology

    DOEpatents

    Ricco, Antonio J.; Manginell, Ronald P.; Huber, Robert J.

    1998-01-01

    A combustible gas sensor that uses a resistively heated, noble metal-coated, micromachined polycrystalline Si filament to calorimetrically detect the presence and concentration of combustible gases. A thin catalytic Pt film was deposited by CVD from the precursor Pt(acac).sub.2 onto microfilaments resistively heated to approximately 500 .degree. C.; Pt deposits only on the hot filament. The filaments tested to date are 2 .mu.m thick .times.10 .mu.m wide .times.100, 250, 500, or 1000 .mu.m-long polycrystalline Si; some are overcoated with a 0.25 .mu.m-thick protective CVD Si.sub.3 N.sub.4 layer.

  18. Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography

    NASA Astrophysics Data System (ADS)

    Medalia, Ohad; Weber, Igor; Frangakis, Achilleas S.; Nicastro, Daniela; Gerisch, Günther; Baumeister, Wolfgang

    2002-11-01

    Electron tomography of vitrified cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment.

  19. Nematic Liquid Crystal Alignment Behaviors between Crossed Stretched Miropolymer Filaments with Anchoring Effects

    NASA Astrophysics Data System (ADS)

    Fujikake, Hideo; Murashige, Takeshi; Sato, Hiroto; Kikuchi, Hiroshi; Kurita, Taiichiro; Sato, Fumio

    2006-04-01

    We observed the molecular alignment of a liquid crystal (LC) induced by crossing two stretched micropolymer filaments between glass substrates and confirmed its light modulation property. The two microfilaments, which were extracted from a cellulose cloth by stretching it in advance, had surface molecular alignment and stabilized nematic LC alignment between the microfilaments crossed with a small angle. In the fabricated LC cell, a spatially-uniform LC planar alignment is achieved in the area of a filament interval of less than 60 μm. By polarizing microscopy observation of the isotropic-to-nematic wetting transition of the LC material between the polymer filaments, it was confirmed that the stable LC alignment area is formed by the surface anchoring of the filaments. When external voltages were applied to the obtained uniformed alignment LC area, a characteristic periodic electrooptic property was confirmed on the basis of electrically-controlled birefringence under the alignment control of the in-plane anchoring of the filaments.

  20. Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

    PubMed

    Sisson, J C; Field, C; Ventura, R; Royou, A; Sullivan, W

    2000-11-13

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

  1. Triggering signaling pathways using F-actin self-organization.

    PubMed

    Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

    2016-10-04

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

  2. Triggering signaling pathways using F-actin self-organization

    PubMed Central

    Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

    2016-01-01

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

  3. The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

    PubMed

    Cabello, G; Galaz, S; Botella, L; Calaf, G; Pacheco, M; Stockert, J C; Villanueva, A; Cañete, M; Juarranz, A

    2003-09-01

    We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

  4. Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization

    PubMed Central

    Sisson, John C.; Field, Christine; Ventura, Richard; Royou, Anne; Sullivan, William

    2000-01-01

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion. PMID:11076973

  5. 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

  6. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    NASA Astrophysics Data System (ADS)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  7. Single-molecule imaging of {beta}-actin mRNAs in the cytoplasm of a living cell

    SciTech Connect

    Yamagishi, Mai; Ishihama, Yo; Shirasaki, Yoshitaka; Kurama, Hideki; Funatsu, Takashi

    2009-04-15

    {beta}-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most {beta}-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D{sub MACRO}) at the leading edge was 0.3 {mu}m{sup 2}/s. On the other hand, D{sub MACRO} in the perinuclear region was 0.02 {mu}m{sup 2}/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize {beta}-actin mRNAs, led to an increase in D{sub MACRO} to 0.2 {mu}m{sup 2}/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of {beta}-actin mRNA.

  8. Plant toxin β-ODAP activates integrin β1 and focal adhesion: A critical pathway to cause neurolathyrism

    PubMed Central

    Tan, Rui-Yue; Xing, Geng-Yan; Zhou, Guang-Ming; Li, Feng-Min; Hu, Wen-Tao; Lambein, Fernand; Xiong, Jun-Lan; Zhang, Sheng-Xiang; Kong, Hai-Yan; Zhu, Hao; Li, Zhi-Xiao; Xiong, You-Cai

    2017-01-01

    Neurolathyrism is a unique neurodegeneration disease caused by β-N-oxalyl-L-α, β- diaminopropionic (β-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, β-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of β1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change. PMID:28094806

  9. Distribution of cytoskeletal structures and organelles of the host cell during evolution of the intracellular parasitism by Trypanosoma cruzi.

    PubMed

    Carvalho, T M; Ferreira, A G; Coimbra, E S; Rosestolato, C T; De Souza, W

    1999-07-01

    The distribution of microtubules, microfilaments, mitochondria, Golgi complex and endosomes/lysosomes was analyzed in Vero cells allowed to interact for different periods of time with the pathogenic protozoan Trypanosoma cruzi and observed by confocal laser scanning microscopy. Microtubules were revealed using a mouse monoclonal anti-alpha-tubulin antibody. Actin filaments were revealed using phalloidin-rhodamine. To identify mitochondria, endosomes/lysosomes and the Golgi complex the cells were labelled with Rhodamine 123, Lucifer yellow and C6-NBD-ceramide, respectively. During cell invasion actin filaments concentrate at the site of parasite penetration in some, but not in all cells, probably depending upon the mechanism used by the trypomastigote form to penetrate into the host cells. Following internalization the trypomastigote form gradually changes into the amastigote form, disruption of the parasitophorous vacuole membrane takes place and the amastigote form enters in direct contact with host cell structures and organelles, and starts to divide. The presence of the parasite in the cytoplasm of the host cell did not induce significant changes in the distribution of actin filaments, microtubules, the Golgi complex, mitochondria and endosomes/lysosomes during the first 48 h of infection. Amastigote forms were seen close to the microtubules. After 72 h of interaction, the number of microtubules and microfilaments around the parasites was reduced and lysosomes and mitochondria were seen in between the parasites.

  10. Cytoskeleton Modifications and Autophagy Induction in TCam-2 Seminoma Cells Exposed to Simulated Microgravity

    PubMed Central

    Ferranti, Francesca; Caruso, Maria; Cammarota, Marcella; Fabrizi, Cinzia; Fumagalli, Lorenzo; Schiraldi, Chiara; Catizone, Angela

    2014-01-01

    The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one. PMID:25140323

  11. Filtration deformability of rabbit pulmonary macrophage.

    PubMed

    Smith, C M; Tukey, D P; Mundshenk, D; Krivit, W; White, J G; Repine, J E; Hoidal, J R

    1982-04-01

    Rabbit PAM deformability was evaluated by positive-pressure filtration through Nucleopore membranes of well-specified pore diameter. The PAM filtration method was standardized and was influenced by apparatus variations (pore size, flow rate, cell concentration), environment (temperature, pH, divalent cations, protein concentration), and differences in PAM cell volume. The influence of phagocyte function on filtration deformability was evaluated by exposing PAMs to pharmacologic and physiologic agents with somewhat exclusive influences on phagocyte physiology. Agents that interact with microfilament contractile protein (N-ethylmaleimide, cytochalasin B) altered deformability profoundly, but no effect was observed with agents interacting with microtubules (vinblastine, colchicine). Agents that cause general PAM activation (phorbol myristate acetate) or stimulate chemotaxis (F-Met-Leu-Phe) increased deformability. On the contrary, PAM deformability was not changed by phagocytosis of Staphylococcus aureus or latex beads. Pharmacologic agents that alter PAM adhesion (aspirin, indomethacin, physiologic dose hydrocortisone) or inhibit glycolysis (2-deoxyglucose) had no influence on filtration deformability. Filtration PAM deformability reflects passive whole cell rigidity, which appears to be determined by the state of polymerization of the actin-myosin microfilament complex.

  12. Actin depolymerization affects stress-induced translational activity of potato tuber tissue

    PubMed

    Morelli; Zhou; Yu; Lu; Vayda

    1998-04-01

    Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

  13. Single-molecule imaging of beta-actin mRNAs in the cytoplasm of a living cell.

    PubMed

    Yamagishi, Mai; Ishihama, Yo; Shirasaki, Yoshitaka; Kurama, Hideki; Funatsu, Takashi

    2009-04-15

    Beta-actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most beta-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D(MACRO)) at the leading edge was 0.3 microm(2)/s. On the other hand, D(MACRO) in the perinuclear region was 0.02 microm(2)/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize beta-actin mRNAs, led to an increase in D(MACRO) to 0.2 microm(2)/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of beta-actin mRNA.

  14. The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

    PubMed Central

    Braet, Filip; Spector, Ilan; Shochet, Nava; Crews, Phillip; Higa, Tatsuo; Menu, Eline; de Zanger, Ronald; Wisse, Eddie

    2002-01-01

    Background Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. Results Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. Conclusion (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs. PMID:11914125

  15. The cells of the rat gastric groove and cardia. An ultrastructural and carbohydrate histochemical study, with special reference to the fibrillovesicular cells.

    PubMed

    Wattel, W; Geuze, J J

    1978-01-31

    The distal wall of the groove between the rat forestomach and glandular stomach is lined with a special type of columnar cells (CCGG) and with fibrillovesicular cells (FVC). The cardiac glands contain cardiac mucosa (CMC) and serous cells (CSC). The CCGG contain small mucous granules and special vesicles and tubules. The CMC are filled with large mucous granules and resemble mucous neck cells. The CSC are filled with large proteinaceous granules. The FVC are characterized by long microvilli, apical bundles of microfilaments and a complex "tubulovesicular system". The pattern of 3H-thymidine incorporation and the presence of immature and transitional forms indicate a possible origin of all the cell types concerned from a common undifferentiated precursor. The membranes of the tubulovesicular system of FVC as well as the apical cell membrane were reactive to Thiéry's carbohydrate stain. However, lanthanum tracing of the extracellular space and ultrastructural stereoscopy did not reveal a permanent continuity between both membrane systems. The absence of 3H-thymidine label showed that FVC were not proliferative. The structural characteristics of FVC do not account for a secretory, resorptive or receptive function. The special arrangement of microfilaments and the tubulovesicular system suggests an ability to fast changes in surface area.

  16. Effect of cytochalasin D on the growth, encystation, and multinucleation of Entamoeba invadens.

    PubMed

    Makioka, A; Kumagai, M; Ohtomo, H; Kobayashi, S; Takeuchi, T

    2000-07-01

    The effect of cytochalasin D, a specific inhibitor of microfilaments, on the growth, encystation, and multinucleation of Entamoeba invadens was examined. Cytochalasin D blocked the growth of axenic E. invadens strain IP-1 in a dose-dependent manner, which suggests that the drug is effective against this species of Entamoeba as well as against E. histolytica strain HM1: IMSS as previously demonstrated. Encystation of E. invadens as induced in vitro was also inhibited by cytochalasin D. This is the first evidence of the participation of microfilaments in the encystation process. Concentrations of cytochalasin D effective for the inhibition of encystation were lower than those effective for the inhibition of growth. Trophozoites grown with cytochalasin D became multinucleate; more than three nuclei per cell were observed in 71% of trophozoites grown in the presence of the drug as opposed to only 5% of those grown in the absence of the drug. Also, trophozoites grown with cytochalasin D produced multinucleate cysts following their transfer to encystation medium. Encystation with cytochalasin D was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Also, encystation without cytochalasin D was less frequently observed among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Thus, the multinucleation of trophozoites induced by cytochalasin D had an inhibitory effect on their encystation.

  17. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    SciTech Connect

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  18. Establishment of a Functional Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcription Complex Involves the Cytoskeleton

    PubMed Central

    Bukrinskaya, Alissa; Brichacek, Beda; Mann, Angela; Stevenson, Mario

    1998-01-01

    After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex. PMID:9841925

  19. A Rho-signaling pathway mediates cortical granule translocation in the sea urchin oocyte.

    PubMed

    Covián-Nares, Fernando; Martínez-Cadena, Guadalupe; López-Godínez, Juana; Voronina, Ekaterina; Wessel, Gary M; García-Soto, Jesús

    2004-03-01

    Cortical granules are secretory vesicles of the egg that play a fundamental role in preventing polyspermy at fertilization. In the sea urchin egg, they localize directly beneath the plasma membrane forming a compact monolayer and, upon fertilization, undergo a Ca(2+)-dependent exocytosis. Cortical granules form during early oogenesis and, during maturation, translocate from the cytosol to the oocyte cortex in a microfilament-mediated process. We tested the hypothesis that these cortical granule dynamics were regulated by Rho, a GTPase of the Ras superfamily. We observed that Rho is synthesized early in oogenesis, mainly in a soluble form. At the end of maturation, however, Rho associates with cortical granules. Inhibition of Rho with the C3 transferase from C. botulinum blocks cortical granule translocation and microfilaments undergo a significant disorganization. A similar effect is observed by GGTI-286, a geranylgeranyl transferase inhibitor, suggesting that the association of Rho with the cortical granules is indispensable for its function. In contrast, the anchorage of the cortical granules in the cortex, as well as their fusion at fertilization, are Rho-independent processes. We conclude that Rho association with the cortical granules is a critical regulatory step in their translocation to the egg cortex.

  20. Ceramic nanoparticle assemblies with tailored shapes and tailored chemistries via biosculpting and shape-preserving inorganic conversion.

    PubMed

    Dickerson, M B; Naik, R R; Sarosi, P M; Agarwal, G; Stone, M O; Sandhage, K H

    2005-01-01

    A novel biosynthetic paradigm is introduced for fabricating three-dimensional (3-D) ceramic nanoparticle assemblies with tailored shapes and tailored chemistries: biosculpting and shape-preserving inorganic conversion (BaSIC). Biosculpting refers to the use of biomolecules that direct the precipitation of ceramic nanoparticles to form a continuous 3-D structure with a tailored shape. We used a peptide derived from a diatom (a type of unicellular algae) to biosculpt silica nanoparticle based assemblies that, in turn, were converted into a new (nonsilica) composition via a shape-preserving gas/silica displacement reaction. Interwoven, microfilamentary silica structures were prepared by exposing a peptide, derived from the silaffin-1A protein of the diatom Cylindrotheca fusiformis, to a tetramethylorthosilicate solution under a linear shear flow condition. Subsequent exposure of the silica microfilaments to magnesium gas at 900 degrees C resulted in conversion into nanocrystalline magnesium oxide microfilaments with a retention of fine (submicrometer) features. Fluid(gas or liquid)/silica displacement reactions leading to a variety of other oxides have also been identified. This hybrid (biogenic/synthetic) approach opens the door to biosculpted ceramic microcomponents with multifarious tailored shapes and compositions for a wide range of environmental, aerospace, biomedical, chemical, telecommunications, automotive, manufacturing, and defense applications.

  1. Microtubule stability affects the unique motility of F-actin in Marchantia polymorpha.

    PubMed

    Era, Atsuko; Kutsuna, Natsumaro; Higaki, Takumi; Hasezawa, Seiichiro; Nakano, Akihiko; Ueda, Takashi

    2013-01-01

    Actin microfilaments play crucial roles in diverse plant functions. Some specific cellular processes require interaction between F-actin and microtubules, and it is believed that there are direct or indirect connections between F-actin and microtubules. We previously reported that actin microfilaments exhibit unique dynamic motility in cells of the liverwort, Marchantia polymorpha; the relevance of this activity to microtubules has not been explored. To examine whether the dynamics of F-actin in M. polymorpha were somehow regulated by microtubules, we investigated the effects of stabilization or destabilization of microtubules on dynamics of actin bundles, which were visualized by Lifeact-Venus. To our surprise, both stabilization and destabilization of microtubules exerted similar effects on F-actin motility; apparent sliding movement of F-actin in M. polymorpha cells was accelerated by both oryzalin and paclitaxel, with the effect of paclitaxel more evident than that of oryzalin. Immunofluorescence staining revealed that some F-actin bundles were arrayed along with microtubules in M. polymorpha thallus cells. These results suggest that microtubules play regulatory roles in the unique F-actin dynamics in M. polymorpha.

  2. New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish.

    PubMed

    Hartigan, A; Estensoro, I; Vancová, M; Bílý, T; Patra, S; Eszterbauer, E; Holzer, A S

    2016-12-16

    Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein- actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique "dancing" behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari's microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins.

  3. The organelle of differentiation in embryos: the cell state splitter.

    PubMed

    Gordon, Natalie K; Gordon, Richard

    2016-03-10

    The cell state splitter is a membraneless organelle at the apical end of each epithelial cell in a developing embryo. It consists of a microfilament ring and an intermediate filament ring subtending a microtubule mat. The microtubules and microfilament ring are in mechanical opposition as in a tensegrity structure. The cell state splitter is bistable, perturbations causing it to contract or expand radially. The intermediate filament ring provides metastability against small perturbations. Once this snap-through organelle is triggered, it initiates signal transduction to the nucleus, which changes gene expression in one of two readied manners, causing its cell to undergo a step of determination and subsequent differentiation. The cell state splitter also triggers the cell state splitters of adjacent cells to respond, resulting in a differentiation wave. Embryogenesis may be represented then as a bifurcating differentiation tree, each edge representing one cell type. In combination with the differentiation waves they propagate, cell state splitters explain the spatiotemporal course of differentiation in the developing embryo. This review is excerpted from and elaborates on "Embryogenesis Explained" (World Scientific Publishing, Singapore, 2016).

  4. Transport of Motor Proteins along Microtubules: A Study by Optical Trapping Method and Analysis of Data

    NASA Astrophysics Data System (ADS)

    McFarlane, Angelique

    The cellular transportation is fundamental for cell function. Under this transportation, organelles bind to motor proteins. These proteins, then move along cellular microfilaments such as microtubules. The optical trapping technique is a method that allows us to monitor the movement of molecular motors along their tracks. In this method, motor proteins are absorbed by micro-sized beads. The beads are captured by the laser and placed close to the microfilaments. Consequently, the motor proteins bind to the track and move along them. This motion can be recorded and analyzed. In this work, we have analyzed many produced trajectories resulted from the motion of a single kinesin along microtubules. We present the design of the experiment, the method of recording and extracting data, as well as the factors that need to be considered to obtain accurate results. Finally, we calculated some of the physical properties resulted from kinesin movement in our experiment. Our outcomes are compatible with previously reported results. I acknowledge the support of NJSGC 2016 during this project. This work was conducted under the supervision of Dr. Mitra Feizabadi.

  5. F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis

    PubMed Central

    Gao, Ying; Mruk, Dolores D.; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    During the release of sperm at spermiation, a biologically active F5-peptide, which can disrupt the Sertoli cell tight junction (TJ) permeability barrier, is produced at the site of the degenerating apical ES (ectoplasmic specialization). This peptide coordinates the events of spermiation and blood-testis barrier (BTB) remodeling at stage VIII of the epithelial cycle, creating a local apical ES-BTB axis to coordinate cellular events across the epithelium. The mechanism(s) by which F5-peptide perturbs BTB restructuring, and its involvement in apical ES dynamics remain unknown. F5-peptide, besides perturbing BTB integrity, was shown to induce germ cell release from the epithelium following its efficient in vivo overexpression in the testis. Overexpression of F5-peptide caused disorganization of actin- and microtubule (MT)-based cytoskeletons, mediated by altering the spatiotemporal expression of actin binding/regulatory proteins in the seminiferous epithelium. F5-peptide perturbed the ability of actin microfilaments and/or MTs from converting between their bundled and unbundled/defragmented configuration, thereby perturbing adhesion between spermatids and Sertoli cells. Since apical ES and basal ES/BTB are interconnected through the underlying cytoskeletal networks, this thus provides an efficient and novel mechanism to coordinate different cellular events across the epithelium during spermatogenesis through changes in the organization of actin microfilaments and MTs. These findings also illustrate the potential of F5-peptide being a male contraceptive peptide for men. PMID:27611949

  6. New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish

    PubMed Central

    Hartigan, A.; Estensoro, I.; Vancová, M.; Bílý, T.; Patra, S.; Eszterbauer, E.; Holzer, A. S.

    2016-01-01

    Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein– actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique “dancing” behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari’s microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins. PMID:27982057

  7. Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation.

    PubMed

    Tavakolinejad, Alireza; Rabbani, Mohsen; Janmaleki, Mohsen

    2015-08-21

    Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells.

  8. Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis

    SciTech Connect

    Krieger, J.N.; Ravdin, J.I.; Rein, M.F.

    1985-12-01

    The cytopathogenic mechanisms of Trichomonas vaginalis have been debated since the 1940s. We examined the following three proposed pathogenic mechanisms: contact-dependent extracellular killing, cytophagocytosis, and extracellular cytotoxins. Serial observations of Chinese hamster ovary (CHO) cell monolayers exposed to trichomonads revealed that (i) trichomonads form clumps, (ii) the clumps adhere to cells in culture, and (iii) monolayer destruction occurs only in areas of contact with T. vaginalis. Kinetic analysis of target cell killing by trichomonads revealed that the probability of CHO cell death was related to the probability of contact with T. vaginalis, supporting the observation by microscopy that trichomonads kill cells only by direct contact. Simultaneous studies of /sup 111/indium oxine label release from CHO cells and trypan blue dye exclusion demonstrated that T. vaginalis kills target cells without phagocytosis. Filtrates of trichomonad cultures or from media in which trichomonads were killing CHO cells had no effect on CHO cell monolayers, indicating that trichomonads do not kill cells by a cell-free or secreted cytotoxin. The microfilament inhibitor cytochalasin D (10 micrograms/ml) inhibited trichomonad killing of CHO cell monolayers by 80% (P less than 0.0001). In contrast, the microtubule inhibitor vinblastine (10(-6) M) caused only 17% inhibition of trichomonad destruction of CHO cell monolayers (P less than 0.020), whereas colchicine (10(-6) M) had no effect. T. vaginalis kills target cells by direct contact without phagocytosis. This event requires intact trichomonad microfilament function; microtubule function appears not to be essential.

  9. Exposure of Human Lung Cancer Cells to 8-Chloro-Adenosine Induces G2/M Arrest and Mitotic Catastrophe1

    PubMed Central

    Zhang, Hong-Yu; Gu, Yan-Yan; Li, Zeng-Gang; Jia, Yu-Hong; Yuan, Lan; Li, Shu-Yan; An, Guo-Shun; Ni, Ju-Hua; Jia, Hong-Ti

    2004-01-01

    Abstract 8-Chloro-adenosine (8-Cl-Ado) is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt) and H1299 (p53-depleted) to 8-Cl-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-Cl-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe. PMID:15720807

  10. Mechanisms involved in the blood-testis barrier increased permeability induced by EMP.

    PubMed

    Wang, Xiao-Wu; Ding, Gui-Rong; Shi, Chang-Hong; Zeng, Li-Hua; Liu, Jun-Ye; Li, Jing; Zhao, Tao; Chen, Yong-Bin; Guo, Guo-Zhen

    2010-09-30

    The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  11. 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.

  12. Basal epithelial cells of human prostate gland are not myoepithelial cells. A comparative immunohistochemical and ultrastructural study with the human salivary gland.

    PubMed Central

    Srigley, J. R.; Dardick, I.; Hartwick, R. W.; Klotz, L.

    1990-01-01

    The hypothesis that basal epithelial cells of the human prostate are of myoepithelial origin was investigated using immunohistochemical and ultrastructural methodologies. The immunohistologic analyses show significant phenotypic differences between prostatic basal cells and myoepithelial cells of the salivary gland. Although both cell types stain intensely with the 312C8-1 monoclonal antibody, only true myoepithelial cells demonstrated significant amounts of muscle-specific actin as decorated by the HHF35 monoclonal antibody. Furthermore, using double-labeling experiments, the prostatic basal cells were strongly decorated with a fluorescein-tagged basal cell-specific keratin but were negative with the rhodamine-tagged phalloidin, a chemical that binds specifically to actin microfilaments. Ultrastructural studies also showed an absence of thin microfilament bundles, dense bodies, and micropinocytotic vesicles in the prostatic basal cells. The current investigations show that the prostatic acini do not have a basal myoepithelium. Although some authors have suggested a stem cell role for prostatic basal cells, the weight of experimental work argues against this hypothesis. The exact role of the basal epithelial cells of the prostate is not known, although they may serve endocrine, paracrine, or other regulatory functions and may be involved in modulating signals between prostatic stroma and epithelium. Images Figure 3 Figure 1 Figure 2 Figure 4 PMID:1691595

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  14. Altered osteoblast structure and function in parabolic flight

    NASA Astrophysics Data System (ADS)

    Zhong-Quan, Dai; Ying-Hui, Li; Fen, Yang; Bai, Ding; Ying-Jun, Tan

    Introduction Bone loss has a significant impact on astronauts during spaceflight being one of the main obstacles preventing interplanetary missions However the exact mechanism is not well understood In the present study we investigated the effects of acute gravitational changes generated by parabolic flight on the structure and function of osteoblasts ROS17 2 8 carried by airbus A300 Methods The alteration of microfilament cytoskeleton was observed by the Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I immunofluorescence stain ALP activity and expression COL1A1 expression osteocalcin secrete which presenting the osteoblast function were detected by modified calcium and cobalt method RT-PCR and radioimmunity methods respectively Results The changed gravity induced the reorganization of microfilament cytoskeleton of osteoblast After 3 hours parabolic flight F-actin of osteoblast cytoskeleton became more thickness and directivity whereas G-actin reduced and relatively concentrated at the edge of nucleus observed by confocal fluorescence microscopy This phenomenon is identical with structure alternation observed in hypergravity but the osteoblast function decrease The excretion of osteocalcin the activity and mRNA expression of ALP decrease but the COL1A1 expression has no changes These results were similar to the changes in simulated or real microgravity Conclusion Above results suggest that short time gravity alternative change induce osteoblast structure and function

  15. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

  16. Effects of parabolic flight on the cytoskeleton in cultured cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Yang, F.; Li, Y. H.; Dai, Z. Q.; Nie, J. L.; Tan, Y. J.; Yu, J. R.

    As intracellular load-bearing structure cytoskeleton is hypothesized to play a crucial role in gravity perception and transduction of cells Recent data show that the cytoskeleton including actin microfilaments and microtubules is involved in modulating both the electrical activity and mechanical activity of myocardium Using fluorescence-labeling of cells with specific antibodies or agentsLwe found discontinued abruption of microtubules and enhanced polymerization of filamentous F actin in neonatal rat cardiac myocytes after exposure to the acute gravitational changes micro- and hyper-gravity in parabolic flight By staining of globular monomeric G actin and F-actin with Alexa Fluor conjugated DNase I and Texas red-phalloidin respectively confocal microscopy demonstrated more prominent structure of F-actin and decreased cytosolic G-actin in flight cells implying a shift in the F G equilibrium in favor of F-actin Using specific antibody against phosphorylated activated forms of extracellular signal-regulated kinase ERK and focal adhesion kinase FAK we found that active ERK is co-localized with reorganized F-actin in flight cells while active FAK did not show evident collateral distribution with actin cytoskeleton indicating that ERK but not FAK might be involved in parabolic flight-induced polymerization of F-actin These results suggest that gravitational changes induced by parabolic flight substantially affected the distribution and organization of the actin microfilaments and microtubules in cultured cardiac myocytes and ERK might participate in the

  17. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    PubMed

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

  18. Plasmolysis: Loss of Turgor and Beyond

    PubMed Central

    Lang, Ingeborg; Sassmann, Stefan; Schmidt, Brigitte; Komis, George

    2014-01-01

    Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole “overrules” the stabilization by cortical cytoskeletal elements. PMID:27135521

  19. Plasmolysis: Loss of Turgor and Beyond.

    PubMed

    Lang, Ingeborg; Sassmann, Stefan; Schmidt, Brigitte; Komis, George

    2014-11-26

    Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.

  20. Mammalian cells exposed to ionizing radiation: Structural and biochemical aspects.

    PubMed

    Sabanero, Myrna; Azorín-Vega, Juan Carlos; Flores-Villavicencio, Lérida Liss; Castruita-Dominguez, J Pedro; Vallejo, Miguel Angel; Barbosa-Sabanero, Gloria; Cordova-Fraga, Teodoro; Sosa-Aquino, Modesto

    2016-02-01

    Acute or chronic exposure to ionizing radiation is a factor that may be hazardous to health. It has been reported that exposure to low doses of radiation (less than 50 mSv/year) and subsequently exposure to high doses produces greater effects in people. It has been reported that people who have been exposed to low doses of radiation (less than 50 mSv/year) and subsequently are exposed to high doses, have greater effects. However, at a molecular and biochemical level, it is an unknown alteration. This study, analyzes the susceptibility of a biological system (HeLa ATCC CCL-2 human cervix cancer cell line) to ionizing radiation (6 and 60 mSv/90 s). Our research considers multiple variables such as: total protein profile, mitochondrial metabolic activity (XTT assay), cell viability (Trypan blue exclusion assay), cytoskeleton (actin microfilaments), nuclei (DAPI), and genomic DNA. The results indicate, that cells exposed to ionizing radiation show structural alterations in nuclear phenotype and aneuploidy, further disruption in the tight junctions and consequently on the distribution of actin microfilaments. Similar alterations were observed in cells treated with a genotoxic agent (200 μM H2O2/1h). In conclusion, this multi-criteria assessment enables precise comparisons of the effects of radiation between various line cells. However, it is necessary to determine stress markers for integration of the effects of ionizing radiation.

  1. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin.

    PubMed Central

    Bernardini, M L; Mounier, J; d'Hauteville, H; Coquis-Rondon, M; Sansonetti, P J

    1989-01-01

    The capacity of Shigella to spread within the cytosol of infected epithelial cells and to infect adjacent cells is critical for the development of infection foci, which lead to mucosal abscesses. Shigella is a nonmotile microorganism that appears to utilize host cell microfilaments to generate intra- as well as intercellular movements, since this movement was inhibited by cytochalasin D and involvement of F-actin was demonstrated by direct labeling of infected cells with the specific dye N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin. Such movements led to the formation of extracellular protrusions, which may explain cell to cell spread. icsA, a locus necessary for intra- and intercellular spread, was identified on the Shigella flexneri virulence plasmid pWR100. This locus was cloned and shown to express a 120-kDa outer membrane protein, which plays an important role in the interactions established between host cell microfilaments and the bacterial surface, thus leading to intracellular movement. Images PMID:2542950

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  3. Gravity sensing mechanisms in plant cells.

    PubMed

    Sievers, A

    1991-07-01

    Sensing of gravity is essential for the survival of plant seedlings. Therefore it is understandable that gravistimulation of only 0.5 sec-duration causes a graviresponse. The earliest graviresponses could be measured within seconds as alterations in membrane potentials of the statocytes in the root cap. Root statocytes are polarly organized. From a 6-day microgravity (10(-3) - 10(-4) g) experiment in the Spacelab D1 Mission it has been concluded that the observed polar differentiation is a result of a genetically prepatterned developmental program. Statoliths, the sedimentable organelles of statocytes, are surrounded by actin filaments which partly keep them in position. Under 6 min of microgravity during parabolic flights of rockets it could be demonstrated that the statoliths moved in the opposite direction to the initial gravity vector. It is concluded that shearing forces are exerted by microfilaments. It is supposed that the change of the position of statoliths is transmitted to gravisensitive structures of the statocytes (ER, plasma membrane) via microfilaments. As graviperception is influenced by calcium ions, it is suggested that these interactions regulate the activity of ion channels and/or pumps in the membranes thus initiating the graviresponse chain. In the case of cytoplasmic streaming in Chara rhizoids, the endogenous difference between the opposing streaming directions is diminished under microgravity during the flights of rockets. Possibly, shear stresses are affected by gravity, thus inducing gravity-related differences in the streaming velocities via actin filaments.

  4. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

    Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

  5. Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

    PubMed Central

    Yamashiro, Shigeko; Yamakita, Yoshihiko; Ono, Shoichiro; Matsumura, Fumio

    1998-01-01

    Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery. PMID:9571235

  6. Chirality of the cytoskeleton in the origins of cellular asymmetry.

    PubMed

    Satir, Peter

    2016-12-19

    Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior-posterior cell and tissue axis.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

  7. Heavy metal effects on cellular shape changes, cleavage, and larval development of the marine gastropod mollusk, (Ilyanassa obsoleta Say)

    SciTech Connect

    Conrad, G.W.

    1988-07-01

    The spawning areas for many marine invertebrates are in intertidal zones which can be exposed to surface water run-off containing heavy metals. The cellular shape changes and cleavage patterns of Ilyanassa embryos greatly resemble those of bivalve mollusks, such as Mytilus edulis, that occur in the same intertidal areas. Determining the concentrations of heavy metals tolerated by the molluscan embryos inhabiting such clam and mussel beds therefore is of some economic significance. Moreover, such research may providedata on the heavy metal effects on the cytoskeleton. There is increasing evidence that components of the cytoskeleton, directly or indirectly, are targets for toxic agents. Polar lobe formation is a cellular shape change that resembles cytokinesis. It is seen in the fertilized eggs of many marine mollusks. Recent data with inorganic and organic Ca/sup 2 +/ antagonists suggest that both polar lobe formation and cytokinesis utilize Ca/sup 2 +/ released from sequestered, intracellular sites. Both of these cellular constrictions are associated with microfilaments and are preceded by activation steps requiring microtubules. The data presented below suggest that several heavy metals affect the microfilament-dependent steps.

  8. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: Identification of novel biomarkers of effect

    SciTech Connect

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

    2009-10-15

    In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

  9. Actin Polymerization Is Essential for Pollen Tube GrowthV⃞

    PubMed Central

    Vidali, Luis; McKenna, Sylvester T.; Hepler, Peter K.

    2001-01-01

    Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a ∼50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a ∼100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1–4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming. PMID:11514633

  10. Arabidopsis FH1 Formin Affects Cotyledon Pavement Cell Shape by Modulating Cytoskeleton Dynamics.

    PubMed

    Rosero, Amparo; Oulehlová, Denisa; Stillerová, Lenka; Schiebertová, Petra; Grunt, Michal; Žárský, Viktor; Cvrčková, Fatima

    2016-03-01

    Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. The mTOR-FAK mechanotransduction signaling axis for focal adhesion maturation and cell proliferation

    PubMed Central

    Lee, Fan-Yen; Zhen, Yen-Yi; Yuen, Chun-Man; Fan, Raymond; Chen, Yen-Ta; Sheu, Jiunn-Jye; Chen, Yi-Ling; Wang, Ching-Jen; Sun, Cheuk-Kwan; Yip, Hon-Kan

    2017-01-01

    Background: Mechanotransduction (MTD) is an important physiopathological signalling pathway associated with cardiovascular disease such as hypertension. Phosphorylation of focal adhesion kinase (FAK) is a MTD-sensing protein. This study tested the hypothesis that mTOR-FAK MTD signaling axis was crucial for focal adhesion (FA) maturation and cell proliferation. Methods: Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling. Results: After demonstrating a failure in FAK phosphorylation after microfilament depolymerization, we attempted to identify the upstream regulator out of three kinases known to be activated in pressure-stimulated MTD [i.e., GSK-3β, Akt, and mTORC1 (mammalian target of rapamycin complex 1)]. Of the three specific inhibitors, only rapamycin, an inhibitor of mTORC1, was found to inhibit FAK phosphorylation, suggesting that mTORC1 is the upstream regulator in shock-wave-elicited FAK phosphorylation. Moreover, mTOR and its readout protein S6K were found to be activated by shock-wave stimulation. On the other hand, microscopic examination revealed not only MTD-induced increase in the number of actin stress fibers, but also alternative subcellular localization of mTORC1 as vesicle-like inclusions on microfilaments. Besides, rapamycin was found to destruct the granular pattern of mTORC1, while dissociation between F-actin and mTORC1 was noted after cytochalasin D administration. Since mTORC1 and FAK are essential for cell proliferation, we performed proliferation assay for mesenchymal stem cell (MSC) with and without shock-wave administration/rapamycin treatment/FAK depletion. The results demonstrated significant enhancement of cell proliferation after shock-wave stimulation but remarkable suppression after rapamycin and siFAK treatment. Conclusion: Our findings suggest not only a co-ordinated regulation of FAK phosphorylation by mTORC1 and microfilaments, but also the participation of mTORC1-FAK signalling in MSC proliferation. PMID:28469768

  12. A multi-component parallel-plate flow chamber system for studying the effect of exercise-induced wall shear stress on endothelial cells.

    PubMed

    Wang, Yan-Xia; Xiang, Cheng; Liu, Bo; Zhu, Yong; Luan, Yong; Liu, Shu-Tian; Qin, Kai-Rong

    2016-12-28

    In vivo studies have demonstrated that reasonable exercise training can improve endothelial function. To confirm the key role of wall shear stress induced by exercise on endothelial cells, and to understand how wall shear stress affects the structure and the function of endothelial cells, it is crucial to design and fabricate an in vitro multi-component parallel-plate flow chamber system which can closely replicate exercise-induced wall shear stress waveforms in artery. The in vivo wall shear stress waveforms from the common carotid artery of a healthy volunteer in resting and immediately after 30 min acute aerobic cycling exercise were first calculated by measuring the inner diameter and the center-line blood flow velocity with a color Doppler ultrasound. According to the above in vivo wall shear stress waveforms, we designed and fabricated a parallel-plate flow chamber system with appropriate components based on a lumped parameter hemodynamics model. To validate the feasibility of this system, human umbilical vein endothelial cells (HUVECs) line were cultured within the parallel-plate flow chamber under abovementioned two types of wall shear stress waveforms and the intracellular actin microfilaments and nitric oxide (NO) production level were evaluated using fluorescence microscope. Our results show that the trends of resting and exercise-induced wall shear stress waveforms, especially the maximal, minimal and mean wall shear stress as well as oscillatory shear index, generated by the parallel-plate flow chamber system are similar to those acquired from the common carotid artery. In addition, the cellular experiments demonstrate that the actin microfilaments and the production of NO within cells exposed to the two different wall shear stress waveforms exhibit different dynamic behaviors; there are larger numbers of actin microfilaments and higher level NO in cells exposed in exercise-induced wall shear stress condition than resting wall shear stress condition

  13. Mitochondrial dynamics and optical conformation changes in DsRed as studied by Fourier imaging correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Senning, Eric Nicolas

    Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6--1.19 mum) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 mum. The versatility of

  14. Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Ksiazek, Dorota; Brandstetter, Hans; Israel, Lars; Bourenkov, Gleb P; Katchalova, Galina; Janssen, Klaus-Peter; Bartunik, Hans D; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2003-09-01

    Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

  15. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Mavoungou, Chrystelle; Israel, Lars; Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.

  16. Perversions with a twist

    PubMed Central

    Silva, Pedro E. S.; Trigueiros, Joao L.; Trindade, Ana C.; Simoes, Ricardo; Dias, Ricardo G.; Godinho, Maria Helena; de Abreu, Fernao Vistulo

    2016-01-01

    Perversions connecting two helices with symmetric handedness are a common occurrence in nature, for example in tendrils. These defects can be found in our day life decorating ribbon gifts or when plants use tendrils to attach to a support. Perversions arise when clamped elastic filaments coil into a helical shape but have to conserve zero overall twist. We investigate whether other types of perversions exist and if they display different properties. Here we show mathematically and experimentally that a continuous range of different perversions can exist and present different geometries. Experimentally, different perversions were generated using micro electrospun fibres. Our experimental results also confirm that these perversions behave differently upon release and adopt different final configurations. These results also demonstrate that it is possible to control on demand the formation and shape of microfilaments, in particular, of electrospun fibres by using ultraviolet light. PMID:27025549

  17. Guiding cellular activity with polarized light.

    PubMed

    Constant, Colin; Bergano, Andrea; Sugaya, Kiminobu; Dogariu, Aristide

    2017-07-03

    Actin, cytoskeleton protein forming microfilaments, play a crucial role in cellular motility. Here we show that exposure to very low levels of polarized light guide their orientation in-vivo within the live cell. Using a simple model to describe the role of actin-filament orientation in directional cellular motion, we demonstrate that the actin polymerization/depolymerization mechanism develops primarily along this direction and, under certain conditions, can lead to guidance of the cell movement. Our results also show a dose dependent increase in actin activity in direct correspondence to the level of laser irradiance. We found that total expression of Tau protein, which stabilize microtubules, was decreased by the irradiance, indicating that exposure to the light may change the activity of kinase, leading to increased cell activity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Nanocapillarity-mediated magnetic assembly of nanoparticles into ultraflexible filaments and reconfigurable networks

    PubMed Central

    Bharti, Bhuvnesh; Fameau, Anne-Laure; Rubinstein, Michael; Velev, Orlin D.

    2016-01-01

    The fabrication of multifunctional materials with tunable structure and properties requires programmed binding of their building blocks1,2. For example, particles organized in long-ranged structures by external fields3,4 can be bound permanently into stiff chains through electrostatic or van der Waals attraction4,5, or into flexible chains through soft molecular linkers such as surface-grafted DNA or polymers6–11. Here, we show that capillarity-mediated binding between magnetic nanoparticles coated with a liquid lipid shell can be used for the assembly of ultraflexible microfilaments and network structures. These filaments can be magnetically regenerated on mechanical damage, owing to the fluidity of the capillary bridges between nanoparticles and their reversible binding on contact. Nanocapillary forces offer opportunities for assembling dynamically reconfigurable multifunctional materials that could find applications as micromanipulators, microbots with ultrasoft joints, or magnetically self-repairing gels. PMID:26237128

  19. Interrogating the Bioactive Pharmacophore of the Latrunculin Chemotype by Investigating the Metabolites of Two Taxonomically Unrelated Sponges

    PubMed Central

    Amagata, Taro; Johnson, Tyler A.; Cichewicz, Robert H.; Tenney, Karen; Mooberry, Susan L.; Media, Joseph; Edelstein, Matthew; Valeriote, Frederick A.; Crews, Phillip

    2009-01-01

    This study involved a campaign to isolate and study additional latrunculin analogs from two taxonomically unrelated sponges, Cacospongia mycofijiensis and Negombata magnifica. A total of 13 latrunculin analogs were obtained by four different ways, reisolation (1–4), our repository (5–6), new derivatives (7–12), and a synthetic analog (7a). The structures of the new metabolites were elucidated based on a combination of comprehensive 1D and 2D NMR analysis, application of DFT calculations, and the preparation of acetonide derivative 7a. The cytotoxicities against both murine and human cancer cell lines observed for 1, 2, 7, 7a, 8, 9, and 12 were significant and the IC50 value range was 0.5–10 μM. Among the cytotoxic derivatives, compound 9 did not exhibit microfilament-disrupting activity at 5 μM. The implications of this observation and the value of further therapeutic study on key latrunculin derivatives are discussed. PMID:18942825

  20. Contraction of isolated brush borders from the intestinal epithelium

    PubMed Central

    1976-01-01

    Brush borders isolated from epithelial cells from the small intestine of neonatal rats are able to contract in the presence of ATP and Mg2+; Ca2+ is not required. Contraction is characterized by a pinching-in of the plasma membrane in the region of the zonula adherens and a subsequent rounding of the brush borders. No movement or consistent shortening of the microvilli is observed. The contraction appears to involve the 5- to 7-nm diameter microfilaments in the terminal web which associate with the zonula adherens. These filaments bind heavy meromyosin as do the actin core filaments of the microvilli. A model for contraction is presented in which, in the intact cell, terminal web filaments and core filaments interact to produce shortening of the microvilli. PMID:783170

  1. Characterization of Glass Fiber Separator Material for Lithium Batteries

    NASA Technical Reports Server (NTRS)

    Subbarao, S.; Frank, H.

    1984-01-01

    Characterization studies were carried out on a glass fiber paper that is currently employed as a separator material for some LiSOCl2 primary cells. The material is of the non-woven type made from microfilaments of E-type glass and contains an ethyl acrylate binder. Results from extraction studies and tensile testing revealed that the binder content and tensile strength of the paper were significantly less than values specified by the manufacturer. Scanning electron micrographs revealed the presence of clusters of impurities many of which were high in iron content. Results of emission spectroscopy revealed high overall levels of iron and leaching, followed by atomic absorption measurements, revealed that essentially all of this iron is soluble in SOCl2.

  2. Nanocapillarity-mediated magnetic assembly of nanoparticles into ultraflexible filaments and reconfigurable networks

    NASA Astrophysics Data System (ADS)

    Bharti, Bhuvnesh; Fameau, Anne-Laure; Rubinstein, Michael; Velev, Orlin D.

    2015-11-01

    The fabrication of multifunctional materials with tunable structure and properties requires programmed binding of their building blocks. For example, particles organized in long-ranged structures by external fields can be bound permanently into stiff chains through electrostatic or van der Waals attraction, or into flexible chains through soft molecular linkers such as surface-grafted DNA or polymers. Here, we show that capillarity-mediated binding between magnetic nanoparticles coated with a liquid lipid shell can be used for the assembly of ultraflexible microfilaments and network structures. These filaments can be magnetically regenerated on mechanical damage, owing to the fluidity of the capillary bridges between nanoparticles and their reversible binding on contact. Nanocapillary forces offer opportunities for assembling dynamically reconfigurable multifunctional materials that could find applications as micromanipulators, microbots with ultrasoft joints, or magnetically self-repairing gels.

  3. Interactions between glycolytic enzymes and components of the cytomatrix

    PubMed Central

    Masters, C.

    1984-01-01

    Evidence is provided that enzymes absorb to cellular structures in a wide range of tissues. In particular, the interactions between glycolytic enzymes and the microfilaments of the cytoplasm are described. The relevance of these interactions to the compartmentation of carbohydrate metabolism is discussed. Examples are given of the variations in degree of binding during alteration of tissue metabolism and, for individual glycolytic enzymes, during fetal development and differentiation. Overall, these data support the concept that metabolic activities in the cytoplasm have an organized structure. Just as the structural elements of the cytosolic compartment have evolved with the capacity to assemble and disassemble in response to the changing requirements of the organism, so the metabolic elements appear to have evolved a parallel system that provides for the appropriate positioning of an energy-producing sequence in relation to the specific, dynamic requirements of the cytoskeleton. PMID:6746730

  4. Millijoule femtosecond micro-Bessel beams for ultra-high aspect ratio machining.

    PubMed

    Mitra, Sambit; Chanal, Margaux; Clady, Raphaël; Mouskeftaras, Alexandros; Grojo, David

    2015-08-20

    We report on a functional experimental design for Bessel beam generation capable of handling high-energy ultrashort pulses (up to 1.2 mJ per pulse of 50 fs duration). This allows us to deliver intensities exceeding the breakdown threshold for air or any dielectric along controlled micro-filaments with lengths exceeding 4 mm. It represents an unprecedented upscaling in comparison to recent femtosecond Bessel beam micromachining experiments. We produce void microchannels through glass substrates to demonstrate that aspect ratios exceeding 1200∶1 can be achieved by using single high-intensity pulses. This demonstration must lead to new methodologies for deep-drilling and high-speed cutting applications.

  5. Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

    PubMed

    Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

    2016-01-01

    In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

  6. Septin 6 localizes to microtubules in neuronal dendrites.

    PubMed

    Moon, Il Soo; Lee, Hyunsook; Walikonis, Randall S

    2013-03-01

    In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs.

  7. Action of cytochalasin D on DNA synthesis in cells in culture

    SciTech Connect

    Glushankova, N.A.

    1986-10-01

    To solve the problem of the effect of changes in the actin cytoskeleton on DNA replication during the action of cytochalasins, the effect of long-term incubation of normal cells with cytochalasin D (CCD), which selectively destroys the microfilament system but does not affect transport of sugars, was investigated. Incorporation of labeled thymidine into mononuclear and binuclear cells in the presence of CCD and after its removal by rinsing also was studied separately. To investigate DNA synthesis the method of autoradiography with /sup 3/H-thymidine was used. A culture of mouse fibroblasts of the BALB/3T3 line and a secondary culture of fibroblasts obtained by trypsinization of mouse embryos (MEF) were used. On incubation of MEF and 3T3 cells, gradual inhibition of DNA synthesis is observed. The results obtained indicate that structural changes in the active cytoskeleton can abruptly and reversibly disturb passage of the normal cell through the cycle.

  8. Mesothelial glandular structures within pseudosarcomatous proliferative funiculitis--a diagnostic pitfall: report of 17 cases.

    PubMed

    Michal, Michal; Hes, Ondrej; Kazakov, Dmitry V

    2008-01-01

    We describe 17 cases of distinct benign pseudomalignant mesothelial proliferations, involving the spermatic cord. All cases revealed necrosis. The areas adjacent to the necrotic tissue comprised a cellular spindle cell proliferation with a haphazard arrangement of the myofibroblasts that in many areas revealed transitions into plump oval epithelioid cells and into cells with genuine epithelial appearances arranged in linear cords and often luminized into small microcysts. These epithelial cells formed isolated groups with glandular structures arising on the myofibroblastic background. Glandular structures were often situated deeply in the stroma of the spermatic cords. All cellular elements were strongly positive with AE1/AE3 antibody. All myofibroblasts stained with SM-actin antibody. Ultrastructurally, the spindle cells displayed features of myofibroblasts including actin microfilaments, as did the plump epithelioid cells that, additionally, had desmosomes, and the cords of the epithelial cells including those forming glandular structures had characteristics of mesothelias including the characteristic microvilli.

  9. Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

    NASA Astrophysics Data System (ADS)

    Collart-Dutilleul, Pierre-Yves; Panayotov, Ivan; Secret, Emilie; Cunin, Frédérique; Gergely, Csilla; Cuisinier, Frédéric; Martin, Marta

    2014-10-01

    The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions.

  10. Plastic contamination in the decapod crustacean Nephrops norvegicus (Linnaeus, 1758).

    PubMed

    Murray, Fiona; Cowie, Phillip Rhys

    2011-06-01

    The aim of this study was to determine the extent Nephrops consumes plastics in the Clyde Sea and if this intake occurs through their diet. Plastic contamination was found to be high in Nephrops, 83% of the animals sampled contained plastics (predominately filaments) in their stomachs. Tightly tangled balls of plastic strands were found in 62% of the animals studied but were least prevalent in animals which had recently moulted. No significant difference in plastic load was observed between males and females. Raman spectroscopy indicated that some of the microfilaments identified from gut contents could be sourced to fishing waste. Nephrops fed fish seeded with strands of polypropylene rope were found to ingest but not to excrete the strands. The fishery for Norway lobster, Nephrops norvegicus, is the most valuable in Scotland and the high prevalence of plastics in Nephrops may have implications for the health of the stock.

  11. Statistical organelle dissection of Arabidopsis guard cells using image database LIPS.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Hosokawa, Yoichiroh; Akita, Kae; Ebine, Kazuo; Ueda, Takashi; Kondo, Noriaki; Hasezawa, Seiichiro

    2012-01-01

    To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

  12. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton.

    PubMed

    Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R

    2014-05-01

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  13. Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts

    NASA Technical Reports Server (NTRS)

    Eckes, B.; Dogic, D.; Colucci-Guyon, E.; Wang, N.; Maniotis, A.; Ingber, D.; Merckling, A.; Langa, F.; Aumailley, M.; Delouvee, A.; Koteliansky, V.; Babinet, C.; Krieg, T.

    1998-01-01

    Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

  14. Two Molecules of Lobophorolide Cooperate to Stabilize an Actin Dimer Using Both Their 'Ring' and 'Tail' Region

    SciTech Connect

    Blain, J.; Mok, Y; Kubanek, J; Allingham, J

    2010-01-01

    Actin filament-disrupting marine macrolides are promising templates from which to design therapeutics against cancer and other diseases that co-opt the actin cytoskeleton. Typically, these macrolides form either a 1:1 or 2:1 actin-macrolide complex where their aliphatic side chain, or 'tail', has been reported to convey the major determinant of cytotoxicity. We now report the structure of the marine macrolide lobophorolide bound to actin with a unique 2:2 stoichiometry in which two lobophorolide molecules cooperate to form a dimerization interface that is composed entirely of the macrolide 'ring' region, and each molecule of lobophorolide interacts with both actin subunits via their ring and tail regions to tether the subunits together. This binding mode imposes multiple barriers against microfilament stability and holds important implications for development of actin-targeting drugs and the evolution of macrolide biosynthetic enzymes.

  15. The cytoskeleton of digitonin-treated rat hepatocytes.

    PubMed Central

    Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

    1980-01-01

    Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes. Images PMID:6997878

  16. Spata6 is required for normal assembly of the sperm connecting piece and tight head-tail conjunction.

    PubMed

    Yuan, Shuiqiao; Stratton, Clifford J; Bao, Jianqiang; Zheng, Huili; Bhetwal, Bhupal P; Yanagimachi, Ryuzo; Yan, Wei

    2015-02-03

    "Pinhead sperm," or "acephalic sperm," a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

  17. JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

    PubMed Central

    Díaz-Chiguer, Dylan L; Hernández-Luis, Francisco; Nogueda-Torres, Benjamín; Castillo, Rafael; Reynoso-Ducoing, Olivia; Hernández-Campos, Alicia; Ambrosio, Javier R

    2014-01-01

    Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target. PMID:25317703

  18. The effect of length and diameter on the resistivity of bromine intercalated graphite fibers

    NASA Technical Reports Server (NTRS)

    Gaier, James R.

    1989-01-01

    The resistivity of bromine intercalated graphite fibers has been shown to vary with both the diameter and the length of the fibers. This is due to bromine depletion from the fiber surface. Model calculations assuming a 1.0 micron bromine depletion zone for P-100, and 3.0 microns for vapor-grown graphite fibers fit the respective diameter dependence of their resistivities quite well. Length dependence data imply a bromine depletion zone along the length of P-100 fibers which is also a few microns, but that of vapor grown fibers appears to be as large as 300 microns. Despite these values, microfilaments, which are much smaller than the expected depletion zones, do form residual bromine intercalation compounds with resistivities about one-half of their pristine value.

  19. Adhesion to and invasion of cultured human cells by Bartonella bacilliformis.

    PubMed Central

    Hill, E M; Raji, A; Valenzuela, M S; Garcia, F; Hoover, R

    1992-01-01

    Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers. The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells. Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells. There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae. Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles. These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis. Images PMID:1398917

  20. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    SciTech Connect

    Lyi, Sangbom Michael; Tan, Min Jie Alvin Parrish, Colin R.

    2014-05-15

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  1. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    PubMed Central

    Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R.

    2014-01-01

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes. PMID:24889253

  2. In vitro phagocytosis and intracellular survival of Campylobacter jejuni with phagocytes

    SciTech Connect

    Kiehlbauch, J.A.

    1986-01-01

    In vitro phagocytosis and intracellular survival of Campylobacter jejuni was studied using three types of mononuclear phagocytes: a J774G8 peritoneal macrophage line, resident BABL/c peritoneal macrophages and human peripheral blood monocytes. In phagocytosis assays using CFU determinations, phagocytosis increased steadily over an 8 hr time period. Results obtained using a /sup 51/Cr assay indicated no consistent significant difference between phagocytosis of C. jejuni between the three mononuclear phagocytes or PMN's and that maximum infection occurred prior to 0.5 hr and maintained throughout the 4 hr assay. Further investigation of the mechanism of attachment and entry of C. jejuni revealed this process required the expenditure of energy by the phagocyte, but was not inhibited by inhibitors of microfilament functions. In addition, phagocytosis was enhanced by the presence of 20% FCS,

  3. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells

    PubMed Central

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W.; Kopnin, Pavel

    2016-01-01

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells. PMID:27683037

  4. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

    PubMed

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W; Kopnin, Pavel

    2016-11-08

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

  5. Regulation of Steroid Hormone Biosynthesis by the Cytoskeleton

    PubMed Central

    Li, Donghui

    2009-01-01

    Steroid hormones are synthesized in response to signaling cascades initiated by the trophic peptide hormones derived from the anterior pituitary. The mechanisms by which these peptide hormones regulate steroid hormone production are multifaceted and include controlling the transcription of steroidogenic genes, regulating cholesterol (substrate) uptake and transport, modulating steroidogenic enzyme activity, and controlling electron availability. Cytoskeletal polymers such as microfilaments and microtubules have also been implicated in regulating steroidogenesis. Of note, steroidogenesis is a multi-step process that occurs in two organelles, the endoplasmic reticulum (ER) and the mitochondrion. However, the precise mechanism by which substrates are delivered back and forth between these two organelles is unknown. In this review we will discuss the role of components of the cytoskeleton in conferring optimal steroidogenic potential. Finally, we present data that identifying a novel mechanism by which sphingosine-1-phosphate induces mitochondrial trafficking to promote steroidogenesis. PMID:18726632

  6. The effect of length and diameter on the resistivity of bromine intercalated graphite fibers

    NASA Technical Reports Server (NTRS)

    Gaier, James R.

    1989-01-01

    The resistivity of bromine intercalated graphite fibers has been shown to vary with both the diameter and the length of the fibers. This is due to bromine depletion from the fiber surface. Model calculations assuming a 1.0 micron bromine depletion zone for P-100, and 3.0 microns for vapor-grown graphite fibers fit the respective diameter dependence of their resistivities quite well. Length dependence data imply a bromine depletion zone along the length of P-100 fibers which is also a few microns, but that of vapor grown fibers appears to be as large as 300 microns. Despite these values, microfilaments, which are much smaller than the expected depletion zones, do form residual bromine intercalation compounds with resistivities about one-half of their pristine value.

  7. The role of P-cadherin in skin biology and skin pathology: lessons from the hair follicle.

    PubMed

    Samuelov, Liat; Sprecher, Eli; Paus, Ralf

    2015-06-01

    Adherens junctions (AJs) are one of the major intercellular junctions in various epithelia including the epidermis and the follicular epithelium. AJs connect the cell surface to the actin cytoskeleton and comprise classic transmembrane cadherins, such as P-cadherin, armadillo family proteins, and actin microfilaments. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in two allelic autosomal recessive disorders: hypotrichosis with juvenile macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly, and macular dystrophy (EEM) syndromes. Both syndromes feature sparse hair heralding progressive macular dystrophy. EEM syndrome is characterized in addition by ectodermal and limb defects. Recent studies have demonstrated that, together with its involvement in cell-cell adhesion, P-cadherin plays a crucial role in regulating cell signaling, malignant transformation, and other major intercellular processes. Here, we review the roles of P-cadherin in skin and hair biology, with emphasize on human hair growth, cycling and pigmentation.

  8. Mechanisms of polar lobe formation in fertilized eggs of molluscs.

    PubMed

    Conrad, G W; Schantz, A R; Patron, R R

    1990-01-01

    Polar lobe formation in the marine mudsnail, Ilyanassa obsoleta, involves formation of a microtubule-, microfilament-dependent furrow that constricts at two rates, then stops and relaxes. Some artificial seawater mixtures allow relatively normal development, facilitate insertion of microelectrode tips, and prevent artifactual bleb formation during such punctures. Membrane events may affect formation of polar lobe constrictions: (1) Brief treatment with digitonin prevents constrictions, but not cytokinesis per se, and the suppression of constrictions is permanent. Tomatine (but not tomatidine) and filipin act similarly, although filipin often also stops cytokinesis as well. (2) Responses to digitonin, tomatine, and filipin occur with little change in membrane potential. (3) Adhesion to substrata in response to brief treatment at low pH prevents both constrictions and cytokinesis. (4) Adhesion to substrata via polylysine allows both constrictions and cytokinesis, but embryos are smaller in volume and develop abnormally. Formation of lobe constrictions may be sensitive to perturbations of the plasma membrane.

  9. Actin dynamics in mouse fibroblasts in microgravity

    NASA Astrophysics Data System (ADS)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  10. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology.

  11. Myosin superfamily: The multi-functional and irreplaceable factors in spermatogenesis and testicular tumors.

    PubMed

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-15

    Spermatogenesis is a fundamental process in sexual development and reproduction, in which the diploid spermatogonia transform into haploid mature spermatozoa. This process is under the regulation of multiple factors and pathway. Myosin has been implicated in various aspects during spermatogenesis. Myosins constitute a diverse superfamily of actin-based molecular motors that translocate along microfilament in an ATP-dependent manner, and six kinds of myosins have been proved that function during spermatogenesis. In mitosis and meiosis, myosins play an important role in spindle assembly and positioning, karyokinesis and cytokinesis. During spermiogenesis, myosins participate in acrosomal formation, nuclear morphogenesis, mitochondrial translocation and spermatid individualization. In this review, we summarize current understanding of the functions of myosin in spermatogenesis and some reproductive system diseases such as testicular tumors and prostate cancer, and discuss the roles of possible upstream molecules which regulate myosin in these processes.

  12. The role of the cytoskeleton during oriented microfibril deposition. I. Elucidation of the possible interaction between microtubules and cellulose synthetic complexes.

    PubMed

    Seagull, R W

    1983-05-01

    A detailed analysis of changes in the cytoskeletal organization during cell elongation and oriented microfibril deposition has been done in the four plant species, clover (Trifolium repens), radish (Raphanus sativus), corn (Zea mays), and sorghum (Sorghum vulgare). Microtubules of variable lengths were found in all the cells examined. Some grouping of microtubules was observed with inter microtubule distances ranging from 14 to 40 nm. Single microfilaments were often observed between parallel microtubules. During cell elongation, microtubule frequency (No./microns) was maintained, thus indicating that microtubules must be formed continuously. The parallel orientation of wall microfibrils is disrupted as they deviate around plasmodesmata and pit-fields; however the cortical microtubules, thought to be influencing microfibril orientation, exhibit no consistent deviation around pit-fields. These observations are used to argue that cortical microtubules cannot influence microfibril orientation through a direct association with cellulose synthetic complexes via microtubule cross bridges.

  13. Plant actin cytoskeleton re-modeling by plant parasitic nematodes.

    PubMed

    Engler, Janice de Almeida; Rodiuc, Natalia; Smertenko, Andrei; Abad, Pierre

    2010-03-01

    The cytoskeleton is an important component of the plant's defense mechanism against the attack of pathogenic organisms. Plants however, are defenseless against parasitic root-knot and cyst nematodes and respond to the invasion by the development of a special feeding site that supplies the parasite with nutrients required for the completion of its life cycle. Recent studies of nematode invasion under treatment with cytoskeletal drugs and in mutant plants where normal functions of the cytoskeleton have been affected, demonstrate the importance of the cytoskeleton in the establishment of a feeding site and successful nematode reproduction. It appears that in the case of microfilaments, nematodes hijack the intracellular machinery that regulates actin dynamics and modulate the organization and properties of the actin filament network. Intervening with this process reduces the nematode infection efficiency and inhibits its life cycle. This discovery uncovers a new pathway that can be exploited for the protection of plants against nematodes.

  14. Neuronal migration in the murine rostral migratory stream requires serum response factor

    PubMed Central

    Alberti, Siegfried; Krause, Sven M.; Kretz, Oliver; Philippar, Ulrike; Lemberger, Thomas; Casanova, Emilio; Wiebel, Franziska F.; Schwarz, Heinz; Frotscher, Michael; Schütz, Günther; Nordheim, Alfred

    2005-01-01

    The central nervous system is fundamentally dependent on guided cell migration, both during development and in adulthood. We report an absolute requirement of the transcription factor serum response factor (SRF) for neuronal migration in the mouse forebrain. Conditional, late-prenatal deletion of Srf causes neurons to accumulate ectopically at the subventricular zone (SVZ), a prime neurogenic region in the brain. SRF-deficient cells of the SVZ exhibit impaired tangential chain migration along the rostral migratory stream into the olfactory bulb. SVZ explants display retarded chain migration in vitro. Regarding target genes, SRF deficiency impairs expression of the β-actin and gelsolin genes, accompanied by reduced cytoskeletal actin fiber density. At the posttranslational level, cofilin, a key regulator of actin dynamics, displays dramatically elevated inhibitory phosphorylation at Ser-3. Our studies indicate that SRF-controlled gene expression directs both the structure and dynamics of the actin microfilament, thereby determining cell-autonomous neuronal migration. PMID:15837932

  15. Metabolic and cytoskeletal modulation of transferrin receptor mobility in mitogen-activated human lymphocytes.

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

    The transferrin receptors which appear on mitogen-activated human peripheral blood lymphocytes were found by the use of immunofluorescence techniques to display temperature-dependent patching and capping reactions upon binding of transferrin. Lateral mobility of ligand-occupied membrane sites was accompanied by both shedding and endocytosis of receptor-transferrin complexes. In the presence of sodium azide or the microfilament inhibitor cytochalasin B, cap formation and shedding were markedly inhibited. In contrast, endocytosis of patched receptor-ligand complexes was inhibited by azide and microtubule inhibitors, including colchicine, vinblastine and vincristine. Co-capping experiments performed to elucidate further the alterations in membrane configuration involved in these reactions failed to reveal any topographical relationship between transferrin receptors and lectin-binding sites in these cells. These studied indicate that temperature-dependent mobility of transferrin receptors upon mitogen-activated peripheral blood lymphocytes is dependent upon the integrity of the cytoskeletal system and metabolic function of the cell. PMID:6258830

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

    NASA Astrophysics Data System (ADS)

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

    2005-08-01

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

  17. Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP).

    PubMed

    Hecht, N B

    2000-06-01

    Functions ranging from RNA transport and translational regulation to DNA rearrangement and repair have been proposed for the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP). TB-RBP is primarily in the nuclei of male germ cells during meiosis and in the cytoplasm of male cells after metaphase I of meiosis. Based on its shift in subcellular locations as germ cells differentiate and its binding to microtubules and microfilaments, a model is presented proposing an involvement of TB-RBP in mRNA transport from nucleus to cytoplasm and in the sharing of mRNAs transcribed from the sex chromosomes by movement through intercellular bridges of germ cells.

  18. Role of Intermediate Filaments in Vesicular Traffic

    PubMed Central

    Margiotta, Azzurra; Bucci, Cecilia

    2016-01-01

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway. PMID:27120621

  19. Cytoskeleton and cell wall function in penetration resistance.

    PubMed

    Hardham, Adrienne R; Jones, David A; Takemoto, Daigo

    2007-08-01

    Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.

  20. Fine structure and RNA synthesis of Tetrahymena during cytochalasin B inhibition of phagocytosis.

    PubMed

    Nilsson, J R

    1977-01-01

    Cytochalasin B inhibits the formation of normal-sized food vacuoles in Tetrahymena but the cells do not starve. Treated cells differ from starved cells in that they retain a high rate of incorporation of tritiated uridine. Large numbers of smaller vacuoles, about 1 micrometer in diameter, are formed, presumably by pinocytic activity of the cytopharyngeal membrane. This effect may perhaps be due to interference with the mechanism by which food vacuoles are sealed off at the cytostome, in which microfilaments may participate. Inhibited organisms may form tubes continuous with the cytopharynx instead of separate food vacuoles. It is not clear, however, why the formation of the small vacuoles is resistant to the drug.

  1. Cytoskeletal components of Beta vulgaris root hairs in altered gravity fields

    NASA Astrophysics Data System (ADS)

    Shevchenko, G. V.

    Root hairs of Beta vulgaris are protrusions from rhizodermal cells and are characterised by plagiotropic growth. The roles of the cytoskeleton and of gravity in this growth process are being studied with the help of a clinostat. Through the use of immunocytochemical and fluorescent staining methods which reveal microtubules (MTs) and microfilaments (MFs), it was found that these cytoskeletal components of the root hairs of 4-day-old seedlings of B. vulgaris were affected by clinorotation at 2 r.p.m. In control conditions, MTs were found to be distributed evenly throughout the root hair, and an intense fluorescence due to MFs was observed at the tip of the hairs. With clinorotation, however, MTs became distributed at random, though no redistribution of MFs was observed. The latter finding conforms to the idea that MFs are responsible for tip growth. That MTs are more sensitive to altered gravity conditions is presently being tested.

  2. Effects of cytochalasin B on cell to cell adhesion and cellular shape of embryo mesoderm cells in vitro.

    PubMed Central

    Chamorro, C A; de Paz Cabello, P; Fernandez, J G; Villar, J M

    1986-01-01

    The effects of cytochalasin B on chick embryo mesoderm cells cultured in vitro were studied by scanning electron microscopy. The untreated cells showed numerous filopodia and lamellipodia and they were flattened onto the coverslip. Several cellular clusters were observed in each culture. The treated cells did not show filopodia, they had a rounded shape and cellular clusters were not present on the coverslip. These alterations are discussed on the basis of the actions of cytochalasin B on the micrcfilamentous cytoskeleton and the role proposed for microfilaments in filopodia formation, cellular shape and locomotion. It was observed that the mechanisms of cell to cell adhesion of chick embryo mesoderm cells were not the same as the mechanisms of cell adhesion to artificial substrates. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:3693072

  3. Erythrocyte adducin: A structural regulator of the red blood cell membrane

    PubMed Central

    Franco, T.; Low, P.S.

    2012-01-01

    Adducin is an α, β heterotetramer that performs multiple important functions in the human erythrocyte membrane. First, adducin forms a bridge that connects the spectrin–actin junctional complex to band 3, the major membrane-spanning protein in the bilayer. Rupture of this bridge leads to membrane instability and spontaneous fragmentation. Second, adducin caps the fast growing (barbed) end of actin filaments, preventing the tetradecameric protofilaments from elongating into macroscopic F-actin microfilaments. Third, adducin stabilizes the association between actin and spectrin, assuring that the junctional complex remains intact during the mechanical distortions experienced by the circulating cell. And finally, adducin responds to stimuli that may be important in regulating the global properties of the cell, possibly including cation transport, cell morphology and membrane deformability. The text below summarizes the structural properties of adducin, its multiple functions in erythrocytes, and the consequences of engineered deletions of each of adducin subunits in transgenic mice. PMID:20655268

  4. PLEC — EDRN Public Portal

    Cancer.gov

    Plectin is a prominent member of an important family of structurally and in part functionally related proteins, termed plakins or cytolinkers, that are capable of interlinking different elements of the cytoskeleton. PLEC1 interlinks intermediate filaments with microtubules and microfilaments and anchors intermediate filaments to desmosomes or hemidesmosomes. It could also bind muscle proteins such as actin to membrane complexes in muscle. There is evidence that PLEC1 may be involved not only in the cross-linking and stabilization of cytoskeletal intermediate filaments network, but also in the regulation of their dynamics. Defects in PLEC1 are the cause of several severe skin and muscle disorders (epidermolysis bullosa simplex with pyloric atresia, epidermolysis bullosa simplex with muscular dystrophy, and epidermolysis bullosa simplex Ogna type). PLEC1 is Widely expressed, with highest levels in muscle, heart, placenta and spinal cord.

  5. Mechanics of Individual Keratin Bundles in Living Cells

    PubMed Central

    Nolting, Jens-Friedrich; Möbius, Wiebke; Köster, Sarah

    2014-01-01

    Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed. PMID:25468348

  6. Effect of the intermediate filament inhibitor IDPN on steroid secretion by frog adrenal glands.

    PubMed

    Feuilloley, M; Netchitailo, P; Delarue, C; Leboulenger, F; Benyamina, M; Vaudry, H

    1988-01-01

    In order to determine the role of intermediate filaments in adrenal steroidogenesis, we have studied the effect of IDPN (beta-beta'iminodipropionitrile), an intermediate filaments perturbing agent, on corticosteroid secretion by frog interrenal glands in vitro. A 6-h administration of IDPN (10(-3) M) did not affect the spontaneous release of corticosterone and aldosterone. While IDPN did not alter the response of adrenal fragments to ACTH, the drug caused a marked decrease in angiotensin II-induced stimulation of corticosterone and aldosterone production. These results indicate that, in contrast to microfilaments, which play an important role in spontaneous steroidogenesis, intermediate filaments are not required for basal corticosteroid secretion but are involved in the mechanism of action of angiotensin in frog adrenocortical cells.

  7. Rho-Signaling-Directed YAP/TAZ Activity Underlies the Long-Term Survival and Expansion of Human Embryonic Stem Cells.

    PubMed

    Ohgushi, Masatoshi; Minaguchi, Maki; Sasai, Yoshiki

    2015-10-01

    Human embryonic stem cells (hESCs) can survive and proliferate for an extended period of time in culture, but unlike that of tumor-derived cells, this form of cellular immortality does not depend on genomic aberrations. In this study, we sought to elucidate the molecular basis of this long-term growth property of hESCs. We found that the survival of hESCs depends on the small GTPase Rho and its activator AKAP-Lbc. We show that AKAP-Lbc/Rho signaling sustains the nuclear function of the transcriptional cofactors YAP and TAZ by modulating actin microfilament organization. By inducing reprogramming and differentiation, we found that dependency on this Rho signaling pathway is associated with the pluripotent state. Thus, our findings show that the capacity of hESCs to undergo long-term expansion in vitro is intrinsically coupled to their cellular identity through interconnected molecular circuits that link cell survival to pluripotency.

  8. Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts

    NASA Technical Reports Server (NTRS)

    Eckes, B.; Dogic, D.; Colucci-Guyon, E.; Wang, N.; Maniotis, A.; Ingber, D.; Merckling, A.; Langa, F.; Aumailley, M.; Delouvee, A.; hide

    1998-01-01

    Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

  9. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction.

    PubMed

    Brown, S; Levinson, W; Spudich, J A

    1976-01-01

    Treatment of chick embryo fibroblasts with 0.5% Triton X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This "Triton cytoskeleton" consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.

  10. Controlled drop emission by wetting properties in driven liquid filaments.

    PubMed

    Ledesma-Aguilar, R; Nistal, R; Hernández-Machado, A; Pagonabarraga, I

    2011-05-01

    The controlled formation of micrometre-sized drops is of great importance to many technological applications. Here we present a wetting-based destabilization mechanism of forced microfilaments on either hydrophilic or hydrophobic stripes that leads to the periodic emission of droplets. The drop emission mechanism is triggered above the maximum critical forcing at which wetting, capillarity, viscous friction and gravity can balance to sustain a stable driven contact line. The corresponding critical filament velocity is predicted as a function of the static wetting angle, which can be tuned through the substrate behaviour, and shows a strong dependence on the filament size. This sensitivity explains the qualitative difference in the critical velocity between hydrophilic and hydrophobic stripes, and accounts for previous experimental results of splashing solids. We demonstrate that this mechanism can be used to control independently the drop size and emission period, opening the possibility of highly monodisperse and flexible drop production techniques in open microfluidic geometries.

  11. Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

    PubMed

    Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

    2015-04-01

    The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

  12. [ALPHA-ACTININS AND SIGNAL TRANSDUCTION PATHWAYS].

    PubMed

    Panyushev, N V; Tentler, D G

    2015-01-01

    Involvement of actin cytoskeleton proteins in signal transduction from cell surface to the nucleus, including regulation of transcription factors activity, has now been supported by a lot of experimental data. Here-with, cytoskeletal proteins may have different functions than ones they execute in the cytoplasm. Particularly, alpha-actinin 4 stabilizing actin microfilaments in the cytoplasm can translocate to the nucleus and change the activity of several transcription factors. Despite the lack of nuclear import signal and DNA binding domain, alpha-actinin 4 can bind to promoter sequences, and co-activate NF-κB-dependent transcription. Selective regulation of NF-κB gene targets may indicate involvement of alpha-actinin 4 in determining the specificity of cell response to NF-κB activation in cells of different types.

  13. Gravity and the cell: Intracellular structures and Stokes sedimentation

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1977-01-01

    Plant and certain animal embryos appear to be responsive to the gravity vector during early stages of development. The convection of particle sedimentation as the basis for the sensing of gravity is investigated using the cells of wheat seedlings, amphibian embryos, and mammals. Exploration of the mammalian cell for sedimenting particles reveals that their existence is unlikely, especially in the presence of a network of microtubules and microfilaments considered to be responsible for intracellular organization. Destruction of these structures renders the cell susceptible to accelerations several times g. Large dense particles, such as chromosomes, nucleoli, and cytoplasmic organelles are acted upon by forces much larger than that due to gravity, and their positions in the cell appear to be insensitive to gravity.

  14. Mechanotransduction across the cell surface and through the cytoskeleton

    NASA Technical Reports Server (NTRS)

    Wang, N.; Butler, J. P.; Ingber, D. E.

    1993-01-01

    Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

  15. Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface.

    PubMed

    Godman, G C; Miranda, A F; Deitch, A D; Tanenbaum, S W

    1975-03-01

    The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.

  16. rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.

    PubMed

    Deretic, D; Huber, L A; Ransom, N; Mancini, M; Simons, K; Papermaster, D S

    1995-01-01

    Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.

  17. Traffic of Secondary Metabolites to Cell Surface in the Red Alga Laurencia dendroidea Depends on a Two-Step Transport by the Cytoskeleton

    PubMed Central

    Reis, Vanessa M.; Oliveira, Louisi S.; Passos, Raoni M. F.; Viana, Nathan B.; Mermelstein, Cláudia; Sant'Anna, Celso; Pereira, Renato C.; Paradas, Wladimir C.; Thompson, Fabiano L.; Amado-Filho, Gilberto M.; Salgado, Leonardo T.

    2013-01-01

    In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the

  18. Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins.

    PubMed

    Girard, P R; Nerem, R M

    1995-04-01

    Flow-related shear stress has been shown to modulate endothelial cell structure and function including F-actin microfilament organization. Focal adhesion-associated proteins such as vinculin, talin, and specific integrins may play a role in the modulation of these cytoskeletal and morphological changes. Double-label immunofluorescence studies indicated that, in static culture, alpha 5 beta 1 fibronectin receptors (alpha 5 beta 1 FNRs) and alpha v beta 3 vitronectin receptors (alpha v beta 3 VNRs) were found predominantly in the peripheral regions of bovine aortic endothelial cells (BAECs) corresponding to the localization of vinculin, talin, and actin microfilament terminations. In response to shear stress, concomitant with cell elongation and the appearance of stress fibers aligned with the direction of flow, there was a prominent localization of vinculin and alpha v beta 3 VNRs as the "upstream" end of the cells. Stress fiber terminations were clearly evident at these concentrations of focal adhesion-associated proteins. These data suggest that the upstream concentration of these proteins may direct shear stress-induced stress fiber formation and may function in the alignment of the fibers in the direction of flow. Levels of surface alpha v beta 3 VNRs were found to decrease in response to flow, possibly reflecting the decrease in numbers of "downstream" receptors. Unlike the arrangement of vinculin and alpha v beta 3 VNRs observed following exposure to flow, talin and alpha 5 beta 1 FNRs, in addition to being localized at the upstream end of the cell, were also evenly distributed throughout the rest of the cell. Surface levels of alpha 5 beta 1 FNRs increased in response to shear stress, perhaps providing an increased adherence of BAECs to the extracellular matrix through these receptors. These data suggest that focal adhesion-associated proteins play specific roles in the response of BAECs to shear stress.

  19. A novel multifunctional biomedical material based on polyacrylonitrile: Preparation and characterization.

    PubMed

    Wu, Huan-ling; Bremner, David H; Li, He-yu; Shi, Qi-quan; Wu, Jun-zi; Xiao, Rui-qiu; Zhu, Li-min

    2016-05-01

    Wet spun microfibers have great potential in the design of multifunctional controlled release materials. Curcumin (Cur) and vitamin E acetate (Vit. E Ac) were used as a model drug system to evaluate the potential application of the drug-loaded microfiber system for enhanced delivery. The drugs and polyacrylonitrile (PAN) were blended together and spun to produce the target drug-loaded microfiber using an improved wet-spinning method and then the microfibers were successfully woven into fabrics. Morphological, mechanical properties, thermal behavior, drug release performance characteristics, and cytocompatibility were determined. The drug-loaded microfiber had a lobed "kidney" shape with a height of 50-100 μm and width of 100-200 μm. The addition of Cur and Vit. E Ac had a great influence on the surface and cross section structure of the microfiber, leading to a rough surface having microvoids. X-ray diffraction and Fourier transform infrared spectroscopy indicated that the drugs were successfully encapsulated and dispersed evenly in the microfilament fiber. After drug loading, the mechanical performance of the microfilament changed, with the breaking strength improved slightly, but the tensile elongation increased significantly. Thermogravimetric results showed that the drug load had no apparent adverse effect on the thermal properties of the microfibers. However, drug release from the fiber, as determined through in-vitro experiments, is relatively low and this property is maintained over time. Furthermore, in-vitro cytocompatibility testing showed that no cytotoxicity on the L929 cells was found up to 5% and 10% respectively of the theoretical drug loading content (TDLC) of curcumin and vitamin E acetate. This study provides reference data to aid the development of multifunctional textiles and to explore their use in the biomedical material field. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Expression of Rho GTPases family in melanoma cells and its influence on cytoskeleton and migration.

    PubMed

    Wen, Si-Jian; Zhang, Wei; Ni, Na-Na; Wu, Qiong; Wang, Xiao-Po; Lin, You-Kun; Sun, Jian-Fang

    2017-05-02

    Rho GTPases family members influenced the filopodia, lamellipodia, stress fiber and adhesion plaque of melanoma cells through regulating cytoskeleton recombination. The role of Rho GTPases family in the migration and invasion of melanoma and its molecular mechanism were explored. The morphological difference between three types of melanoma cells (M14, A375 and MV3) and human melanocyte (MC) was observed by the Hoffman microscope. Cells were stained by phalloidin labeled by rhodamine. The differences between 4 types of cells in filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent) were observed under the super-high resolution microscope. The migration ability of 4 types of cells was detected by Transwell migration assay. QPCR was used to detect the mRNA transcription level of Rho GTPases family. WB was adopted to detect the expression of RhoD and DIAPH2 proteins. There were significant differences in filopodia, lamellipodia, stress fiber and adhesion plaque between MC and 3 types of melanoma cells (M14, A375 and MV3). MC did not have stress fiber or adhesion plaque, while M14, A375 and MV3 had stress fiber and adhesion plaque. All 4 types of cells had thin and short filopodia. MV3 had fewer but thicker stress fibers than the latter two. Transwell migration test indicated the followings: M14 and A375 had a similar high migration rate; the migration rate of MV3 was slightly low; MC did not have the ability of transmembrane migration. QPCR results of Rho GTPases family in 4 types of cells showed the change corresponding to immunofluorescence. WB results showed that RhoD was barely expressed in M14, A375 or MV3. DIAPH2, the downstream effector molecule of RhoD, had the corresponding change. Rho GTPases influences the migration and invasion of melanoma cells through regulating filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent).

  1. Immunochemical demonstration of tropomyosin in the neurofibrillary pathology of Alzheimer's disease.

    PubMed Central

    Galloway, P. G.; Mulvihill, P.; Siedlak, S.; Mijares, M.; Kawai, M.; Padget, H.; Kim, R.; Perry, G.

    1990-01-01

    The focus of research on the neurofibrillary pathology (NFP) of Alzheimer disease has been not only to determine the component forming the paired helical filaments but also to determine whether they result from abnormal processes affecting a single protein. Therefore, although these studies have lead to controversy concerning the respective contribution of components of microtubules and neurofilaments, there has been essentially no consideration of whether other cytoskeletal systems might also be involved and of what are the common features for the incorporated components. Particularly relevant to this issue is our finding that several antisera raised to either skeletal or smooth muscle tropomyosin, a microfilament component, intensely recognize NFP. These antibodies continue to recognize NFP after affinity purification to tropomyosin or paired helical filament fractions. We show that the antibodies do not recognize NFP due to cross-reactivity with the previously identified NFP components related to neurofilaments and microtubules, tau, and MAP2, or neurofilament proteins because the antisera did not recognize these proteins on immunoblots or were not adsorbable by the proteins. Ultrastructural analysis of the immunoreaction showed that tropomyosin-related epitopes were clustered rather than uniformly distributed along paired helical and straight filaments. Although the distribution suggests that tropomyosin is an NFP-associated protein, its retention by paired helical and straight filaments after detergent extraction indicates that it is an integral component strongly and specifically associated with the filaments characteristic of NFP. These findings indicate that NFP involves the three primary neuronal cytoskeletal filament systems, microtubules, neurofilaments, as well as microfilaments, and therefore that NFP probably results from the reorganization of these normal filaments that interact to comprise the cytomatrix and may continue this interaction under the

  2. Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

    PubMed Central

    Angrisano, Fiona; Delves, Michael J.; Zuccala, Elizabeth S.; Turnbull, Lynne; Dekiwadia, Chaitali; Olshina, Maya A.; Marapana, Danushka S.; Wong, Wilson; Mollard, Vanessa; Bradin, Clare H.; Tonkin, Christopher J.; Gunning, Peter W.; Ralph, Stuart A.; Whitchurch, Cynthia B.; Sinden, Robert E.; Cowman, Alan F.; McFadden, Geoffrey I.; Baum, Jake

    2012-01-01

    Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu. PMID:22389687

  3. The Role of the Cytoskeleton and Myosin-Vc in the Targeting of KCa3.1 to the Basolateral Membrane of Polarized Epithelial Cells

    PubMed Central

    Farquhar, Rachel E.; Rodrigues, Ely; Hamilton, Kirk L.

    2017-01-01

    Understanding the targeting of KCa3.1 to the basolateral membrane (BLM) of polarized epithelial cells is still emerging. Here, we examined the role of the cytoskeleton (microtubules and microfilaments) and Myosin-Vc (Myo-Vc) in the targeting of KCa3.1 in Fischer rat thyroid epithelial cells. We used a pharmacological approach with immunoblot (for the BLM expression of KCa3.1), Ussing chamber (functional BLM expression of KCa3.1) and siRNA experiments. The actin cytoskeleton inhibitors cytochalasin D (10 μM, 5 h) and latrunculin A (10 μM, 5 h) reduced the targeting of KCa3.1 to the BLM by 88 ± 4 and 70 ± 5%, respectively. Colchicine (10 μM, 5 h) a microtubule inhibitor reduced targeting of KCa3.1 to the BLM by 63 ± 7% and decreased 1-EBIO-stimulated KCa3.1 K+ current by 46 ± 18%, compared with control cells. ML9 (10 μM, 5 h), an inhibitor of myosin light chain kinase, decreased targeting of the channel by 83 ± 2% and reduced K+ current by 54 ± 8% compared to control cells. Inhibiting Myo-V with 2,3-butanedione monoxime (10 mM, 5 h) reduced targeting of the channel to the BLM by 58 ± 5% and decreased the stimulated current of KCa3.1 by 48 ± 12% compared with control cells. Finally, using siRNA for Myo-Vc, we demonstrated that knockdown of Myo-Vc reduced the BLM expression of KCa3.1 by 44 ± 7% and KCa3.1 K+ current by 1.04 ± 0.14 μA compared with control cells. These data suggest that the microtubule and microfilament cytoskeleton and Myo-Vc are critical for the targeting of KCa3.1. PMID:28101059

  4. Traffic of secondary metabolites to cell surface in the red alga Laurencia dendroidea depends on a two-step transport by the cytoskeleton.

    PubMed

    Reis, Vanessa M; Oliveira, Louisi S; Passos, Raoni M F; Viana, Nathan B; Mermelstein, Cláudia; Sant'anna, Celso; Pereira, Renato C; Paradas, Wladimir C; Thompson, Fabiano L; Amado-Filho, Gilberto M; Salgado, Leonardo T

    2013-01-01

    In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the

  5. Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

    PubMed Central

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Wang, Ching C.; Perez Rangel, Armando; Camacho Nuez, Minerva; Garcia Tovar, Carlos; Ayala-Sumuano, Jorge Tonatiuh; Luna-Arias, Juan Pedro; Hernandez, Jose Manuel

    2010-01-01

    Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. Methodology and Principal Findings In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. Conclusions and Significance Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and

  6. Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress.

    PubMed

    Nunes, Paula; Roth, Isabelle; Meda, Paolo; Féraille, Eric; Brown, Dennis; Hasler, Udo

    2015-06-16

    Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.

  7. Effects of hypergravity on adipose-derived stem cell morphology, mechanical property and proliferation

    SciTech Connect

    Tavakolinejad, Alireza; Rabbani, Mohsen; Janmaleki, Mohsen

    2015-08-21

    Alteration in specific inertial conditions can lead to changes in morphology, proliferation, mechanical properties and cytoskeleton of cells. In this report, the effects of hypergravity on morphology of Adipose-Derived Stem Cells (ADSCs) are indicated. ADSCs were repeatedly exposed to discontinuous hypergravity conditions of 10 g, 20 g, 40 g and 60 g by utilizing centrifuge (three times of 20 min exposure, with an interval of 40 min at 1 g). Cell morphology in terms of length, width and cell elongation index and cytoskeleton of actin filaments and microtubules were analyzed by image processing. Consistent changes observed in cell elongation index as morphological change. Moreover, cell proliferation was assessed and mechanical properties of cells in case of elastic modulus of cells were evaluated by Atomic Force Microscopy. Increase in proliferation and decrease in elastic modulus of cells are further results of this study. Staining ADSC was done to show changes in cytoskeleton of the cells associated to hypergravity condition specifically in microfilament and microtubule components. After exposing to hypergravity, significant changes were observed in microfilaments and microtubule density as components of cytoskeleton. It was concluded that there could be a relationship between changes in morphology and MFs as the main component of the cells. - Highlights: • Hypergravity (10 g, 20 g, 40 g and 60 g) affects on adipose derived stem cells (ADSCs). • ADSCs after exposure to the hypergravity are more slender. • The height of ADSCs increases in all test groups comparing their control group. • Hypergravity decreases ADSCs modulus of elasticity and cell actin fiber content. • Hypergravity enhances proliferation rate of ADSCs.

  8. The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding

    PubMed Central

    Marchiando, Amanda M.; Shen, Le; Graham, W. Vallen; Edelblum, Karen L.; Duckworth, Carrie A.; Guan, Yanfang; Montrose, Marshall H.; Turner, Jerrold R.; Watson, Alastair J.M.

    2011-01-01

    BACKGROUND & AIMS Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance, as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. METHODS We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein1–ZO-1. After injection of high doses of TNF (7.5µg, i.p.), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. RESULTS Changes detected included redistribution of the tight junction proteins ZO-1 and occluding to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. CONCLUSIONS Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function. PMID:21237166

  9. Enterocyte cytoskeleton changes are crucial for enhanced translocation of nonpathogenic Escherichia coli across metabolically stressed gut epithelia.

    PubMed

    Nazli, Aisha; Wang, Arthur; Steen, Oren; Prescott, David; Lu, Jun; Perdue, Mary H; Söderholm, Johan D; Sherman, Philip M; McKay, Derek M

    2006-01-01

    Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenic Escherichia coli (strain HB101, 10(6) CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP+E. coli HB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells.

  10. Antiproliferative and antimetastatic action of quercetin on A549 non-small cell lung cancer cells through its effect on the cytoskeleton.

    PubMed

    Klimaszewska-Wiśniewska, Anna; Hałas-Wiśniewska, Marta; Izdebska, Magdalena; Gagat, Maciej; Grzanka, Alina; Grzanka, Dariusz

    2017-03-01

    To our knowledge, this study is the first to investigate the effect of the dietary flavonoid quercetin on the main cytoskeletal elements, namely microfilaments, microtubules and vimentin intermediate filaments, as well as cytoskeleton-driven processes in A549 non-small cell lung cancer cells. The methyl-thiazol-diphenyl-tetrazolium assay, annexin V/propidium iodide test, electron microscopic examination, cell cycle analysis based on DNA content, real-time PCR assays, in vitro scratch wound-healing assay, fluorescence staining of F-actin, β-tubulin and vimentin were performed to assess the effects of quercetin on A549 cells. Our results showed that quercetin triggered BCL2/BAX-mediated apoptosis, as well as necrosis and mitotic catastrophe, and inhibited the migratory potential of A549 cells. The disassembling effect of quercetin on microfilaments, microtubules and vimentin filaments along with its inhibitory impact on vimentin and N-cadherin expression might account for the decreased migration of A549 cells in response to quercetin treatment. We also suggest that the possible mechanism underlying quercetin-induced mitotic catastrophe involves the perturbation of mitotic microtubules leading to monopolar spindle formation, and, consequently, to the failure of cytokinesis. We further propose that cytokinesis failure could also be a result of the depletion of actin filaments by quercetin. These findings are important to our further understanding of the detailed mechanism of the antitumor activity of quercetin and render this flavonoid a potentially useful candidate for combination therapy with conventional antimicrotubule drugs, nucleic acid-directed agents or novel cytoskeletal-directed agents. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Cannabinoid Receptor Type 1 (CB1) Activation Inhibits Small GTPase RhoA Activity and Regulates Motility of Prostate Carcinoma Cells

    PubMed Central

    Gomez-Granados, Ana Doris; Tang, Alan T.; Pfeiffer, Adam W.; Williams, Carol L.; Campbell, William B.

    2012-01-01

    The cannabinoid receptor type 1 (CB1) is a G protein-coupled receptor that is activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). The CB1 and its endogenous and synthetic agonists are emerging as therapeutic targets in several cancers due to their ability to suppress carcinoma cell invasion and migration. However, the mechanisms that the CB1 regulates cell motility are not well understood. In this study, we examined the molecular mechanisms that diminish cell migration upon the CB1 activation in prostate carcinoma cells. The CB1 activation with the agonist WIN55212 significantly diminishes the small GTPase RhoA activity but modestly increases the Rac1 and Cdc42 activity. The diminished RhoA activity is accompanied by the loss of actin/myosin microfilaments, cell spreading, and cell migration. Interestingly, the CB1 inactivation with the selective CB1 antagonist AM251 significantly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes cell migration. This finding suggests that endogenously produced EC activate the CB1, resulting in chronic repression of RhoA activity and cell migration. Consistent with this possibility, RhoA activity is significantly diminished by the exogenous application of AEA but not by 2-AG in PC-3 cells (cells with very low AEA hydrolysis). Pretreatment of cells with a monoacylglycerol lipase inhibitor, JZL184, which blocks 2-AG hydrolysis, decreases the RhoA activity. These results indicate the unique CB1 signaling and support the model that EC, through their autocrine activation of CB1 and subsequent repression of RhoA activity, suppress migration in prostate carcinoma cells. PMID:22087025

  12. Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

    PubMed

    Watanabe, Tomoko; Thayil, Anisha; Jesacher, Alexander; Grieve, Kate; Debarre, Delphine; Wilson, Tony; Booth, Martin; Srinivas, Shankar

    2010-06-03

    Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images. We have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours. LD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

  13. Structures linking the myonemes, endoplasmic reticulum, and surface membranes in the contractile ciliate Vorticella.

    PubMed

    Allen, R D

    1973-02-01

    An electron microscope investigation of the interface between the myonemes of Vorticella convallaria and their associated endoplasmic reticulum (ER) has revealed structures of a complex morphology linking these two organelles. These structures are named "linkage complexes". Each complex contains a spindle-shaped midpiece which lies in a groove of the ER membrane. Microfilaments splay out from the tips of the midpiece and may come in contact with the inner alveolar sac membrane. Three to six raillike structures lie on each side of the midpiece and parallel it. The ER membrane appears to pass through the sides of the rails. In the lumen of the ER these rails are associated with a meshwork of filaments. A cradle of five rods lies within the groove under the midpiece. The ER membrane also passes through these rods which contact the same meshwork. In the scopular region and in the stalk the microfilaments from the midpiece form a bundle which passes into the lumen of modified basal bodies. These basal bodies are connected to the alveolar sac which, in the stalk, passes as a flattened tube along its length. The parts of the dissociated linkage complex are scattered throughout the spasmoneme of the stalk along membranes of the intraspasmonemal tubules. Thus, both stalk and body contractile bundles have linkage complexes that link their associated membrane systems to the microfibrils and, in turn, connect this membrane-microfibrillar interface to the pellicular membranes. The arrangement of the linkage complex suggests an involvement in the control of the transport of calcium ions between ER and microfibrils, and possibly the transfer of a message from the surface membranes to the sites of calcium release to trigger myonemal contraction.

  14. PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton.

    PubMed

    Bianchi, Massimiliano G; Rotoli, Bianca Maria; Dall'Asta, Valeria; Gazzola, Gian C; Gatti, Rita; Bussolati, Ovidio

    2006-04-01

    The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or

  15. Fertilization Induces a Transient Exposure of Phosphatidylserine in Mouse Eggs

    PubMed Central

    Curia, Claudio A.; Ernesto, Juan I.; Stein, Paula; Busso, Dolores; Schultz, Richard M.; Cuasnicu, Patricia S.; Cohen, Débora J.

    2013-01-01

    Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca2+ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca2+ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca2+ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca2+ ionophore, suggesting that the Ca2+ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca2+ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis. PMID:23951277

  16. Isolation and Contraction of the Stress Fiber

    PubMed Central

    Katoh, Kazuo; Kano, Yumiko; Masuda, Michitaka; Onishi, Hirofumi; Fujiwara, Keigi

    1998-01-01

    Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle. PMID:9658180

  17. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

    NASA Technical Reports Server (NTRS)

    Sims, J. R.; Karp, S.; Ingber, D. E.

    1992-01-01

    Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

  18. Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

    NASA Astrophysics Data System (ADS)

    Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui

    2011-01-01

    Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure

  19. STRUCTURES LINKING THE MYONEMES, ENDOPLASMIC RETICULUM, AND SURFACE MEMBRANES IN THE CONTRACTILE CILIATE VORTICELLA

    PubMed Central

    Allen, Richard D.

    1973-01-01

    An electron microscope investigation of the interface between the myonemes of Vorticella convallaria and their associated endoplasmic reticulum (ER) has revealed structures of a complex morphology linking these two organelles. These structures are named "linkage complexes". Each complex contains a spindle-shaped midpiece which lies in a groove of the ER membrane. Microfilaments splay out from the tips of the midpiece and may come in contact with the inner alveolar sac membrane. Three to six raillike structures lie on each side of the midpiece and parallel it. The ER membrane appears to pass through the sides of the rails. In the lumen of the ER these rails are associated with a meshwork of filaments. A cradle of five rods lies within the groove under the midpiece. The ER membrane also passes through these rods which contact the same meshwork. In the scopular region and in the stalk the microfilaments from the midpiece form a bundle which passes into the lumen of modified basal bodies. These basal bodies are connected to the alveolar sac which, in the stalk, passes as a flattened tube along its length. The parts of the dissociated linkage complex are scattered throughout the spasmoneme of the stalk along membranes of the intraspasmonemal tubules. Thus, both stalk and body contractile bundles have linkage complexes that link their associated membrane systems to the microfibrils and, in turn, connect this membrane-microfibrillar interface to the pellicular membranes. The arrangement of the linkage complex suggests an involvement in the control of the transport of calcium ions between ER and microfibrils, and possibly the transfer of a message from the surface membranes to the sites of calcium release to trigger myonemal contraction. PMID:4630196

  20. Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface

    PubMed Central

    1975-01-01

    The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane. PMID:168210

  1. Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

    PubMed Central

    Nunes, Paula; Roth, Isabelle; Meda, Paolo; Féraille, Eric; Brown, Dennis; Hasler, Udo

    2015-01-01

    Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking. PMID:26045497

  2. Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes.

    PubMed

    Gellynck, K; Shah, R; Deng, D; Parkar, M; Liu, W; Knowles, J C; Buxton, P

    2013-06-29

    Static compressive stress can influence the matrix, which subsequently affects cell behaviour and the cell's ability to further transform the matrix. This study aimed to assess response to static compressive stress at different stages of osteoblast differentiation and assess the cell cytoskeleton's role as a conduit of matrix-derived stimuli. Mouse bone marrow mesenchymal stem cells (MSCs) (D1 ORL UVA), osteoblastic cells (MC3T3-E1) and post-osteoblast/pre-osteocyte-like cells (MLO-A5) were seeded in hydrated and compressed collagen gels. Contraction was quantified macroscopically, and cell morphology, survival, differentiation and mineralisation assessed using confocal microscopy, alamarBlue® assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and histological stains, respectively. Confocal microscopy demonstrated cell shape changes and favourable microfilament organisation with static compressive stress of the collagen matrix; furthermore, cell survival was greater compared to the hydrated gels. The stage of osteoblast differentiation determined the degree of matrix contraction, with MSCs demonstrating the greatest amount. Introduction of microfilament disrupting inhibitors confirmed that pre-stress and tensegrity forces were under the influence of gel density, and there was increased survival and differentiation of the cells within the compressed collagen compared to the hydrated collagen. There was also relative stiffening and differentiation with time of the compressed cell-seeded collagen, allowing for greater manipulation. In conclusion, the combined collagen chemistry and increased density of the microenvironment can promote upregulation of osteogenic genes and mineralisation; MSCs can facilitate matrix contraction to form an engineered membrane with the potential to serve as a 'pseudo-periosteum' in the regeneration of bone defects.

  3. Vascular smooth muscle contraction evoked by cell volume modulation: role of the cytoskeleton network.

    PubMed

    Koltsova, Svetlana V; Gusakova, Svetlana V; Anfinogenova, Yana J; Baskakov, Mikhail B; Orlov, Sergei N

    2008-01-01

    Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.

  4. Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus1[OPEN

    PubMed Central

    Inada, Noriko; Higaki, Takumi; Hasezawa, Seiichiro

    2016-01-01

    Actin-depolymerizing factors (ADFs) are conserved proteins that function in regulating the structure and dynamics of actin microfilaments in eukaryotes. In this study, we present evidence that Arabidopsis (Arabidopsis thaliana) subclass I ADFs, particularly ADF4, functions as a susceptibility factor for an adapted powdery mildew fungus. The null mutant of ADF4 significantly increased resistance against the adapted powdery mildew fungus Golovinomyces orontii. The degree of resistance was further enhanced in transgenic plants in which the expression of all subclass I ADFs (i.e. ADF1–ADF4) was suppressed. Microscopic observations revealed that the enhanced resistance of adf4 and ADF1-4 knockdown plants (ADF1-4Ri) was associated with the accumulation of hydrogen peroxide and cell death specific to G. orontii-infected cells. The increased resistance and accumulation of hydrogen peroxide in ADF1-4Ri were suppressed by the introduction of mutations in the salicylic acid- and jasmonic acid-signaling pathways but not by a mutation in the ethylene-signaling pathway. Quantification by microscopic images detected an increase in the level of actin microfilament bundling in ADF1-4Ri but not in adf4 at early G. orontii infection time points. Interestingly, complementation analysis revealed that nuclear localization of ADF4 was crucial for susceptibility to G. orontii. Based on its G. orontii-infected-cell-specific phenotype, we suggest that subclass I ADFs are susceptibility factors that function in a direct interaction between the host plant and the powdery mildew fungus. PMID:26747284

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

    PubMed Central

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

    1993-01-01

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

  6. Evidence that translocation of collagen fibril segments plays a role in early intrinsic tendon repair.

    PubMed

    Gunn, J Stephen; Ehrlich, H Paul

    2012-02-01

    Severed tendon repair advances with either a scar through extrinsic repair or regeneration through intrinsic repair. The authors examined whether intrinsic tendon repair reintroduces embryonic fibrillogenesis, whereby preformed collagen fibril segments are incorporated into growing collagen fibers at wound edges. Isolated tendons from 10-day-old chicken embryos were suspended in 1 mg/ml of the antibiotic gentamicin for 90 days, which released fibril segments that were fluorescently tagged with rhodamine. Tendons isolated from 14-day-old chicken embryos were wounded to half their diameter and then maintained as explants in stationary organ culture. Fluorescent-tagged fibril segments were introduced to wounded tendon explants in the presence of high concentrations of neomycin, an antibiotic; cycloheximide, a protein synthesis inhibitor; cytochalasin D, a disruptor of microfilaments; and colchicine, a disruptor of microtubules. At 24 hours, explants were viewed by means of fluorescent microscopy. Untreated, wounded tendon explants showed the translocation of fluorescent-tagged fibril segments from the explant surface to accumulation at wound edges. In the presence of high concentrations of neomycin, cytochalasin D, or colchicine, fluorescent-tagged fibril segments failed to accumulate at wound edges and were retained on the explant surface. Inhibition of protein synthesis by cycloheximide did not alter the accumulation of fluorescent-tagged fibril segments at wound edges. Inhibiting fluorescent-tagged fibril segment accumulation by antibiotics is consistent with their role in releasing fibril segments. Experimental findings show fibril segment translocation and accumulation at wound edges involves microfilaments and microtubules, but not protein synthesis. The experiments support the hypothesis that intrinsic tendon repair advances through the incorporation of fibril segments at wound edges.

  7. Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus.

    PubMed

    Inada, Noriko; Higaki, Takumi; Hasezawa, Seiichiro

    2016-03-01

    Actin-depolymerizing factors (ADFs) are conserved proteins that function in regulating the structure and dynamics of actin microfilaments in eukaryotes. In this study, we present evidence that Arabidopsis (Arabidopsis thaliana) subclass I ADFs, particularly ADF4, functions as a susceptibility factor for an adapted powdery mildew fungus. The null mutant of ADF4 significantly increased resistance against the adapted powdery mildew fungus Golovinomyces orontii. The degree of resistance was further enhanced in transgenic plants in which the expression of all subclass I ADFs (i.e. ADF1-ADF4) was suppressed. Microscopic observations revealed that the enhanced resistance of adf4 and ADF1-4 knockdown plants (ADF1-4Ri) was associated with the accumulation of hydrogen peroxide and cell death specific to G. orontii-infected cells. The increased resistance and accumulation of hydrogen peroxide in ADF1-4Ri were suppressed by the introduction of mutations in the salicylic acid- and jasmonic acid-signaling pathways but not by a mutation in the ethylene-signaling pathway. Quantification by microscopic images detected an increase in the level of actin microfilament bundling in ADF1-4Ri but not in adf4 at early G. orontii infection time points. Interestingly, complementation analysis revealed that nuclear localization of ADF4 was crucial for susceptibility to G. orontii. Based on its G. orontii-infected-cell-specific phenotype, we suggest that subclass I ADFs are susceptibility factors that function in a direct interaction between the host plant and the powdery mildew fungus.

  8. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

    NASA Technical Reports Server (NTRS)

    Sims, J. R.; Karp, S.; Ingber, D. E.

    1992-01-01

    Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

  9. [Effects of direct current electric field on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the mechanisms].

    PubMed

    Liu, Jie; Ren, Xi; Guo, Xiaowei; Sun, Huanbo; Tang, Yong; Luo, Zhenghui; Zhang, Qiong; Zhang, Dongxia; Huang, Yuesheng; Zhang, Jiaping

    2016-04-01

    To explore the effects of direct current electric fields on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the related mechanisms. Twelve neonatal BALB/c mice were divided into 4 batches. The skin on the back of 3 neonatal mice in each batch was obtained to culture fibroblasts. Fibroblasts of the second passage were inoculated in 27 square cover slips with the concentration of 5 × 10(4) cells per mL. (1) Experiment 1. Six square cover slips inoculated with fibroblasts of the second passage were divided into electric field group (EF) and sham electric field group (SEF), with 3 cover slips in each group. The cover slips were put in live cell imaging workstation. The cells in group EF was treated with electric power with EF intensity of 200 mV/mm, while simulating process without actual power was given to SEF group (the same below) for 6 h. Cell proliferation rate was subsequently counted. (2) Experiment 2. Six cover slips were divided and underwent the same processes as in experiment 1. Cell movement locus within EF hour (EFH) 6, direction change of cell migration at EFH 0 (immediately), 1, 2, 3, 4, 5, and 6 which was denoted as cos(α), cell migration velocity within EFH 6, direction change of long axis of cell within EFH 6, and direction change of cell arrangement at EFH 0, 1, 2, 3, 4, 5, and 6 which was denoted as polarity value cos[2(θ-90)] were observed under live cell imaging workstation. After EFH 6, the morphological changes in microtubules and microfilaments were observed with immunofluorescent staining. (3) Experiment 3. Six cover slips were divided into cytochalasin D group (treated with 1 μmol/L cytochalasin D for 10 min) and colchicine group (treated with 5 μmol/L colchicine for 10 min), with 3 cover slips in each group. The morphological changes in microfilaments and microtubules were observed with the same method as in experiment 2. (4) Experiment 4. Nine cover slips were divided into control group (no

  10. Influenza virus morphogenesis and budding

    PubMed Central

    Nayak, Debi P.; Balogun, Rilwan A.; Yamada, Hiroshi; Zhou, Z. Hong; Barman, Subrata

    2009-01-01

    Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major subviral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins. Budding processes involve bud initiation, bud growth and bud release. Presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in

  11. Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

    NASA Astrophysics Data System (ADS)

    He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

    Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS

  12. Cellular basis of gravity resistance in plants

    NASA Astrophysics Data System (ADS)

    Hoson, Takayuki; Matsumoto, Shouhei; Inui, Kenichi; Zhang, Yan; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Takashi

    Mechanical resistance to the gravitational force is a principal gravity response in plants distinct from gravitropism. In the final step of gravity resistance, plants increase the rigidity of their cell walls via modifications to the cell wall metabolism and apoplastic environment. We studied cellular events that are related to the cell wall changes under hypergravity conditions produced by centrifugation. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of stem organs. In Arabidopsis tubulin mutants, the percentage of cells with longitudinal microtubules was high even at 1 g, and it was further increased by hypergravity. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1 g, and the degree of twisting phenotype was intensified under hypergravity conditions. The left-handed helical growth mutants had right-handed microtubule arrays, whereas the right-handed mutant had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions suppressed both the twisting phenotype and reorientation of microtubules in tubulin mutants. These results support the hypothesis that cortical microtubules play an es-sential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by hypergravity. Actin microfilaments, in addition to microtubules, may be involved in gravity resistance. The nucleus of epidermal cells of azuki bean epicotyls, which is present almost in the center of the cell at 1 g, was displaced to the cell bottom by increasing the magnitude of gravity. Cytochalasin D stimulated the sedimentation by hypergravity of the nu-cleus, suggesting that the positioning of the nucleus is regulated by actin microfilaments, which is

  13. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  14. Some structures and processes of human epithelial cells involved in uptake of enterohemorrhagic Escherichia coli O157:H7 strains.

    PubMed Central

    Oelschlaeger, T A; Barrett, T J; Kopecko, D J

    1994-01-01

    Several enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 isolated from patients with hemorrhagic colitis, ischemic colitis, or hemolytic uremic syndrome were all found to be able to invade certain human epithelial cell lines in vitro. Their ability to gain entry into epithelial cells was compared with those of known invasive Shigella flexneri and Salmonella typhi strains and the noninvasive E. coli strain HB101 in invasion assays utilizing gentamicin to kill extracellular bacteria. All EHEC strains under investigation were efficiently internalized into T24 bladder and HCT-8 ileocecal cells. In striking contrast to shigellae, the same EHEC strains were not taken up into human embryonic intestinal INT407 cells or HEp-2 cells any more than the noninvasive E. coli strain HB101. The mechanism(s) of EHEC internalization was characterized by comparing the invasion efficiencies in the absence and presence of a variety of inhibitors acting on structures and processes of prokaryotic or eukaryotic cells. Also, wild-type, plasmid-containing EHEC strains were compared with their plasmid-cured isogenic derivative strains to determine if plasmid genes affect invasion ability. Plasmid-cured EHEC invaded as well as wild-type EHEC, indicating that invasion ability is chromosomally encoded. Inhibition of bacterial protein synthesis by simultaneous addition of bacteria and chloramphenicol to the monolayer blocked EHEC uptake dramatically, suggesting the presence of an invasion protein(s) with a short half-life. Studies utilizing inhibitors which act on eukaryotic cells demonstrated a strong dependence on microfilaments in the process of uptake of all EHEC strains into both T24 and HCT-8 cells. In general, depolymerization of microtubules as well as inhibition of receptor-mediated endocytosis reduced the efficiency of EHEC invasion of T24 cells, whereas interference with endosome acidification reduced EHEC entry into only HCT-8 cells. Taxol-induced stabilization of

  15. Structure of the F–actin–tropomyosin complex

    PubMed Central

    von der Ecken, Julian; Müller, Mirco; Lehman, William; Manstein, Dietmar J.; Penczek, Pawel A.; Raunser, Stefan

    2015-01-01

    Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss1, familial thoracic aortic aneurysms and dissections2, and multiple variations of myopathies3. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin4,5. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin6. Although crystal structures for monomeric actin (G-actin) are available7, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 ångstroms in complex with tropomyosin at a resolution of 6.5ångstroms, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify the density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined actin–tropomyosin–myosin structure8 reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong

  16. Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

    PubMed Central

    Ma, Yi-ming; Han, Wei; Li, Jia; Hu, Li-hong; Zhou, Yu-bo

    2015-01-01

    Aim: To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms. Methods: Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye. Results: Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis. Conclusion: Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro. PMID:25832431

  17. Structure of the F-actin-tropomyosin complex.

    PubMed

    von der Ecken, Julian; Müller, Mirco; Lehman, William; Manstein, Dietmar J; Penczek, Pawel A; Raunser, Stefan

    2015-03-05

    Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted

  18. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  19. Structure and function of a G-actin sequestering protein with a vital role in malaria oocyst development inside the mosquito vector.

    PubMed

    Hliscs, Marion; Sattler, Julia M; Tempel, Wolfram; Artz, Jennifer D; Dong, Aiping; Hui, Raymond; Matuschewski, Kai; Schüler, Herwig

    2010-04-09

    Cyclase-associated proteins (CAPs) are evolutionary conserved G-actin-binding proteins that regulate microfilament turnover. CAPs have a modular structure consisting of an N-terminal adenylate cyclase binding domain, a central proline-rich segment, and a C-terminal actin binding domain. Protozoan parasites of the phylum Apicomplexa, such as Cryptosporidium and the malaria parasite Plasmodium, express small CAP orthologs with homology to the C-terminal actin binding domain (C-CAP). Here, we demonstrate by reverse genetics that C-CAP is dispensable for the pathogenic Plasmodium blood stages. However, c-cap(-) parasites display a complete defect in oocyst development in the insect vector. By trans-species complementation we show that the Cryptosporidium parvum ortholog complements the Plasmodium gene functions. Purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization. The crystal structure of C. parvum C-CAP shows two monomers with a right-handed beta-helical fold intercalated at their C termini to form the putative physiological dimer. Our results reveal a specific vital role for an apicomplexan G-actin-binding protein during sporogony, the parasite replication phase that precedes formation of malaria transmission stages. This study also exemplifies how Plasmodium reverse genetics combined with biochemical and structural analyses of orthologous proteins can offer a fast track toward systematic gene characterization in apicomplexan parasites.

  20. Morphological characterization of a newly established human osteosarcoma cell line, HS-Os-1, revealing its distinct osteoblastic nature.

    PubMed

    Sonobe, H; Mizobuchi, H; Manabe, Y; Furihata, M; Iwata, J; Hikita, T; Oka, T; Ohtsuki, Y; Goto, T

    1991-01-01

    A newly established human osteosarcoma cell line, HS-Os-1, from an osteoblastic tumor arising in the left humerus of an 11-year-old girl was morphologically characterized in vitro and in vivo. HS-Os-1 cells in a monolayer have been maintained for more than 2 years since the initial cultivation, and were round or polygonal in shape with marked pleomorphism. Their cytoplasm was strongly positive for specific markers of osteoblasts, such as alkaline phosphatase and osteocalcin. Tumors induced in nude mice by HS-Os-1 cell inoculation at passage 12 or 23 revealed typical histological features of osteoblastic osteosarcoma, similar to those observed in the original tumor, producing prominent osteoid matrix with calcification. Ultrastructurally, HS-Os-1 cells in vitro and tumor cells in vivo showed similar well-developed, markedly dilated rough endoplasmic reticulum, polysomes and microfilaments in their cytoplasm. Additionally, many collagen fibers associated with deposition of electron-dense material were detected in the stroma featuring osteoid matrix. Thus, the HS-Os-1 cell line was shown to exhibit its osteoblastic nature in vitro and in vivo, and therefore might become an extremely useful tool for various pathomorphological investigations on human osteosarcomas.

  1. GPCRs and actin-cytoskeleton dynamics.

    PubMed

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

  2. Hyphal ontogeny in Neurospora crassa: a model organism for all seasons.

    PubMed

    Riquelme, Meritxell; Martínez-Núñez, Leonora

    2016-01-01

    Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus Neurospora crassa develops a variety of cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa and the emerging questions that we believe should be addressed.

  3. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  4. Nanochips of Tantalum Oxide Nanodots as artificial-microenvironments for monitoring Ovarian cancer progressiveness

    NASA Astrophysics Data System (ADS)

    Dhawan, Udesh; Wang, Ssu-Meng; Chu, Ying Hao; Huang, Guewha S.; Lin, Yan Ren; Hung, Yao Ching; Chen, Wen Liang

    2016-08-01

    Nanotopography modulates cell characteristics and cell behavior. Nanotopological cues can be exploited to investigate the in-vivo modulation of cell characteristics by the cellular microenvironment. However, the studies explaining the modulation of tumor cell characteristics and identifying the transition step in cancer progressiveness are scarce. Here, we engineered nanochips comprising of Tantalum oxide nanodot arrays of 10, 50, 100 and 200 nm as artificial microenvironments to study the modulation of cancer cell behavior. Clinical samples of different types of Ovarian cancer at different stages were obtained, primary cultures were established and then seeded on different nanochips. Immunofluorescence (IF) was performed to compare the morphologies and cell characteristics. Indices corresponding to cell characteristics were defined. A statistical comparison of the cell characteristics in response to the nanochips was performed. The cells displayed differential growth parameters. Morphology, Viability, focal adhesions, microfilament bundles and cell area were modulated by the nanochips which can be used as a measure to study the cancer progressiveness. The ease of fabrication of nanochips ensures mass-production. The ability of the nanochips to act as artificial microenvironments and modulate cell behavior may lead to further prospects in the markerless monitoring of the progressiveness and ultimately, improving the prognosis of Ovarian cancer.

  5. Heavy metals health risk assessment for population via consumption of food crops and fruits in Owerri, South Eastern, Nigeria

    PubMed Central

    2012-01-01

    Background This study assessed lead, cadmium, and nickel level in food crops, fruits and soil samples from Ohaji and Umuagwo and Owerri in South Eastern Nigeria and estimated the potential health risks of metals. Samples were washed, oven-dried at 70–80°C for 24 h and powdered. Samples were digested with perchloric acid and nitric acid. Metals were analysed with Unicam Atomic Absorption Spectrophotometer. Result The concentration of Pb, Cd, and Ni in Ohaji exceeded the maximum allowable concentrations for agricultural soil as recommended by EU. Lead, Cd, and Ni in the food crops were highest in Oryza sativa, Glycine max, and Pentabacta microfila respectively. Highest levels of Pb, Cd, and Ni, in fruits were detected in Canarium schweinfurthii, Citrus reticulata, Ananas comosus respectively. The true lead and cadmium intake for the rice based meal were 3.53 and 0.034 g/kg respectively. Whereas the true intake of lead and cadmium for the cassava based meal were 19.42 and 0.049 g/kg respectively. Conclusion Local food stuff commonly available in South Eastern Nigeria villages may contribute to the body burden of heavy metal. This is of public health importance. PMID:22853175

  6. A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

    PubMed Central

    Lim, Pei Jin; Chu, Justin Jang Hann

    2014-01-01

    Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals. PMID:24587455

  7. Calorimetric gas sensor

    DOEpatents

    Ricco, A.J.; Hughes, R.C.; Smith, J.H.; Moreno, D.J.; Manginell, R.P.; Senturia, S.D.; Huber, R.J.

    1998-11-10

    A combustible gas sensor is described that uses a resistively heated, noble metal-coated, micromachined polycrystalline Si filament to calorimetrically detect the presence and concentration of combustible gases. The filaments tested to date are 2 {micro}m thick {times} 10{micro}m wide {times} 100, 250, 500, or 1000 {micro}m-long polycrystalline Si; some are overcoated with a 0.25 {micro}m-thick protective CVD Si{sub 3}N{sub 4} layer. A thin catalytic Pt film was deposited by CVD from the precursor Pt(acac){sub 2} onto microfilaments resistively heated to approximately 500 C; Pt deposits only on the hot filament. Using a constant-resistance-mode feedback circuit, Pt-coated filaments operating at ca. 300 C (35 mW input power) respond linearly, in terms of the change in supply current required to maintain constant resistance (temperature), to H{sub 2} concentrations between 100 ppm and 1% in an 80/20 N{sub 2}/O{sub 2} mixture. Other catalytic materials can also be used. 11 figs.

  8. Calorimetric gas sensor

    DOEpatents

    Ricco, Antonio J.; Hughes, Robert C.; Smith, James H.; Moreno, Daniel J.; Manginell, Ronald P.; Senturia, Stephen D.; Huber, Robert J.

    1998-01-01

    A combustible gas sensor that uses a resistively heated, noble metal-coated, micromachined polycrystalline Si filament to calorimetrically detect the presence and concentration of combustible gases. The filaments tested to date are 2 .mu.m thick.times.10 .mu.m wide.times.100, 250, 500, or 1000 .mu.m-long polycrystalline Si; some are overcoated with a 0.25 .mu.m-thick protective CVD Si.sub.3 N.sub.4 layer. A thin catalytic Pt film was deposited by CVD from the precursor Pt(acac).sub.2 onto microfilaments resistively heated to approximately 500.degree. C.; Pt deposits only on the hot filament. Using a constant-resistance-mode feedback circuit, Pt-coated filaments operating at ca. 300.degree. C. (35 mW input power) respond linearly, in terms of the change in supply current required to maintain constant resistance (temperature), to H.sub.2 concentrations between 100 ppm and 1% in an 80/20 N.sub.2 /O.sub.2 mixture. Other catalytic materials can also be used.

  9. Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets.

    PubMed

    Volkmann, D; Buchen, B; Hejnowicz, Z; Tewinkel, M; Sievers, A

    1991-01-01

    During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Charo globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. l0(-4) g (i.e. microgravity) during the parabolic flight (lasting for 301-390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 micrometers and 3.6 micrometers in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.

  10. Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets.

    PubMed

    Volkmann, D; Buchen, B; Hejnowicz, Z; Tewinkel, M; Sievers, A

    1991-09-01

    During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Chara globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. 10(-4) · g (i.e. microgravity) during the parabolic flight (lasting for 301-390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 μm and 3.6 μm in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.

  11. Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia).

    PubMed

    Nakashima, Keisuke; Nishino, Atsuo; Hirose, Euichi

    2011-08-01

    A postanal tail is a major synapomorphy of the phylum Chordata, which is composed of three subphyla: Vertebrata, Cephalochordata, and Tunicata (Urochordata). Among tunicates, appendicularians are the only group that retains the tail in the adult, and the adult tail functions in locomotion and feeding in combination with a cellulose-based house structure. Given the phylogenetic position of tunicates, the appendicularian adult tail may possess ancestral features of the chordate tail. We assess the ultrastructural development of the tail epidermis of the appendicularian Oikopleura dioica. The epidermis of the larval tail is enclosed by the larval envelope, which is a thin sheet similar to the outer tunic layer of ascidian larvae. The epidermis of the adult tail seems to bear no tunic-like cellulosic integuments, and the tail fin is a simple folding of the epidermis. Every epidermal cell, except for the triangular cells at the edge of the tail fin, has a conspicuous matrix layer of fibrous content in the apical cytoplasm without enclosing membranes. The epidermis of the larval tail does not have a fibrous matrix layer, suggesting the production of the layer during larval development and metamorphosis. Zonulae adhaerentes firmly bind the epidermal cells of the adult tail to one another, and the dense microfilaments lining the cell borders constitute a mechanical support for the cell membranes. The intracellular matrix, cell junctions, and cytoskeletons probably make the tail epidermis a tough, flexible shell supporting the active beating of the oikopleuran adult tail.

  12. Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia)

    NASA Astrophysics Data System (ADS)

    Nakashima, Keisuke; Nishino, Atsuo; Hirose, Euichi

    2011-08-01

    A postanal tail is a major synapomorphy of the phylum Chordata, which is composed of three subphyla: Vertebrata, Cephalochordata, and Tunicata (Urochordata). Among tunicates, appendicularians are the only group that retains the tail in the adult, and the adult tail functions in locomotion and feeding in combination with a cellulose-based house structure. Given the phylogenetic position of tunicates, the appendicularian adult tail may possess ancestral features of the chordate tail. We assess the ultrastructural development of the tail epidermis of the appendicularian Oikopleura dioica. The epidermis of the larval tail is enclosed by the larval envelope, which is a thin sheet similar to the outer tunic layer of ascidian larvae. The epidermis of the adult tail seems to bear no tunic-like cellulosic integuments, and the tail fin is a simple folding of the epidermis. Every epidermal cell, except for the triangular cells at the edge of the tail fin, has a conspicuous matrix layer of fibrous content in the apical cytoplasm without enclosing membranes. The epidermis of the larval tail does not have a fibrous matrix layer, suggesting the production of the layer during larval development and metamorphosis. Zonulae adhaerentes firmly bind the epidermal cells of the adult tail to one another, and the dense microfilaments lining the cell borders constitute a mechanical support for the cell membranes. The intracellular matrix, cell junctions, and cytoskeletons probably make the tail epidermis a tough, flexible shell supporting the active beating of the oikopleuran adult tail.

  13. Ultraviolet A radiation transiently disrupts gap junctional communication in human keratinocytes.

    PubMed

    Provost, Nicolas; Moreau, Marielle; Leturque, Armelle; Nizard, Carine

    2003-01-01

    Ultraviolet A (UVA) (320-400 nm) radiation is known to cause cutaneous aging and skin cancer. We studied the effect of UVA (365 nm) radiation on the human epidermis by focusing on keratinocyte gap junction-mediated intercellular communication (GJIC). We observed a dose-dependent 10-fold decrease in GJIC induced by UVA in normal human keratinocytes. This decrease in GJIC was associated with time-dependent internalization of connexin43 (Cx43). UVA radiation also damaged the actin cytoskeleton, as shown by microfilament disappearance. Importantly, the decrease in GJIC was transient when keratinocytes were irradiated with 10 J/cm(2) UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVA irradiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43 localization and phosphorylation were differently regulated by the two treatments. This suggests that at least two different pathways may mediate the observed fall in GJIC. These findings identify keratinocyte GJIC as a new UVA target that might sensitize human skin to photoaging and cancer formation.

  14. Apoptosis-linked gene 2 promotes breast cancer growth and metastasis by regulating the cytoskeleton.

    PubMed

    Qin, Juan; Li, Dengwen; Zhou, Yunqiang; Xie, Songbo; Du, Xin; Hao, Ziwei; Liu, Ruming; Liu, Xinqi; Liu, Min; Zhou, Jun

    2017-01-10

    Breast cancer is the most prevalent cancer in women. Although it begins as local disease, breast cancer frequently metastasizes to the lymph nodes and distant organs. Therefore, novel therapeutic targets are needed for the management of this disease. Apoptosis-linked gene 2 (ALG-2) is a calcium-binding protein crucial for diverse physiological processes and has recently been implicated in cancer development. However, it remains unclear whether this protein is involved in the pathogenesis of breast cancer. Here, we demonstrate that the expression of ALG-2 is significantly upregulated in breast cancer tissues and is correlated with clinicopathological characteristics indicative of tumor malignancy. Our data further show that ALG-2 stimulates breast cancer growth and metastasis in mice. ALG-2 also promotes breast cancer cell proliferation, survival, and motility in vitro. Mechanistic data reveal that ALG-2 disrupts the localization of centrosome proteins, resulting in spindle multipolarity and chromosome missegregation. In addition, ALG-2 drives the polarization and migration of breast cancer cells by facilitating the rearrangement of microtubules and microfilaments. These findings reveal a critical role for ALG-2 in the pathogenesis of breast cancer and have important implications for its diagnosis and therapy.

  15. An update on the pharmacokinetics and pharmacodynamics of alisertib, a selective Aurora kinase A inhibitor.

    PubMed

    Durlacher, Cameron T; Li, Zhi-Ling; Chen, Xiao-Wu; He, Zhi-Xu; Zhou, Shu-Feng

    2016-06-01

    Human Aurora kinases, including Aurora kinase A (AURKA), B (AURKB), and C (AURKC), play an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. AURKA and AURKB are key regulators of mitosis and centrosome via polymerizing microfilaments and controlling chromatid segregation. In particular, AURKA plays critical roles in the regulation of mitotic entry, centrosome function, bipolar spindle assembly, and chromosome segregation. AURKA has been found to be overexpressed in various solid and haematological cancers and has been linked with poor prognosis. Its important role in cancer initiation, growth, and metastasis has brought the focus to search for potent and selective AURKA inhibitors for cancer treatment. MLN8237, also known as alisertib, is one selective AURKA inhibitor that has shown remarkable anticancer effects in preclinical studies. Alisertib exhibits favourable pharmacokinetic properties. Alisertib has generally showed good partial response rates of 4-52% and good safety profiles in Phase I and II trials when it is solely administered as well as combined with cytotoxic chemotherapeutic drugs. Recently, the multicentre, randomized Phase III study of alisertib in patients with relapsed or refractory peripheral T-cell lymphoma has been discontinued due to unsatisfactory efficacy. The low risk of side effects, accessibility, and effectiveness of alisertib makes it a new promising anticancer therapy and further mechanistic and clinical studies are warranted.

  16. Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Yamamoto, Kazuyoshi; Kiss, John Z.

    2002-01-01

    The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

  17. Role of cytoskeleton in differentiation of gravisensitive root sites in simulated microgravity

    NASA Astrophysics Data System (ADS)

    Shevchenko, G.; Kordyum, E.

    Cytoskeleton is known to be one of the elements participating in signaling reactions caused by altered gravity in plant cells. Up to date actin microfilaments (MFs) are considered mainly in respect of their involvement in statolith movement and tubulin microtubules (MTs) are investigated in respect of their participation in gravitropic plant growth response (root bending). But there are numerous data evidencing that the role of cytoskeleton in plant gravisensing is far more complex. To contribute to the issue the novel approach is proposed. In particular, since gravity is persistent factor through plant evolution it is suggested to compare the arrangement of cytoskeletal elements at the consequent developmental stages of graviperceiving (root cap) and gravireacting (cell in elongation zone) root sites both in stationary control and simulated microgravity. Special emphasis is given to MF dynamics in the process of statocyte differentiation and establishing statocyte polarity while developing from cells of root cap meristem. MTs are going to be elucidated in epidermal and cortex root cell lines originating from meristem of proper root. Root of Beta vulgaris seedling is used as an object. Methods of cytoskeleton immunohistochemistry, cytoskeleton inhibitors, plant mutant on cytoskeleton genes as well as blockers of auxin transport are intended to be applied. It is anticipated that data will be collected on the influence caused by simulated microgravity on cytoskeleton involvement in the development of plant gravisensing organs. Such an approach will not only widen our knowledge about cytoskeleton role in plant development but also in plant gravireaction.

  18. Complement-induced equine neutrophil adhesiveness and aggregation.

    PubMed

    Slauson, D O; Skrabalak, D S; Neilsen, N R; Zwahlen, R D

    1987-05-01

    Equine neutrophils (PMN) were isolated from citrated normal blood by density gradient separation on Ficoll-Hypaque to greater than 96% purity and 98% viability and an average of 3.78 x 10(7) PMN/ml. The agonist C5a des Arg was used in serial dilutions of whole zymosan-activated equine plasma (ZAP) or was partially purified from ZAP by column chromatography. Purified equine PMN exhibited rapid aggregation following incubation with C5a des Arg which was further dependent on the availability of divalent cations, especially Mg++. The microfilament disruptive agent cytochalasin B (5 micrograms/50 microliters) greatly augmented aggregation responses to C5a des Arg. Subaggregating doses of C5a des Arg promoted PMN adhesiveness as assayed on 0.5 x 10 cm borosilicate glass columns containing a 2.0 cm bed of Sephadex G-25. This C5a des Arg-induced increased adhesiveness was inhibitable by prior incubation of the PMN with either non-steroidal (0.065 M phenylbutazone) or steroidal (0.005 M dexamethasone) anti-inflammatory agents. Ultrastructural studies correlated well with functional assays and revealed marked organelle-free lamellipodia formation without PMN-PMN contact at subaggregating doses of the agonist and progressive PMN-PMN contact at aggregating doses. Equine PMN are responsive to C5a des Arg, and induced adhesiveness responses can be manipulated by anti-inflammatory agents.

  19. Myofibroblast secretome and its auto-/paracrine signaling

    PubMed Central

    Bomb, Ritin; Heckle, Mark R.; Sun, Yao; Mancarella, Salvatore; Guntaka, Ramareddy V.; Gerling, Ivan C.; Weber, Karl T.

    2016-01-01

    Summary Myofibroblasts (myoFb) are phenotypically transformed, contractile fibroblast-like cells expressing α-smooth muscle actin microfilaments. They are integral to collagen fibrillogenesis with scar tissue formation at sites of repair irrespective of the etiologic origins of injury or tissue involved. MyoFb can persist long after healing is complete, where their ongoing turnover of collagen accounts for a progressive structural remodeling of an organ (a.k.a. fibrosis, sclerosis or cirrhosis). Such persistent metabolic activity is derived from a secretome consisting of requisite components in the de novo generation of angiotensin (Ang) II. Autocrine and paracrine signaling induced by tissue AngII is expressed via AT1 receptor ligand binding to respectively promote: i) regulation of myoFb collagen synthesis via the fibrogenic cytokine TGF-β1-Smad pathway; and ii) dedifferentiation and protein degradation of atrophic myocytes immobilized and ensnared by fibrillar collagen at sites of scarring. Several cardioprotective strategies in the prevention of fibrosis and involving myofibroblasts are considered. They include: inducing myoFb apoptosis through inactivation of antiapoptotic proteins; AT1 receptor antagonist to interfere with auto-/paracrine myoFb signaling or to induce counterregulatory expression of ACE2; and attacking the AngII-AT1R-TGF-β1-Smad pathway by antibody or the use of triplex-forming oligonucleotides. PMID:26818589

  20. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology

    NASA Technical Reports Server (NTRS)

    Ingber, Donald E.

    2002-01-01

    Great advances have been made in the identification of the soluble angiogenic factors, insoluble extracellular matrix (ECM) molecules, and receptor signaling pathways that mediate control of angiogenesis--the growth of blood capillaries. This review focuses on work that explores how endothelial cells integrate these chemical signals with mechanical cues from their local tissue microenvironment so as to produce functional capillary networks that exhibit specialized form as well as function. These studies have revealed that ECM governs whether an endothelial cell will switch between growth, differentiation, motility, or apoptosis programs in response to a soluble stimulus based on its ability to mechanically resist cell tractional forces and thereby produce cell and cytoskeletal distortion. Transmembrane integrin receptors play a key role in this mechanochemical transduction process because they both organize a cytoskeletal signaling complex within the focal adhesion and preferentially focus mechanical forces on this site. Molecular filaments within the internal cytoskeleton--microfilaments, microtubules, and intermediate filaments--also contribute to the cell's structural and functional response to mechanical stress through their role as discrete support elements within a tensegrity-stabilized cytoskeletal array. Importantly, a similar form of mechanical control also has been shown to be involved in the regulation of contractility in vascular smooth muscle cells and cardiac myocytes. Thus, the mechanism by which cells perform mechanochemical transduction and the implications of these findings for morphogenetic control are discussed in the wider context of vascular development and cardiovascular physiology.

  1. Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization

    PubMed Central

    1983-01-01

    The organization of intermediate filaments in cultured epithelial cells was rapidly and radically affected by intracellularly injected monoclonal antikeratin filament antibodies. Different antibodies had different effects, ranging from an apparent splaying apart of keratin filament bundles to the complete disruption of the keratin filament network. Antibodies were detectable within cells for more than four days after injection. The antibody-induced disruption of keratin filament organization had no light-microscopically discernible effect on microfilament or microtubule organization, cellular morphology, mitosis, the integrity of epithelial sheets, mitotic rate, or cellular reintegration after mitosis. Cell-to-cell adhesion junctions survived keratin filament disruption. However, antibody injected into a keratinocyte-derived cell line, rich in desmosomes, brought on a superfasciculation of keratin filament bundles, which appeared to pull desmosomal junctions together, suggesting that desmosomes can move in the plane of the plasma membrane and may only be 'fixed' by their anchoring to the cytoplasmic filament network. Our observations suggest that keratin filaments are not involved in the establishment or maintenance of cell shape in cultured cells. PMID:6187752

  2. Columella cells revisited: novel structures, novel properties, and a novel gravisensing model

    NASA Technical Reports Server (NTRS)

    Staehelin, L. A.; Zheng, H. Q.; Yoder, T. L.; Smith, J. D.; Todd, P.

    2000-01-01

    A hundred years of research has not produced a clear understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast statoliths in root cap columella cells into a growth response. Most models postulate that the statoliths interact with microfilaments (MF) to transmit signals to the plasma membrane (or ER), or that sedimentation onto these organelles produces the signals. However, no direct evidence for statolith-MF links has been reported, and no asymmetric structures of columella cells have been identified that might explain how a root turned by 90 degrees knows which side is up. To address these and other questions, we have (1) quantitatively examined the effects of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in high-pressure frozen/freeze-substituted roots; and (3) followed the sedimentation dynamics of statolith movements in reoriented root tips. The findings have led to the formulation of a new model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal network. This network is denser in the ER-devoid central region of the cell than in the ER-rich cell cortex and is coupled to receptors in the plasma membrane. Statolith sedimentation is postulated to disrupt the network and its links to receptors in some regions of the cell cortex, while allowing them to reform in other regions and thereby produce a directional signal.

  3. Magnetophoretic Induction of Root Curvature

    NASA Technical Reports Server (NTRS)

    Hasenstein, Karl H.

    1997-01-01

    The last year of the grant period concerned the consolidation of previous experiments to ascertain that the theoretical premise apply not just to root but also to shoots. In addition, we verified that high gradient magnetic fields do not interfere with regular cellular activities. Previous results have established that: (1) intracellular magnetophoresis is possible; and (2) HGMF lead to root curvature. In order to investigate whether HGMF affect the assembly and/or organization of structural proteins, we examined the arrangement of microtubules in roots exposed to HGMF. The cytoskeletal investigations were performed with fomaldehyde-fixed, nonembedded tissue segments that were cut with a vibratome. Microtubules (MTs) were stained with rat anti-yeast tubulin (YOL 1/34) and DTAF-labeled antibody against rat IgG. Microfilaments (MFs) were visualized by incubation in rhodamine-labeled phalloidin. The distribution and arrangement of both components of the cytoskeleton were examined with a confocal microscope. Measurements of growth rates and graviresponse were done using a video-digitizer. Since HGMF repel diamagnetic substances including starch-filled amyloplasts and most The second aspect of the work includes studies of the effect of cytoskeletal inhibitors on MTs and MFs. The analysis of the effect of micotubular inhibitors on the auxin transport in roots showed that there is very little effect of MT-depolymerizing or stabilizing drugs on auxin transport. This is in line with observations that application of such drugs is not immediately affecting the graviresponsiveness of roots.

  4. Persistent Noggin arrests cardiomyocyte morphogenesis and results in early in utero lethality

    PubMed Central

    Simmons, Olga; Snider, Paige; Wang, Jain; Schwartz, Robert J.; Chen, YiPing; Conway, Simon J.

    2014-01-01

    Background Multiple BMP genes are expressed in the developing heart from the initiation to late-differentiation stages, and play pivotal roles in cardiovascular development. In this study, we investigated the requirement of BMP activity in heart development by transgenic over-expression of extracellular BMP antagonist Noggin. Results Using Nkx2.5-Cre to drive lineage-restricted Noggin within cardiomyocyte progenitors, we show persistent Noggin arrests cardiac development at the linear heart stage. This is coupled with a significantly reduced cell proliferation rate, subsequent cardiomyocyte programmed cell death and reduction of downstream intracellular pSMAD1/5/8 expression. Noggin mutants exhibit reduced heartbeat which likely results in subsequent fully penetrant in utero lethality. Significantly, confocal and electron micrographic examination revealed considerably fewer contractile elements, as well as a lack of maturation of actin-myosin microfilaments. Molecular analysis demonstrated that ectopic Noggin-expressing regions in the early heart’s pacemaker region, failed to express the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4), resulting in an overall decrease in Hcn4 levels. Conclusions Combined, our results reveal a novel role for BMP signaling in the progression of heart development from the tubular heart stage to the looped stage via regulation of proliferation and promotion of maturation of the in utero heart’s contractile apparatus and pacemaker. PMID:25428115

  5. Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents.

    PubMed

    Canu, Nadia; Pagano, Ilaria; La Rosa, Luca Rosario; Pellegrino, Marsha; Ciotti, Maria Teresa; Mercanti, Delio; Moretti, Fabiola; Sposato, Valentina; Triaca, Viviana; Petrella, Carla; Maruyama, Ichiro N; Levi, Andrea; Calissano, Pietro

    2017-01-01

    The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modified by NGF, or by drugs that either inhibit ER-to-Golgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death [amyloid β (Aβ)-peptide, staurosporine and rapamycin], albeit via different mechanisms, all caused dissociation of APP/TrkA complexes and increased production of C-terminal fragment (β-CTF) APP fragment. These findings open new perspectives for investigating the interplay between these proteins during neurodegeneration and AD.

  6. The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly

    PubMed Central

    Shwartz, Arkadi; Dhanyasi, Nagaraju; Schejter, Eyal D; Shilo, Ben-Zion

    2016-01-01

    Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. We have used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, we identify several distinct phases in the dynamic construction of thin-filament arrays. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches we have identified Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles. DOI: http://dx.doi.org/10.7554/eLife.16540.001 PMID:27731794

  7. Staufen Recruitment into Stress Granules Does Not Affect Early mRNA Transport in Oligodendrocytes

    PubMed Central

    Thomas, María G.; Tosar, Leandro J. Martinez; Loschi, Mariela; Pasquini, Juana M.; Correale, Jorge; Kindler, Stefan; Boccaccio, Graciela L.

    2005-01-01

    Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response. PMID:15525674

  8. 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.

  9. Induction of neuron-specific tropomyosin mRNAs by nerve growth factor is dependent on morphological differentiation

    PubMed Central

    1993-01-01

    We have examined the expression of brain-specific tropomyosins during neuronal differentiation. Both TmBr-1 and TmBr-3 were shown to be neuron specific. TmBr-1 and TmBr-3 mRNA levels increased during the most active phase of neurite outgrowth in the developing rat cerebellum. In PC12 cells stimulated by nerve growth factor (NGF) to differentiate to the neuronal phenotype, TmBr-1 and TmBr-3 levels increased with an increasing degree of morphological differentiation. Induction of TmBr-1 and TmBr-3 expression only occurred under conditions where PC12 cells were permitted to extend neurites. NGF was unable to maintain levels of TmBr-1 and TmBr-3 with the loss of neuronal phenotype by resuspension of differentiated PC12 cells. The unique cellular expression and regulation in vivo and in vitro of TmBr- 1 and TmBr-3 strongly suggests a critical role of these tropomyosins in neuronal microfilament function. The findings reveal that the induction and maintenance of the neuronal tropomyosins is dependent on morphological differentiation and the maintenance of the neuronal phenotype. PMID:8416988

  10. Fast Shape Evolution of Laser Filaments in the Wake of Femtosecond Driving Pulse

    NASA Astrophysics Data System (ADS)

    Romanov, Dmitri; Levis, Robert

    2013-05-01

    A theoretical model is developed for subnanosecond evolution of highly nonequilibrium, inhomogeneous free-electron gas in a laser filament/microfilament wake channel. The evolution is driven by two interrelated mechanisms: (i) impact ionization of residual neutral atoms inside the channel and on its surface, and (ii) thermal conduction in the electron gas. The simulation results for the cases of weak and moderate initial ionization show crucial importance of incorporating the spread effects, especially as regards the electron temperature. The calculated evolution patterns determine the transient optical and electronic properties of filament wake channels. Accordingly, we propose tracing the wake channel evolution via linear and nonlinear light-scattering experiments. The evolving shape of the electron density distribution can be extracted from longitudinal and/or transverse Fraunhofer diffraction patterns. Complementarily, the evolving temperature distribution may be deduced either from angular-resolved four-wave-mixing experiments or from the spatial-spectral patterns of giant Rabi sidebands. Medium-specific estimates are made for atmospheric-pressure argon gas. In molecular-gas cases, the model can be straightforwardly augmented to incorporate the processes of dissociative recombination and vibrational excitations. Support from the Air Force Office of Scientific Research, Grant No. N00014-10-0293, is gratefully acknowledged.

  11. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    PubMed

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria.

    PubMed

    Chakroun, Maissa; Banyuls, Núria; Bel, Yolanda; Escriche, Baltasar; Ferré, Juan

    2016-06-01

    Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

  13. Calcium and cytoskeleton signaling during cell volume regulation in isolated nematocytes of Aiptasia mutabilis (Cnidaria: Anthozoa).

    PubMed

    Marino, A; La Spada, G

    2007-05-01

    Cell volume regulation has not been completely clarified in Coelenterates. The present investigation focuses on cell volume regulation under anisosmotic conditions, both hyposmotic and hypertonic, and on the underlying signals in nematocytes isolated from the Coelenterate Aiptasia mutabilis living in sea water. Nematocytes, once isolated from acontia, that were submitted to either hyposmotic (35%) and hypertonic shock (45%) show RVD and RVI capabilities, respectively. In order to ascertain the role of Ca2+ in triggering such regulatory mechanisms and the possible involvement of cytoskeleton components, tests were performed by employing either Ca2+ free conditions, Gd3+ as Ca2+ channel blockers, TFP as calmodulin inhibitor, colchicine as microtubule inhibitor and cytochalasin B as microfilament polymerization inhibitor. Results show that isolated nematocytes of A. mutabilis can regulate their volume upon both hyposmotic and hypertonic challenge. Ca2+ both from external medium and from internal stores is needed to perform RVD mechanisms, whereas, intracellular Ca2+ seems to be mainly involved in RVI. Moreover cytoskeletal components may play an important role since a significant RVD and RVI inhibition was observed in treated cells. On the basis of our observations further studies are warranted to further verify the role of signals, including phosphatases and phosphorylases, in cell volume regulation of primitive eukaryotic cells.

  14. Immunohistochemical studies on the poll gland of the dromedary camel (Camelus dromedarius) during the rutting season.

    PubMed

    Ebada, Safwat; Helal, Amr; Alkafafy, Mohamed

    2012-07-01

    The poll glands are subcutaneous exocrine glands located on the back of the neck behind the ears in male camels. The function of poll glands is not well known, though they are thought to play a role during the rutting season. The presence, location and degree of immunolocalization of microfilaments and intermediate filament systems: actin and cytokeratins (Cks) and also S100 protein were studied in the poll glands in sexually mature one-humped camels during the rutting season. These proteins were variably expressed between the epithelia, perialveolar, interalveolar tissue and the periductal tissue. Strong α-smooth muscle actin (α-SMA) immunoreactivity (IR) was displayed by the perialveolar myoepithelial cells, periductal and vascular smooth muscle cells (SMCs), but not in the epithelial cells. Cytokeratin (Ck)-IR was strong in the epithelial lining of the secretory alveoli and excretory ducts, however, the apical blebs of the secretory cells were almost negative. Weak to moderate Ck-IR was observed in the perialveolar myoepithelial cells, but not in the interalveolar tissue or endothelial cells. S100 protein was expressed variably in the epithelial lining of the secretory alveoli. S100-IR was more obvious in the supranuclear region and the apical blebs. Variable reaction was observed in the perialveolar myoepithelial cells, periductal and interductal tissue and endothelial cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

  15. Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models

    PubMed Central

    Schiffer, Mario; Teng, Beina; Gu, Changkyu; Shchedrina, Valentina A.; Kasaikina, Marina; Pham, Vincent A.; Hanke, Nils; Rong, Song; Gueler, Faikah; Schroder, Patricia; Tossidou, Irini; Park, Joon-Keun; Staggs, Lynne; Haller, Hermann; Erschow, Sergej; Hilfiker-Kleiner, Denise; Wei, Changli; Chen, Chuang; Tardi, Nicholas; Hakroush, Samy; Selig, Martin K.; Vasilyev, Aleksandr; Merscher, Sandra; Reiser, Jochen; Sever, Sanja

    2015-01-01

    Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to crosslink actin microfilaments into higher order structures have been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and of CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the significant regenerative potential of injured glomeruli and that targeting the oligomerization cycle of dynamin represents an attractive potential therapeutic target to treat CKD. PMID:25962121

  16. Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

    SciTech Connect

    Parry, G.; Beck, J.C.; Moss, L.; Bartley, J. ); Ojakian, G.K. )

    1990-06-01

    The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. The authors have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembranous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. They suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS0O while colchicine and acrylamide did not. They hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

  17. Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

    PubMed

    Ram, Rashmi; Wescott, Andrew P; Varandas, Katherine; Dirksen, Robert T; Blaxall, Burns C

    2014-01-01

    Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.

  18. The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize.

    PubMed Central

    Blancaflor, E B; Hasenstein, K H

    1997-01-01

    To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots. PMID:11536803

  19. Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin

    PubMed Central

    Wichmann, Heidi; Vocke, Farina; Brinkhoff, Thorsten; Simon, Meinhard; Richter-Landsberg, Christiane

    2015-01-01

    The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3–0.5 µg/mL (1.4–2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca2+-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death. PMID:26633426

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

    PubMed Central

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

    1991-01-01

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

  1. Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation.

    PubMed

    González, R; Sjunnesson, Y C B

    2013-10-01

    Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development.

  2. Robust formulation for the design of tissue engineering scaffolds: A comprehensive study on structural anisotropy, viscoelasticity and degradation of 3D scaffolds fabricated with customized desktop robot based rapid prototyping (DRBRP) system.

    PubMed

    Hoque, M Enamul

    2017-03-01

    This study investigates the scaffolds' structural anisotropy (i.e. the effect of loading direction), viscoelasticity (i.e. the effect of cross head speed or strain rate), and the influence of simulated physiological environment (PBS solution at 37°C) on the mechanical properties. Besides, the in vitro degradation study has also been performed that evaluates the effect of variation in material and lay-down pattern on the scaffolds' degradation kinetics in terms of mass loss, and change in morphological and mechanical properties. Porous three dimensional (3D) scaffolds of polycarprolactone (PCL) and polycarprolactone-polyethylene glycol (PCL-PEG) were developed by laying down the microfilaments directionally layer-by-layer using an in-house built computer-controlled extrusion and deposition process, called desktop robot based rapid prototyping (DRBRP) system. The loading direction, strain rate and physiological environment directly influenced the mechanical properties of the scaffolds. In vitro degradation study demonstrated that both PCL and PCL-PEG scaffolds realized homogeneous hydrolytic degradation via surface erosion resulting in a consistent and predictable mass loss. The linear mass loss caused uniform and linear increase in porosity that accordingly led to the decrease in mechanical properties. The synthetic polymer had the potential to modulate hydrophilicity and/or degradability and consequently, the biomechanical properties of the scaffolds by varying the polymer constituents.

  3. Ultrastructural analysis of contractile cell development in lung microvessels in hyperoxic pulmonary hypertension. Fibroblasts and intermediate cells selectively reorganize nonmuscular segments.

    PubMed Central

    Jones, R.

    1992-01-01

    The current study traces the development of contractile cells in the nonmuscular segments of rat lung microvessels in hyperoxic pulmonary hypertension. New intimal cells first develop into a well-defined layer beneath the endothelium and internal to an elastic lamina. Ultrastructurally, these cells are found to be 1) fibroblasts recruited to the vessel wall from the interstitium and 2) intermediate cells, a population of preexisting vascular cells (structurally between a smooth muscle cell and a pericyte). Early in hyperoxia (days 3 through 7), interstitial fibroblasts migrate and align around the smallest vessels in which an elastic lamina is either absent or fragmentary. These cells then are incorporated into the vessel wall by tropoelastin secretion and the formation of an elastic lamina along their abluminal margin. After day 7, the new mural fibroblasts acquire the features of contractile cells, namely a basal lamina, extensive microfilaments, and dense bodies. In other vessels, as early as day 3 of hyperoxia, intermediate cells within the vessel intima begin to acquire the additional filaments and dense bodies of contractile cells. As hyperoxia continues, each cell pathway gives rise to vessels with distinct intimal or medial layers of contractile cells. In this way, thick-walled 'newly muscularized' vessel segments form adjacent to the capillary bed. Images Figure 1 Figure 5 Figure 6 Figure 7 p1500-a Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:1466406

  4. The myosin family: unconventional roles of actin-dependent molecular motors in immune cells.

    PubMed

    Maravillas-Montero, José L; Santos-Argumedo, Leopoldo

    2012-01-01

    Myosins comprise a family of ATP-dependent motor proteins that are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. Recent phylogenetic analysis places myosins into 35 highly diverse classes. Although these actin-based molecular motors have been characterized extensively, and much is known about their function in different cellular compartments, there is little information available about these molecules in hematopoietic cells. The available data establish that myosins expressed by immune cells are able to support general tasks, such as maintaining plasma membrane tension, moving and secreting vesicles, aiding in endo- and exocytotic processes, and promoting the adhesion and motility of cells. Additionally, however, myosins are involved in highly specialized functions, such as regulating cell activation, IS-induced signaling, and the severing of microfilaments via the control of GTPases. In this review, we summarize the current understanding of myosins in leukocytes, with emphasis on the emerging roles of these molecular motors in immune functions.

  5. Serum non-organ specific autoantibodies in human immunodeficiency virus 1 infection.

    PubMed Central

    Cassani, F; Baffoni, L; Raise, E; Selleri, L; Monti, M; Bonazzi, L; Gritti, F M; Bianchi, F B

    1991-01-01

    Serum samples from 66 seropositive subjects (56 with a history of intravenous drug abuse), including asymptomatic carriers and patients with persistent generalised lymphadenopathy (PGL), AIDS related complex (ARC), and AIDS, were tested by indirect immunofluorescence on rat tissue sections and HEp-2 cells for the presence of antibodies to nuclei, smooth muscle, intermediate filaments (anti-IMF) and microfilaments (anti-MF). Counterimmunoelectrophoresis was also used to detect antibodies to extractable nuclear antigens. Smooth muscle antibodies with the V pattern or antinuclear antibodies, mainly of the speckled type, or anti-IMF, occurred in 35 cases, being widely distributed in all groups. Such an autoantibody response resembles the "viral" autoimmunity described in various infectious diseases and in particular that of non-A, non-B post-transfusion hepatitis. Autoantibodies may be of some prognostic relevance, as the prevalence of smooth muscle antibodies V increased as the disease progressed (asymptomatic carriers 20%, those with PGL 29%, those with ARC 47%, and those with AIDS 63%. In the PGL group autoantibody positivity correlated with the presence of skin anergy. The fact that autoantibodies were more frequently detected in patients with circulating immune complexes suggests that these can contain autoantibodies and the corresponding autoantigens. Images PMID:1671787

  6. Microminiature coaxial cable and methods manufacture

    DOEpatents

    Bongianni, W.L.

    1986-04-08

    A coaxial cable is provided having a ribbon inner conductor surrounded by a dielectric and a circumferential conductor. The coaxial cable may be microminiature comprising a very thin ribbon strip conductor from between 5 to 15 [mu]m thick and from 150 to 200 [mu]m wide, having a surrounding foamed dielectric or parylene applied thereon by a vapor plasma process and an outer conductor of an adhering high conductivity metal vacuum deposited on the dielectric. Alternately the foam dielectric embodiment may have a contiguous parylene coating applied adjacent the inner conductor or the outer conductor or both. Also, the cable may be fabricated by forming a thin ribbon of strip conductive material into an inner conductor, applying thereabout a dielectric by spraying on a solution of polystyrene and polyethylene and then vacuum depositing and adhering high conductivity metal about the dielectric. The cable strength may be increased by adding glass microfilament fibers or glass microballoons to the solution of polystyrene and polyethylene. Further, the outer conductive layer may be applied by electroless deposition in an aqueous solution rather than by vacuum deposition. A thin coating of parylene is preferably applied to the outer conductor to prevent its oxidation and inhibit mechanical abrasion. 2 figs.

  7. Microminiature coaxial cable and methods of manufacture

    DOEpatents

    Bongianni, W.L.

    1983-12-29

    A coaxial cable is provided having a ribbon inner conductor surrounded by a dielectric and a circumferential conductor. The coaxial cable may be microminiature comprising a very thin ribbon strip conductor from between 5 to 15 ..mu..m thick and from 150 to 200 ..mu..m wide, having a surrounding foamed dielectric or parylene applied thereon by a vapor plasma process and an outer conductor of an adhering high conductivity metal vacuum deposited on the dieleectric. Alternately the foam dielectric embodiment may have a contiguous parylene coating applied adjacent the inner conductor or the outer conductor or both. Also, the cable may be fabricated by forming a thin ribbon of strip conductive material into an inner conductor, applying thereabout a dielectric by spraying on a solution of polystyrene and polyethylene and then vacuum depositing and adhering high conductivity metal about the dielectric. The cable strength may be increased by adding glass microfilament fibers or glass microballoons to the solution of polystyrene and polyethylene. Further, the outer conductive layer may be applied by electroless deposition in an aqueous solution rather than by vacuum deposition. A thin coating of parylene is preferably applied to the outer conductor to prevent its oxidation and inhibit mechanical abrasion.

  8. Microminiature coaxial cable and method of manufacture

    DOEpatents

    Bongianni, W.L.

    1989-03-28

    A coaxial cable is provided having a ribbon inner conductor surrounded by a dielectric and a circumferential conductor. The coaxial cable may be microminiature comprising a very thin ribbon strip conductor from between 5 to 15 [mu]m thick and from 150 to 200 [mu]m wide, having a surrounding foamed dielectric or parylene applied thereon by a vapor plasma process and an outer conductor of an adhering high conductivity metal vacuum deposited on the dielectric. Alternately, the foam dielectric embodiment may have a contiguous parylene coating applied adjacent the inner conductor or the outer conductor or both. Also, the cable may be fabricated by forming a thin ribbon of strip conductive material into an inner conductor, applying thereabout a dielectric by spraying on a solution of polystyrene and polyethylene and then vacuum depositing and adhering high conductivity metal about the dielectric. The cable strength may be increased by adding glass microfilament fibers or glass microspheres to the solution of polystyrene and polyethylene. Further, the outer conductive layer may be applied by electroless deposition in an aqueous solution rather than by vacuum deposition. A thin coating of parylene is preferably applied to the outer conductor to prevent its oxidation and inhibit mechanical abrasion. 2 figs.

  9. Microminiature coaxial cable and methods manufacture

    DOEpatents

    Bongianni, Wayne L.

    1986-01-01

    A coaxial cable is provided having a ribbon inner conductor surrounded by a dielectric and a circumferential conductor. The coaxial cable may be microminiature comprising a very thin ribbon strip conductor from between 5 to 15 .mu.m thick and from 150 to 200 .mu.m wide, having a surrounding foamed dielectric or parylene applied thereon by a vapor plasma process and an outer conductor of an adhering high conductivity metal vacuum deposited on the dielectric. Alternately the foam dielectric embodiment may have a contiguous parylene coating applied adjacent the inner conductor or the outer conductor or both. Also, the cable may be fabricated by forming a thin ribbon of strip conductive material into an inner conductor, applying thereabout a dielectric by spraying on a solution of polystyrene and polyethylene and then vacuum depositing and adhering high conductivity metal about the dielectric. The cable strength may be increased by adding glass microfilament fibers or glass microballoons to the solution of polystyrene and polyethylene. Further, the outer conductive layer may be applied by electroless deposition in an aqueous solution rather than by vacuum deposition. A thin coating of parylene is preferably applied to the outer conductor to prevent its oxidation and inhibit mechanical abrasion.

  10. Microminiature coaxial cable and method of manufacture

    DOEpatents

    Bongianni, Wayne L.

    1989-01-01

    A coaxial cable is provided having a ribbon inner conductor surrounded by a dielectric and a circumferential conductor. The coaxial cable may be microminiature comprising a very thin ribbon strip conductor from between 5 to 15 .mu.m thick and from 150 to 200 .mu.m wide, having a surrounding foamed dielectric or parylene applied thereon by a vapor plasma process and an outer conductor of an adhering high conductivity metal vacuum deposited on the dielectric. Alternately, the foam dielectric embodiment may have a contiguous parylene coating applied adjacent the inner conductor or the outer conductor or both. Also, the cable may be fabricated by forming a thin ribbon of strip conductive material into an inner conductor, applying thereabout a dielectric by spraying on a solution of polystyrene and polyethylene and then vacuum depositing and adhering high conductivity metal about the dielectric. The cable strength may be increased by adding glass microfilament fibers or glass microspheres to the solution of polystyrene and polyethylene. Further, the outer conductive layer may be applied by electroless deposition in an aqueous solution rather than by vacuum deposition. A thin coating of parylene is preferably applied to the outer conductor to prevent its oxidation and inhibit mechanical abrasion.

  11. Latrunculin A and Its C-17-O-Carbamates Inhibit Prostate Tumor Cell Invasion and HIF-1 Activation in Breast Tumor Cells⊥

    PubMed Central

    El Sayed, Khalid A.; Shallal, Hassan M.; Khanfar, Mohammad A.; Muralidharan, A.; Awate, Bhushan; Youssef, Diaa T.A.; Liu, Yang; Zhou, Yu-Dong; Nagle, Dale G.; Shah, Girish

    2010-01-01

    The marine-derived macrolides latrunculins A (1) and B, from the Red Sea sponge Negombata magnifica, have been found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety that binds arginine 210 NH. Latrunculin A-17-O-carbamates 2–6 were prepared by reaction with the corresponding isocyanates. Latrunculin A (1) and carbamates 4–6 displayed potent anti-invasive activity against the human highly metastatic human prostate cancer PC-3M cells in a Matrigel™ assay at a concentration range of 50 nM-1 µM. Latrunculin A (1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by three-fold. Carbamates 4 and 5 were two and half and five-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A (1, IC50 6.7 µM) and its 17-O-[N-(benzyl)carbamate (6, IC50 29 µM) suppress hypoxia-induced HIF-1 activation in T47D breast tumor cells. PMID:18298079

  12. Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation [corrected and republished article originally printed in J Cell Biol 1988 May;106(5):1723-34

    PubMed Central

    1988-01-01

    Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation. PMID:3170634

  13. Mechanical behavior in living cells consistent with the tensegrity model

    NASA Technical Reports Server (NTRS)

    Wang, N.; Naruse, K.; Stamenovic, D.; Fredberg, J. J.; Mijailovich, S. M.; Tolic-Norrelykke, I. M.; Polte, T.; Mannix, R.; Ingber, D. E.

    2001-01-01

    Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

  14. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    SciTech Connect

    Brouwer, A.; Knook, D.L.

    1982-10-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA /sup 125/I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA /sup 125/I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA /sup 125/I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA /sup 125/I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA /sup 125/I. The intracellular degradation of CA /sup 125/I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA /sup 125/I occurred within the Kupffer cell lysosomes.

  15. Disruption of the microtubule network alters cellulose deposition and causes major changes in pectin distribution in the cell wall of the green alga, Penium margaritaceum

    PubMed Central

    Domozych, David S.

    2014-01-01

    Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition. PMID:24285826

  16. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  17. Disruption of the Actin Cytoskeleton Results in the Promotion of Gravitropism in Inflorescence Stems and Hypocotyls of Arabidopsis1

    PubMed Central

    Yamamoto, Kazuyoshi; Kiss, John Z.

    2002-01-01

    The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems. PMID:11842170

  18. N-WASP Is Required for Stabilization of Podocyte Foot Processes

    PubMed Central

    Schell, Christoph; Baumhakl, Lisa; Salou, Sarah; Conzelmann, Ann-Christin; Meyer, Charlotte; Helmstädter, Martin; Wrede, Christoph; Grahammer, Florian; Eimer, Stefan; Kerjaschki, Dontscho; Walz, Gerd; Snapper, Scott

    2013-01-01

    Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP–deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP–mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier. PMID:23471198

  19. The Centrosomal Protein C-Nap1 Is Required for Cell Cycle–Regulated Centrosome Cohesion

    PubMed Central

    Mayor, Thibault; Stierhof, York-Dieter; Tanaka, Kayoko; Fry, Andrew M.; Nigg, Erich A.

    2000-01-01

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle–regulated manner. To test this model, we have performed a detailed structure–function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle–regulated structure that mediates centriole–centriole cohesion. PMID:11076968

  20. The Enrichment of Histomonas meleagridis and Its Pathogen-Specific Protein Analysis: A First Step to Shed Light on Its Virulence.

    PubMed

    Pham, Anh Dao Nguyen; Mast, Jan; Magez, Stefan; Goddeeris, Bruno Maria; Carpentier, Sebastien C

    2016-09-01

    Since the discovery of Histomonas meleagridis in 1893, the necessity of isolating pure H. meleagridis has been highlighted over the years in the battle against histomonosis. Insights into the molecular characteristics of this protozoon open possibilities to proper treatment. Axenization of H. meleagridis in vitro cultures cocultured with bacteria has been unsuccessful. Numerous unsuccessful attempts at culturing H. meleagridis axenically have reinforced the assumption that the protozoa had an obligate relationship with certain bacteria originating from the host ceca. Within these perspectives, we enriched H. meleagridis cells from a mono-eukaryotic culture copropagated with host cecal bacteria by flow cytometry. The enrichment of histomonads was confirmed through transmission electron microscopy and two-dimensional gel electrophoresis. For the first time several protein spots were successfully identified. The majority of spots were annotated as cytoskeletal proteins. Actin microfilaments are known to be a key player in cell spreading, cell adhesion, phagocytosis, signal transduction, and several other processes. Together with the identification of superoxide dismutase, the information generated from protein analysis of H. meleagridis may serve as a very first step toward understanding its pathogenesis and virulence.

  1. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms.

    PubMed

    Wieland, T; Faulstich, H

    1978-12-01

    This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.

  2. Fine structure of bacterial adhesion to the epithelial cell membranes of the filiform papillae of tongue and palatine mucosa of rodents: a morphometric, TEM, and HRSEM study.

    PubMed

    Watanabe, Ii-Sei; Ogawa, Koichi; Cury, Diego Pulzatto; Dias, Fernando José; Sosthenes, Marcia Consentino Kronka; Issa, João Paulo Mardegan; Iyomasa, Mamie Mizusaki

    2013-12-01

    The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point-dried, and coated with gold-palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three-dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods. Copyright © 2013 Wiley Periodicals, Inc.

  3. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  4. Translational diffusion of globular proteins in the cytoplasm of cultured muscle cells.

    PubMed Central

    Arrio-Dupont, M; Foucault, G; Vacher, M; Devaux, P F; Cribier, S

    2000-01-01

    Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility. PMID:10653802

  5. Long-distance communication by specialized cellular projections during pigment pattern development and evolution

    PubMed Central

    Eom, Dae Seok; Bain, Emily J; Patterson, Larissa B; Grout, Megan E; Parichy, David M

    2015-01-01

    Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form. DOI: http://dx.doi.org/10.7554/eLife.12401.001 PMID:26701906

  6. The ability of PVX p25 to form RL structures in plant cells is necessary for its function in movement, but not for its suppression of RNA silencing.

    PubMed

    Yan, Fei; Lu, Yuwen; Lin, Lin; Zheng, Hongying; Chen, Jianping

    2012-01-01

    The p25 triple gene block protein of Potato virus X (PVX) is multifunctional, participating in viral movement and acting as a suppressor of RNA silencing. The cell-to-cell movement of PVX is known to depend on the suppression function of p25. GFP-fused p25 accumulates in rod-like (RL) structures with intense fluorescence in cells. By monitoring the location of fluorescence at different times, we have now shown that the RL structure is composed of filaments. P25 mutants without the conditional ability to recover movement function could not form RL structures while the mutants that had the ability did form the structure, suggesting that the ability of p25 to form RL structures is necessary for its function in cell-to-cell movement, but not for its suppressor function. Moreover, chemical inhibition of microfilaments in cells destroyed the formation of the complete RL structure. Additionally, TGBp2 and TGBp3 were recruited into the RL structure, suggesting a relationship between the TGBps in virus movement.

  7. Legume lectins inhibit human parainfluenza virus type 2 infection by interfering with the entry.

    PubMed

    Uematsu, Jun; Koyama, Aoi; Takano, Sayaka; Ura, Yukari; Tanemura, Miho; Kihira, Sahoko; Yamamoto, Hidetaka; Kawano, Mitsuo; Tsurudome, Masato; O'Brien, Myles; Komada, Hiroshi

    2012-07-01

    Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.

  8. Ibuprofen Enhances Recovery from Spinal Cord Injury by Limiting Tissue Loss and Stimulating Axonal Growth

    PubMed Central

    Wang, Xingxing; Budel, Stephane; Baughman, Kenneth; Gould, Grahame; Song, Kang-Ho

    2009-01-01

    Abstract The GTP-binding protein RhoA regulates microfilament dynamics in many cell types and mediates the inhibition of axonal regeneration by myelin and chondroitin sulfate proteoglycans. Unlike most other nonsteroidal anti-inflammatory drugs, ibuprofen suppresses basal RhoA activity (Zhou et al., 2003). A recent report suggested that ibuprofen promotes corticospinal axon regeneration after spinal cord injury (Fu et al., 2007). Here, we confirm that ibuprofen reduces ligand-induced Rho signaling and myelin-induced inhibition of neurite outgrowth in vitro. Following 4 weeks of subcutaneous administration of ibuprofen, beginning 3 days after spinal cord contusion, animals recovered walking function to a greater degree, with twice as many rats achieving a hind limb weight-bearing status. We examined the relative role of tissue sparing, axonal sprouting, and axonal regeneration in the action of ibuprofen. Histologically, ibuprofen-treated animals display an increase in spared tissue without an alteration in astrocytic or microglial reaction. Ibuprofen increases axonal sprouting from serotonergic raphespinal axons, and from rostral corticospinal fibers in the injured spinal cord, but does not permit caudal corticospinal regeneration after spinal contusion. Treatment of mice with complete spinal cord transection demonstrates long-distance raphespinal axon regeneration in the presence of ibuprofen. Thus, administration of ibuprofen improves the recovery of rats from a clinically relevant spinal cord trauma by protecting tissue, stimulating axonal sprouting, and allowing a minor degree of raphespinal regeneration. PMID:19125588

  9. Magnetoreception System in Honeybees (Apis mellifera)

    PubMed Central

    Hsu, Chin-Yuan; Ko, Fu-Yao; Li, Chia-Wei; Fann, Kuni; Lue, Juh-Tzeng

    2007-01-01

    Honeybees (Apis mellifera) undergo iron biomineralization, providing the basis for magnetoreception. We showed earlier the presence of superparamagnetic magnetite in iron granules formed in honeybees, and subscribed to the notion that external magnetic fields may cause expansion or contraction of the superparamagnetic particles in an orientation-specific manner, relaying the signal via cytoskeleton (Hsu and Li 1994). In this study, we established a size-density purification procedure, with which quantitative amount of iron granules was obtained from honey bee trophocytes and characterized; the density of iron granules was determined to be 1.25 g/cm3. While we confirmed the presence of superparamagnetic magnetite in the iron granules, we observed changes in the size of the magnetic granules in the trophycytes upon applying additional magnetic field to the cells. A concomitant release of calcium ion was observed by confocal microscope. This size fluctuation triggered the increase of intracellular Ca+2 , which was inhibited by colchicines and latrunculin B, known to be blockers for microtubule and microfilament syntheses, respectively. The associated cytoskeleton may thus relay the magnetosignal, initiating a neural response. A model for the mechanism of magnetoreception in honeybees is proposed, which may be applicable to most, if not all, magnetotactic organisms. PMID:17460762

  10. Neurofilament dynamics and involvement in neurological disorders.

    PubMed

    Gentil, Benoit J; Tibshirani, Michael; Durham, Heather D

    2015-06-01

    Neurons are extremely polarised cells in which the cytoskeleton, composed of microtubules, microfilaments and neurofilaments, plays a crucial role in maintaining structure and function. Neurofilaments, the 10-nm intermediate filaments of neurons, provide structure and mechanoresistance but also provide a scaffolding for the organization of the nucleus and organelles such as mitochondria and ER. Disruption of neurofilament organization and expression or metabolism of neurofilament proteins is characteristic of certain neurological syndromes including Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth sensorimotor neuropathies and Giant Axonal Neuropathy. Microfluorometric live imaging techniques have been instrumental in revealing the dynamics of neurofilament assembly and transport and their functions in organizing intracellular organelle networks. The insolubility of neurofilament proteins has limited identifying interactors by conventional biochemical techniques but yeast two-hybrid experiments have revealed new roles for oligomeric, nonfilamentous structures including vesicular trafficking. Although having long half-lives, new evidence points to degradation of subunits by the ubiquitin-proteasome system as a mechanism of normal turnover. Although certain E3-ligases ubiquitinating neurofilament proteins have been identified, the overall process of neurofilament degradation is not well understood. We review these mechanisms of neurofilament homeostasis and abnormalities in motor neuron and peripheral nerve disorders. Much remains to discover about the disruption of processes that leads to their pathological aggregation and accumulation and the relevance to pathogenesis. Understanding these mechanisms is crucial for identifying novel therapeutic strategies.

  11. Galectin-3-induced cell spreading and motility relies on distinct signaling mechanisms compared to fibronectin.

    PubMed

    More, Shyam K; Chiplunkar, Shubhada V; Kalraiya, Rajiv D

    2016-05-01

    Secreted galectin-3 often gets incorporated into extracellular matrix and is utilized by cancer cells for spreading, movement, and metastatic dissemination. Here we investigate molecular mechanisms by which galectin-3 brings about these effects and compare it with fibronectin. Imaging of cells spread on fibronectin showed stress fibers throughout cell body, however, galectin-3-induced formation of parallel actin bundles in the lamellipodial region resulting in unique morphological features. FRAP analysis showed that the actin turnover in the lamellipodial region was much higher in cells spread on galectin-3 as compared to that on fibronectin. Rac1 activation is correlated with lamellipodial organization on both the substrates. Activation of Akt and Rac1, the regulators of actin dynamics, show inverse correlation with each other on both galectin-3 and fibronectin. Activation of Erk however, remained similar. Further, inhibition of activation of Akt and Erk inhibited spreading and motility of cells on galectin-3 but not on fibronectin. The results very comprehensively demonstrate distinct signaling pathways that regulate microfilament organization, lamellipodial structures, spreading, and movement of cells plated on galectin-3 as opposed to fibronectin.

  12. Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes.

    PubMed

    Keller, H U; Naef, A; Zimmermann, A

    1984-07-01

    We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.

  13. Columella cells revisited: novel structures, novel properties, and a novel gravisensing model

    NASA Technical Reports Server (NTRS)

    Staehelin, L. A.; Zheng, H. Q.; Yoder, T. L.; Smith, J. D.; Todd, P.

    2000-01-01

    A hundred years of research has not produced a clear understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast statoliths in root cap columella cells into a growth response. Most models postulate that the statoliths interact with microfilaments (MF) to transmit signals to the plasma membrane (or ER), or that sedimentation onto these organelles produces the signals. However, no direct evidence for statolith-MF links has been reported, and no asymmetric structures of columella cells have been identified that might explain how a root turned by 90 degrees knows which side is up. To address these and other questions, we have (1) quantitatively examined the effects of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in high-pressure frozen/freeze-substituted roots; and (3) followed the sedimentation dynamics of statolith movements in reoriented root tips. The findings have led to the formulation of a new model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal network. This network is denser in the ER-devoid central region of the cell than in the ER-rich cell cortex and is coupled to receptors in the plasma membrane. Statolith sedimentation is postulated to disrupt the network and its links to receptors in some regions of the cell cortex, while allowing them to reform in other regions and thereby produce a directional signal.

  14. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro.

    PubMed Central

    Fuki, I V; Kuhn, K M; Lomazov, I R; Rothman, V L; Tuszynski, G P; Iozzo, R V; Swenson, T L; Fisher, E A; Williams, K J

    1997-01-01

    Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo. PMID:9294130

  15. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

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

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region.

  16. 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

  17. [Peculiarities of ultrastructure of excretory system in Bothrioplana semperi (Platyhelminthes, Turbellaria)].

    PubMed

    Kornakova, E E

    2010-01-01

    Ultrastructural study of morphology of cirtocytes and excretory channels was performed in the free living turbellaria Bothrioplana semperi (Turbellaria, Seriata). It has been shown that cirtocytes of this species are formed by two cells--the terminal and the proximal cells of the channel. The fan is composed of two rod rows. The external row goes out from the terminal cell, the internal one is a derivate of the channel proximal cell. Inside each rod of the external row there runs a bundle of microfilaments; it originates in the cytoplasm of the channel proximal cell distal to bases of the external rods. On the internal rod membranes there are noted small electrondense granules disposed separately or fused in the solid layer continuing into a dense "membrane" connecting rods of the external and internal rows. Rare internal leptotrichiae go out from the cirtocyte cavity bottom. External leptotrichiae are absent. The septate desmosome at the level of the terminal cell is absent, but is present in the channel proximal cell at the level of terminal of cilia. The apical surface of the channel cell carries rare short microvilli. The basement membrane of cells of excretory channels forms deep invaginations almost reaching the apical membrane. Epithelium of excretory channels is deprived of cilia. Ultrastructure of cirtocytes and excretory channels of B. semperi is similar to those in representatives of the suborder Proseriata (Seriata). The significance of ultrastructure of the Proseriata cirtocytes, especially of the order of formation of versh, for construction of phylogeny of Platyhelminthes is discussed.

  18. Myosins as fundamental components during tumorigenesis: diverse and indispensable

    PubMed Central

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-01

    Myosin is a kind of actin-based motor protein. As the crucial functions of myosin during tumorigenesis have become increasingly apparent, the profile of myosin in the field of cancer research has also been growing. Eighteen distinct classes of myosins have been discovered in the past twenty years and constitute a diverse superfamily. Various myosins share similar structures. They all convert energy from ATP hydrolysis to exert mechanical stress upon interactions with microfilaments. Ongoing research is increasingly suggesting that at least seven kinds of myosins participate in the formation and development of cancer. Myosins play essential roles in cytokinesis failure, chromosomal and centrosomal amplification, multipolar spindle formation and DNA microsatellite instability. These are all prerequisites of tumor formation. Subsequently, myosins activate various processes of tumor invasion and metastasis development including cell migration, adhesion, protrusion formation, loss of cell polarity and suppression of apoptosis. In this review, we summarize the current understanding of the roles of myosins during tumorigenesis and discuss the factors and mechanisms which may regulate myosins in tumor progression. Furthermore, we put forward a completely new concept of “chromomyosin” to demonstrate the pivotal functions of myosins during karyokinesis and how this acts to optimize the functions of the members of the myosin superfamily. PMID:27121062

  19. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  20. [Participation of the cytoskeleton in the physiology of the endometrium].

    PubMed

    Durán Reyes, G; Hicks, J J

    1995-11-01

    The cytoskeleton in the endometrium, takes part not only in all the mechanic functions of the cell, but because of movement and location of healthy organelles and proteins, it also takes part in the metabolism. The endometrial epithelium, because of its morphology and its supposed cellular homogeneity, has been studied more than the stroma. It is known that intermedium filaments show a characteristic pattern of typical distribution and expression of the cellular type. During pregnancy and pseudopregnancy, in the apical region of the epithelial cells, both, luminal and glandular, there is an abundance of keratin in the basolateral region; while the vimentin is abundant only in the luminal epithelial cells and it increases in the implantation day. In humans and rats, the desmin only expresses during the decidual response. It is considered that intermedium filaments have a role in the polarity changes of the membrane. The microfilaments (MF) are related with the regulation of the cellular morphology and movement. In the luminal epithelium the MF play a role in the transformations of the uterine surface like the microvilli. The microtubules in the endometrium and other organs play an important role in the organelles position like lysosomes, mitochondria and Golgi complex. Also it is proved that take part in the DNA synthesis, because colchicine drug inhibits it.

  1. Cell alterations but no DNA strand breaks induced in vitro by cylindrospermopsin in CHO K1 cells.

    PubMed

    Fessard, Valérie; Bernard, Cécile

    2003-10-01

    Cylindrospermopsin has been involved in some cyanobacterial blooms associated with animal and human intoxications in different countries. Liver is the main target organ even though thymus and kidney are also affected. Its toxic effect has been shown to be induced by protein synthesis inhibition. However, further research about its toxicological potential is required, as revealed by the U.S. Unregulated Contaminant Monitoring Regulation (US UCMR) meeting in 2001. Induction of DNA damage by cylindrospermopsin has been reported by some authors either by a direct effect on DNA or by an indirect effect on associated macromolecules. This study focused on evaluating its in vitro genotoxic potential using the comet assay coupled to various cell alteration measurements. No DNA damage was detected by the alkaline comet assay on Chinese hamster ovary (CHO) K1 cells after 24 h of treatment with cylindrospermopsin concentrations of 0.5 and 1 microg mL(-1). However, inhibition of cell growth was noticed as well as cell blebbing and rounding. These morphological effects were linked to cytoskeletal reorganization (mainly microfilaments) but not to apoptosis. This study concluded that cylindrospermopsin does not obviously react directly with DNA in CHO K1 cells. But the hypothesis of its metabolization into a genotoxic product must be explored further.

  2. Pixuna virus modifies host cell cytoskeleton to secure infection.

    PubMed

    Gil, Pedro Ignacio; Albrieu-Llinás, Guillermo; Mlewski, Estela Cecilia; Monetti, Marina; Fozzatti, Laura; Cuffini, Cecilia; Fernández Romero, José; Kunda, Patricia; Paglini, María Gabriela

    2017-07-18

    Pixuna virus (PIXV) is an enzootic member of the Venezuelan Equine Encephalitis Virus complex and belongs to the New World cluster of alphaviruses. Herein we explore the role of the cellular cytoskeleton during PIXV replication. We first identified that PIXV undergoes an eclipse phase consisting of 4 h followed by 20 h of an exponential phase in Vero cells. The infected cells showed morphological changes due to structural modifications in actin microfilaments (MFs) and microtubules (MTs). Cytoskeleton-binding agents, that alter the architecture and dynamics of MFs and MTs, were used to study the role of cytoskeleton on PIXV replication. The virus production was significantly affected (p < 0.05) after treatment with paclitaxel or nocodazole due to changes in the MTs network. Interestingly, disassembly of MFs with cytochalasin D, at early stage of PIXV replication cycle, significantly increased the virus yields in the extracellular medium (p < 0.005). Furthermore, the stabilization of actin network with jasplakinolide had no effect on virus yields. Our results demonstrate that PIXV relies not only on intact MTs for the efficient production of virus, but also on a dynamic actin network during the early steps of viral replication.

  3. Smooth Brucella strains invade and replicate in human lung epithelial cells without inducing cell death.

    PubMed

    Ferrero, Mariana C; Fossati, Carlos A; Baldi, Pablo C

    2009-04-01

    Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar(A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5-3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity. (c) 2009 Elsevier Masson SAS. All rights reserved.

  4. Tubulin cytoskeleton in elongation zone of Arabidopsis root is affected by clinorotation

    NASA Astrophysics Data System (ADS)

    Shevchenko, G.; Kalinina, Ya.; Kordyum, E.

    Our aim is to find out how clinorotation influences root growth For this purpose we followed the dynamics of tubulin cytoskeleton cortical and endoplasmic microtubules in cells from elongation zone of Arabidopsis roots transfected with GFP-MAP4 3 day old seedlings In distal part of elongation zone in epidermal cells mainly distinct endoplasmic microtubules were observed Prominent cortical microtubules start to be evident in cells in central elongation zone Under clinorotation clusters formed by MAP4 appear in all parts of elongation zone evidencing that microtubule arrangement is somehow distorted there Application of cytochalasin D which disrupts proper functioning of actin cytoskeleton in controls affected mainly the endoplasmic microtubules in cells with isotropic growth where MAP4 was clustered Under clinorotation disruption of actin cytoskeleton by cytochalasin D caused appearance of MAP4 clusters in cells growing anisotropically In those cells cortical microtubules are affected as well as endoplasmic Due to the fact that cortical microtubules are responsible for ordered growth of plant cell and are arranged into a robust structure change of their organization under clinorotation could impact cell growth This proves that cells in elongation zone switching their growth mode from isotropic to anisotropic are rather sensitive to altered gravity The fact that more severe distortion of cortical microtubules was noted in cells with damaged actin microfilaments proves mutually related functioning of actin and tubulin cytoskeletons under clinorotation

  5. Graviresponses in fungi

    NASA Astrophysics Data System (ADS)

    Moore, D.

    Although the orientation of mycelial hyphal growth is usually independent of the gravity vector, individual specialised hyphae can show response to gravity. This is exemplified by the sporangiophore of Phycomyces, but the most striking gravitropic reactions occur in mushroom fruit bodies. During the course of development of a mushroom different tropisms predominate at different times; the young fruit body primordium is positively phototropic, but negative gravitropism later predominates. The switch between tropisms has been associated with meiosis. The spore-bearing tissue is positively gravitropic and responds independently of the stem. Bracket polypores do not show tropisms but exhibit gravimorphogenetic responses: disturbance leads to renewal of growth producing an entirely new fruiting structure. Indications from both clinostat and space flown experiments are that the basic form of the mushroom (overall tissue arrangement of stem, cap, gills, hymenium, veil) is established independently of the gravity vector although maturation, and especially commitment to the meiosis-sporulation pathway, requires the normal gravity vector. The gravity perception mechanism is difficult to identify. The latest results suggest that disturbance of cytoskeletal microfilaments is involved in perception (with nuclei possibly being used as statoliths), and Ca^2+-mediated signal transduction may be involved in directing growth differentials.

  6. THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility.

    PubMed

    Whippo, Craig W; Khurana, Parul; Davis, Phillip A; DeBlasio, Stacy L; DeSloover, Daniel; Staiger, Christopher J; Hangarter, Roger P

    2011-01-11

    Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors and is transduced to the actin cytoskeleton. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane, but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilla development as a regulator of actin architecture. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.

  7. Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor.

    PubMed

    Sato, Y; Wada, M; Kadota, A

    2001-01-01

    Light induced chloroplast movement has been studied as a model system for photoreception and actin microfilament (MF)-based intracellular motilities in plants. Chloroplast photo-accumulation and -avoidance movement is mediated by phytochrome as well as blue light (BL) receptor in the moss Physcomitrella patens. Here we report the discovery of an involvement of a microtubule (MT)-based system in addition to an MF-based system in photorelocation of chloroplasts in this moss. In the dark, MTs provided tracks for rapid movement of chloroplasts in a longitudinal direction and MFs contributed the tracks for slow movement in any direction. We found that phytochrome responses utilized only the MT-based system, while BL responses had an alternative way of moving, either along MTs or MFs. MT-based systems were mediated by both photoreceptors, but chloroplasts showed movements with different velocity and pattern between them. No apparent difference in the behavior of chloroplast movement between the accumulation and avoidance movement was detected in phytochrome responses or BL responses, except for the direction of the movement. The results presented here demonstrate that chloroplasts use both MTs and MFs for motility and that phytochrome and a BL receptor control directional photo-movement of chloroplasts through the differential regulation of these motile systems.

  8. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

    PubMed Central

    Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D.

    2016-01-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  9. A stromal myoid cell line provokes thymic erythropoiesis between 16th to 20th weeks of intrauterine life.

    PubMed

    Tamiolakis, D; Venizelos, J; Kotini, A; Karamanidis, D; Boglou, P; Papadopoulos, N

    2004-02-01

    The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular microenvironment. The thymic RE cells are functionally specialised based on their intrathymic location and this differentiation is modulated by various interaction signals of differentiating thymocytes and other non lymphatic haematopoietic stem cells. To study the role of another cell line in fetal thymic haematopoietic proliferation and differentiation in different stages of development: the stromal myoid cells. Fifteen cases of fetal thymic specimens (4th to 8th weeks: five cases 16th to 20th weeks: five cases and 28th to 32nd weeks: five cases respectively) were studied. Tissue paraffin samples were stained immunohistochemically using (i) a monoclonal antibody recognising alpha-smooth muscle actin, a contractile microfilament expressed exclusively by smooth muscle cells, myofibroblasts and related cells, (ii) a monoclonal antibody glycophorin C recognising the erythropoietic cells. Histology-Embryology Department of Democritus University of Thrace (Alexandroupolis) over ten year period (1991-2001). The number of alpha-smooth muscle actin-positive cells significantly increased during the late second and third trimester of gestation. In the above period a relevant increase in the number of glycophorin C positive cells were observed. Our data suggest that a myoid cell line is involved in the formation of an appropriate microenvironment for homing and proliferation of erythropoietic cells.

  10. PHBV/PAM scaffolds with local oriented structure through UV polymerization for tissue engineering.

    PubMed

    Ke, Yu; Wu, Gang; Wang, Yingjun

    2014-01-01

    Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100  μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

  11. The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize

    NASA Technical Reports Server (NTRS)

    Blancaflor, E. B.; Hasenstein, K. H.

    1997-01-01

    To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

  12. Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

    NASA Technical Reports Server (NTRS)

    Chen, J.; Fabry, B.; Schiffrin, E. L.; Wang, N.; Ingber, D. E. (Principal Investigator)

    2001-01-01

    A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

  13. Resistance of African Green Monkey Kidney Cell Lines to Actinomycin D: Drug Uptake in 37 RC Cells After Persistent Inhibition of Transcription

    PubMed Central

    Benedetto, Arrigo; Cassone, Antonio; Delfini, Carlo

    1979-01-01

    37 RC cells, a cultured line derived from African green monkey kidneys, survived long treatments with actinomycin D (AMD; 0.1 to 0.5 μg/ml) under strong inhibition of ribonucleic acid synthesis and blocking of cell division. One aspect of the complex cellular response to this treatment was a progressive lowering of the influx rate of AMD and, consequently, of its endocellular concentration, leading to a late resurgence of transcription. Overall protein synthesis decreased in AMD-treated cells, but more of the residual protein was exported to the cell surface, a fact associated with the development of numerous strands of endoplasmic reticulum and Golgi bodies in the cytoplasm. The lowering of AMD influx during the treatment was not simply due to the decay of protein synthesis, and there was no evidence for a carrier-mediated transport of the drug. It was paralleled by, but seemingly not related to, modifications in cellular microtubules and microfilaments. The rate of AMD influx was restored to levels comparable to those of untreated cells by short exposure to ethylenediaminetetraacetic acid and trypsin. It is concluded that the changes in plasma membrane of 37 RC cells, creating an obstacle to the influx of AMD after long treatment with this drug, probably consist of an accumulation and/or a different distribution of glycoproteins or other surface components on the cell surface. Images PMID:106777

  14. Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

    PubMed Central

    2013-01-01

    Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

  15. Ultrastructural data on the spore of Myxobolus maculatus n. sp. (phylum Myxozoa), parasite from the Amazonian fish Metynnis maculatus (Teleostei).

    PubMed

    Casal, G; Matos, E; Azevedo, C

    2002-08-29

    Light and electron microscopy studies of a myxosporean, parasitic in the intertubular interstitial tissue of the kidney of the freshwater teleost fish Metynnis maculatus Kner, 1860 (Characidae) from the lower Amazon River (Brazil), are described. We observed polysporic histozoic plasmodia delimited by a double membrane and with several pinocytic channels and containing several life cycle stages, including mature spores. The spore body was of pyriform shape and was 21.0 microm long, 8.9 microm wide and 7.5 microm thick. Elongated-pyriform polar capsules were of equal size (12.7 x 3.2 microm) and contained a polar filament with 14 or 15 coils. The spore features fit those of the genus Myxobolus. Densification of the capsular primordium matrix, which increased in density from the inner core outwards, differentiating at the periphery into small microfilaments measuring 45 nm each, and tubuli arranged in aggregates and dispersed within the capsular matrix of the mature spores, are described. Based on the morphological differences and specificity of the host, we propose the creation of a new species named Myxobolus maculatus n. sp.

  16. Fungal Invasion of Normally Non-Phagocytic Host Cells

    PubMed Central

    Filler, Scott G; Sheppard, Donald C

    2006-01-01

    Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research. PMID:17196036

  17. Differential Effects of Caldesmon on the Intermediate Conformational States of Polymerizing Actin*

    PubMed Central

    Huang, Renjian; Grabarek, Zenon; Wang, Chih-Lueh Albert

    2010-01-01

    The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton. PMID:19889635

  18. Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa.

    PubMed

    Vickerman, Keith; Appleton, Paul L; Clarke, Ken J; Moreira, David

    2005-11-01

    A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.

  19. Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: Formation of intercellular membrane bridges

    SciTech Connect

    Jung, Se-Hui; Park, Jin-Young; Joo, Jung-Hoon; Kim, Young-Myeong; Ha, Kwon-Soo

    2011-07-15

    Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainly comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.

  20. Diameters of microtubules change during rotation of the lipotubuloids of Ornithogalum umbellatum stipule epidermis as a result of varying protofilament monomers sizes and distance between them.

    PubMed

    Kwiatkowska, Maria; Stepiński, Dariusz; Popłońska, Katarzyna

    2009-12-01

    Microtubules in lipotubuloids of the Ornithogalum umbellatum stipule epidermis cells change their diameters depending on the motion of the cytoplasmic domains rich in microtubules and lipid bodies. Microtubules fixed during rotary and progressive motion of the lipotubuloids composed of the same number of protofilaments fall into two populations - wide (43-58 nm) and narrow (24-39 nm) in size. Following blockage of the motion with 2,4-dinitrophenol (DNP), the range of this diversity is smaller, microtubules become a medium-sized population (34-48 nm). When DNP is removed and the motion reactivated, 2 populations of microtubules reappear. Analysis of the structure of the microtubule wall revealed that changes in the microtubule diameters resulted from varying distances between the adjacent protofilaments, and stretching and compression of tubulin subunits in the protofilaments. A supposition has been put forward that the changes in the sizes of O. umbellatum microtubule diameters: 1) are connected with the interactions between microtubules and actin microfilaments lying along these microtubules; 2) can be the driving force of the rotary motion of lipotubuloids.

  1. Nicotine alters bovine oocyte meiosis and affects subsequent embryonic development.

    PubMed

    Liu, Ying; Li, Guang-Peng; White, Kenneth L; Rickords, Lee F; Sessions, Benjamin R; Aston, Kenneth I; Bunch, Thomas D

    2007-11-01

    The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development.

  2. Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Taylor, M.; Sommer, L.; Levine, H.; Anderson, K.; Runco, M.; Kemp, R.

    1999-01-01

    Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions. Copyright 1999 Academic Press.

  3. Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

    PubMed Central

    Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B.; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

    2013-01-01

    Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition. PMID:24098701

  4. Fibroblast growth factor-1-inducible gene FR-17 encodes a nonmuscle alpha-actinin isoform.

    PubMed

    Hsu, D K; Guo, Y; Alberts, G F; Peifley, K A; Winkles, J A

    1996-05-01

    Polypeptide growth factor binding to cell surface receptors activates a cytoplasmic signaling cascade that ultimately promotes the expression of specific nuclear genes. As an approach to investigate the molecular mechanism of fibroblast growth factor (FGF)-1 mitogenic signaling, we have begun to identify and characterize FGF-1-inducible genes in murine NIH 3T3 cells. Here we report that one of these genes, termed FGF-regulated (FR)-17, is predicted to encode a nonmuscle isoform of alpha-actinin, an actin cross-linking protein found along microfilaments and in focal adhesion plaques. FGF-1 induction of alpha-actinin mRNA expression is first detectable at 2 h after mitogen addition and is dependent on the novo RNA and protein synthesis. Maximal alpha-actinin mRNA expression, corresponding to an approximately nineteenfold level of induction, is present after 12 h of FGF-1 stimulation. Western blot analysis indicated that FGF-1-stimulated cells also produce an increased amount of alpha-actinin protein. The FGF-1-related mitogen FGF-2, calf serum, several of the polypeptide growth factors present in serum, and the tumor promoter phorbol myristate acetate can also induce alpha-actinin mRNA expression. Finally, nonmuscle alpha-actinin mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in adult mouse intestine and kidney. These results indicate that nonmuscle alpha-actinin is a serum-, polypeptide growth factor-, and tumor promoter-inducible gene in mouse fibroblasts.

  5. A dynamic podosome-like structure of epithelial cells.

    PubMed

    Spinardi, Laura; Rietdorf, Jens; Nitsch, Lucio; Bono, Maria; Tacchetti, Carlo; Way, Michael; Marchisio, Pier Carlo

    2004-05-01

    Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

  6. In vitro toxicity evaluation of 25-nm anatase TiO2 nanoparticles in immortalized keratinocyte cells.

    PubMed

    Chan, Jin; Ying, Tang; Guang, Yang F; Lin, Li X; Kai, Tang; Fang, Zhang Y; Ting, Ye X; Xing, Lin F; Ji, Yang Y

    2011-12-01

    Titanium dioxide (TiO(2)) nanoparticles (NPs) are massively fabricated and widely used in daily life, and thus potential risk has been posed to human health. However, the mechanism of the interaction between TiO(2) NPs and cells is still unclear. In this study, the interaction of anatase TiO(2) NPs with HaCaT cells is studied in vitro with multi-techniques. The TiO(2) NPs not only insert into cells through endocytic pathway but also penetrate into the cell. The TiO(2) NPs could produce reactive oxygen species (ROS) after dispersion spontaneously. Furthermore, the interaction between TiO(2) NPs and cellular components might also generate ROS. The ROS generation could lead to cellular toxicity if the level of ROS production overwhelms the antioxidant defense. Cytoskeletal components, particularly the microfilaments and microtubules, cause modifications upon exposure to TiO(2) NPs. With all results, the toxicological effects of TiO(2) NPs on HaCaT cell can be simplified into six events.

  7. [Relation of ultrastructural changes of articular cartilage and the arthroscopic classification in osteoarthritic knee].

    PubMed

    Chai, B F

    1992-01-01

    This paper reported the ultrastructural changes found in the diseased articular cartilages of 43 osteoarthritic knee joints, which were assessed according to the "Arthroscopic classification of the articular cartilage". The electron microscopic findings and the arthroscopic classification of the articular lesions were correlated. The lesioned articular cartilage revealed two categories of pathological changes. 1. The changes on the part of the articular chondrocytes comprised (1) The nucleus showed pyknosis and karyorrhexis. (2) The cytoplasm exhibited fat droplets, glycogen granules, and/or microfilaments. Lysosomes also emerged frequently. The mitochondria swelled and the rough-surfaced endoplasmic reticulum dilated and became vesiculated. At the same time there was detachment of cell processes or of the cytoplasmic membrane. The chondrocyte underwent necrosis, contracted and eventually disintegrated into lipid debris. These changes increased in extent and degree with the lesion and the severity went parallel with the sequence of the "Arthroscopic stage classification". 2. The changes on the part of the matrix included appearance of electron-dense lipid debris and numerous, coarse and banded collagen fibrils. They resided both in the pericellular matrix and in the general matrix. Sometimes fibroblast-like cells made their appearance in the matrix. These cells also revealed degenerative changes. All these changes went parallel with the sequence of the "Arthroscopic grade classification".

  8. 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.

  9. Nonspecific cytotoxic cells in fish (Ictalurus punctatus). V. Metabolic requirements of lysis.

    PubMed

    Carlson, R L; Evans, D L; Graves, S S

    1985-01-01

    The mechanisms of lysis of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus) were studied by determining the effects of various inhibitors of cellular metabolism on cytolysis of NC-37 human lymphoma target cells. Inhibition of NCC-mediated lysis by dinitrophenol (DNP) and sodium azide (NaN3) indicated a requirement for cellular energy metabolism. Cytochalasin B, an inhibitor of microfilaments, and monensin, an inhibitor of cellular secretion, both prevented lysis by NCC. Three microtubule inhibitors, vinblastine sulfate, colchicine, and demecolcine, all inhibited target cell lysis. Two divalent cation chelators, EDTA and EGTA, blocked NCC activity. Elimination of both Ca2+ and Mg2+ by EDTA prevented target cell binding and killing. Selective removal of Ca2+ by EGTA prevented killing but did not block target cell binding. These results indicated that nonspecific cytotoxicity in fish is an active process which requires cell movement and an intact secretory apparatus. The mechanisms of cytolysis by NCC were found (except for the requirement of microtubules) to be analogous to those of mammalian NK cells. Combined with morphological studies, these data strongly suggest that a phylogenetic relationship exists between these effector cells.

  10. Morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants.

    PubMed

    Cooley-Andrade, O; Connor, D E; Ma, D D F; Weisel, J W; Parsi, K

    2016-04-01

    To investigate morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants sodium tetradecyl sulfate and polidocanol. Samples of whole blood, isolated leukocytes, platelets, endothelial cells, and fibroblasts were incubated with varying concentrations of sclerosants. Whole blood smears were stained with Giemsa and examined by light and bright field microscopy. Phalloidin and Hoechst stains were used to analyze cytoplasmic and nuclear morphology by fluorescence microscopy. Endothelial cell and fibroblasts were analyzed by live cell imaging. Higher concentrations of sclerosants induced cell lysis. Morphological changes in intact cells were observed at sublytic concentrations of detergents. Low concentration sodium tetradecyl sulfate induced erythrocyte acanthocytosis and macrocytosis, while polidocanol induced Rouleaux formation and increased the population of target cells and stomatocytes. Leukocytes showed swelling, blebbing, vacuolation, and nuclear degradation following exposure to sodium tetradecyl sulfate, while polidocanol induced pseudopodia formation, chromatin condensation, and fragmentation. Platelets exhibited pseudopodia with sodium tetradecyl sulfate and a "fried egg" appearance with polidocanol. Exposure to sodium tetradecyl sulfate resulted in size shrinkage in both endothelial cell and fibroblasts, while endothelial cell developed distinct spindle morphology. Polidocanol induced cytoplasmic microfilament bundles in both endothelial cell and fibroblasts. Patchy chromatin condensation was observed following exposure of fibroblasts to either agent. Detergent sclerosants are biologically active at sublytic concentrations. The observed morphological changes are consistent with cell activation, apoptosis, and oncosis. The cellular response is concentration dependent, cell-specific, and sclerosant specific. © The Author(s) 2015.

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

    PubMed

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

    2006-06-01

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

  12. Autophagy, apoptosis and organelle features during cell exposure to cadmium.

    PubMed

    Vergilio, Cristiane dos Santos; de Melo, Edésio José Tenório

    2013-08-01

    Cadmium (Cd) induces several effects in different tissues, but our knowledge of the toxic effects on organelles is insufficient. To observe the progression of Cd effects on organelle structure and function, HuH-7 cells (human hepatic carcinoma cell line) were exposed to CdCl2 in increasing concentrations (1 microM - 20 microM) and exposure times (2 h - 24 h). During Cd treatment, the cells exhibited a progressive decrease in viability that was both time- and dose-dependent. Cd treated cells displayed progressive morphological changes that included cytoplasm retraction and nuclear condensation preceding a total loss of cell adhesion. Treatment with 10 microM for 12 h led to irreversible damages. Before these drastic and irreparable damages, treated cells (5 microM for 12 h) presented a progressive loss of mitochondrial function and cytoplasm acidification as well as dysfunction and disorganization of microfilaments and endoplasmic reticulum. These damages led to the induction of apoptotic events and an increase in autophagic bodies in the cytoplasm. These results revealed that Cd affects multiple intra-cellular targets that induce alterations in the mitochondria, cytoskeleton, endoplasmic reticulum and acidic compartments, ultimately culminating in cell death via apoptotic and autophagic pathways.

  13. Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

    PubMed Central

    Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

    1989-01-01

    The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes. Images PMID:2545625

  14. [Contractile proteins in chemical signal transduction in plant microspores].

    PubMed

    Roshchina, V V

    2005-01-01

    Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active forms of oxygen hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of amaryllis Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of thus treated microspores demonstrated suppressed development, particularly, for cytochalasin B treatment. At the same time, an increased typical blue fluorescence of certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could reside was observed. In contrast to anticontractile agents, dopamine, serotonin B, and the peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or the peroxides decreased the germination rate. Involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.

  15. Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Ye, Dongmei; Ma, Thomas Y; Said, Hamid M

    2007-11-01

    Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

  16. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

    PubMed

    Finka, Andrija; Saidi, Younousse; Goloubinoff, Pierre; Neuhaus, Jean-Marc; Zrÿd, Jean-Pierre; Schaefer, Didier G

    2008-10-01

    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

  17. Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens.

    PubMed

    Menand, Benoît; Calder, Grant; Dolan, Liam

    2007-01-01

    Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.

  18. Nanosurface design of dental implants for improved cell growth and function

    NASA Astrophysics Data System (ADS)

    Pan, Hsu-An; Hung, Yao-Ching; Chiou, Jin-Chern; Tai, Shih-Ming; Chen, Hsin-Hung; Huang, G. Steven

    2012-08-01

    A strategy was proposed for the topological design of dental implants based on an in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 to 200 nm. MG63 osteoblasts were seeded on nanodot arrays and cultured for 3 days. Cell number, percentage undergoing apoptotic-like cell death, cell adhesion and cytoskeletal organization were evaluated. Nanodots with a diameter of approximately 50 nm enhanced cell number by 44%, minimized apoptotic-like cell death to 2.7%, promoted a 30% increase in microfilament bundles and maximized cell adhesion with a 73% increase in focal adhesions. An enhancement of about 50% in mineralization was observed, determined by von Kossa staining and by Alizarin Red S staining. Therefore, we provide a complete range of nanosurfaces for growing osteoblasts to discriminate their nanoscale environment. Nanodot arrays present an opportunity to positively and negatively modulate cell behavior and maturation. Our results suggest a topological approach which is beneficial for the design of dental implants.

  19. Reciprocal Interactions between Cell Adhesion Molecules of the Immunoglobulin Superfamily and the Cytoskeleton in Neurons.

    PubMed

    Leshchyns'ka, Iryna; Sytnyk, Vladimir

    2016-01-01

    Cell adhesion molecules of the immunoglobulin superfamily (IgSF) including the neural cell adhesion molecule (NCAM) and members of the L1 family of neuronal cell adhesion molecules play important functions in the developing nervous system by regulating formation, growth and branching of neurites, and establishment of the synaptic contacts between neurons. In the mature brain, members of IgSF regulate synapse composition, function, and plasticity required for learning and memory. The intracellular domains of IgSF cell adhesion molecules interact with the components of the cytoskeleton including the submembrane actin-spectrin meshwork, actin microfilaments, and microtubules. In this review, we summarize current data indicating that interactions between IgSF cell adhesion molecules and the cytoskeleton are reciprocal, and that while IgSF cell adhesion molecules regulate the assembly of the cytoskeleton, the cytoskeleton plays an important role in regulation of the functions of IgSF cell adhesion molecules. Reciprocal interactions between NCAM and L1 family members and the cytoskeleton and their role in neuronal differentiation and synapse formation are discussed in detail.

  20. Expression of the proteoglycan syndecan-4 and the mechanism by which it mediates stress fiber formation in folliculostellate cells in the rat anterior pituitary gland.

    PubMed

    Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Tsukada, Takehiro; Ly, Floren; Kikuchi, Motoshi; Yashiro, Takashi

    2012-08-01

    Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.

  1. Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

    NASA Technical Reports Server (NTRS)

    Schatten, Heide

    1996-01-01

    The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

  2. Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique.

    PubMed

    Knight, M M; Idowu, B D; Lee, D A; Bader, D L

    2001-05-01

    This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.

  3. Nanochips of Tantalum Oxide Nanodots as artificial-microenvironments for monitoring Ovarian cancer progressiveness

    PubMed Central

    Dhawan, Udesh; Wang, Ssu-Meng; Chu, Ying Hao; Huang, Guewha S.; Lin, Yan Ren; Hung, Yao Ching; Chen, Wen Liang

    2016-01-01

    Nanotopography modulates cell characteristics and cell behavior. Nanotopological cues can be exploited to investigate the in-vivo modulation of cell characteristics by the cellular microenvironment. However, the studies explaining the modulation of tumor cell characteristics and identifying the transition step in cancer progressiveness are scarce. Here, we engineered nanochips comprising of Tantalum oxide nanodot arrays of 10, 50, 100 and 200 nm as artificial microenvironments to study the modulation of cancer cell behavior. Clinical samples of different types of Ovarian cancer at different stages were obtained, primary cultures were established and then seeded on different nanochips. Immunofluorescence (IF) was performed to compare the morphologies and cell characteristics. Indices corresponding to cell characteristics were defined. A statistical comparison of the cell characteristics in response to the nanochips was performed. The cells displayed differential growth parameters. Morphology, Viability, focal adhesions, microfilament bundles and cell area were modulated by the nanochips which can be used as a measure to study the cancer progressiveness. The ease of fabrication of nanochips ensures mass-production. The ability of the nanochips to act as artificial microenvironments and modulate cell behavior may lead to further prospects in the markerless monitoring of the progressiveness and ultimately, improving the prognosis of Ovarian cancer. PMID:27534915

  4. Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity.

    PubMed

    Lopes Bezerra, Leila M; Filler, Scott G

    2004-03-15

    Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

  5. RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

    PubMed

    Miranda-Rodríguez, Jerónimo Roberto; Salas-Vidal, Enrique; Lomelí, Hilda; Zurita, Mario; Schnabel, Denhi

    2017-01-01

    Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

  6. LIGHT-INDUCED OIL GLOBULE MIGRATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE).

    PubMed

    Peled, Ehud; Pick, Uri; Zarka, Aliza; Shimoni, Eyal; Leu, Stefan; Boussiba, Sammy

    2012-10-01

    Astaxanthin-rich oil globules in Haematococcus pluvialis display rapid light-induced peripheral migration that is unique to this organism and serves to protect the photosynthetic system from excessive light. We observed rapid light-induced peripheral migration that is associated with chlorophyll fluorescence quenching, whereas the recovery was slow. A simple assay to follow globule migration, based on chlorophyll fluorescence level has been developed. Globule migration was induced by high intensity blue light, but not by high intensity red light. The electron transport inhibitor dichlorophenyl-dimethylurea did not inhibit globule migration, whereas the quinone analog (dibromo-methyl-isopropylbenzoquinone), induced globule migration even at low light. Actin microfilament-directed toxins, such as cytochalasin B and latrunculin A, inhibited the light-induced globule migration, whereas toxins against microtubules were ineffective. Electron microscopic (EM) imaging confirmed the cytoplasmic localization and peripheral migration of globules upon exposure to very high light (VHL). Scanning EM of freeze-fractured cells also revealed globules within cytoplasmic bridges traversing the chloroplast, presumably representing the pathway of migration. Close alignments of globules with endoplasmic reticulum (ER) membranes were also observed following VHL illumination. We propose that light-induced globule migration is regulated by the redox state of the photosynthetic electron transport system. Possible mechanisms of actin-based globule migration are discussed.

  7. Glycolytic inhibitors 2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration.

    PubMed

    Feng, Xiaolan; Wang, Pan; Liu, Quanhong; Zhang, Ting; Mai, Bingjie; Wang, Xiaobing

    2015-06-01

    Most cancer cells have the specially increased glycolytic phenotype, which makes this pathway become an attractive therapeutic target. Although glycolytic inhibitor 2-deoxyglucose (2-DG) has been demonstrated to potentiate the cytotoxicity of photodynamic therapy (PDT), the impacts on cell migration after the combined treatment has never been reported yet. The present study aimed to analyze the influence of glycolytic inhibitors 2-DG and 3-bromopyruvate (3-BP) combined with Ce6-PDT on cell motility of Triple Negative Breast Cancer MDA-MB-231 cells. As determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium-bromide-Tetraz-olium (MTT) assay, more decreased cell viability was observed in 2-DG + PDT and 3-BP + PDT groups when compared with either monotherapy. Under optimal conditions, synergistic potentiation on cell membrane destruction and the decline of cell adhesion and cells migratory ability were observed in both 2-DG + PDT and 3-BP + PDT by electron microscope observation (SEM), wound healing and trans-well assays. Besides, serious microfilament network collapses as well as impairment of matrix metalloproteinases-9 (MMP-9) were notably improved after the combined treatments by immunofluorescent staining. These results suggest that 2-DG and 3-BP can both significantly potentiated Ce6-PDT efficacy of cell migration inhibition.

  8. The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons

    PubMed Central

    Leung, Conrad L.; Sun, Dongming; Liem, Ronald K.H.

    1999-01-01

    The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo. PMID:9971739

  9. Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

    PubMed Central

    Chakroun, Maissa; Banyuls, Núria; Bel, Yolanda; Escriche, Baltasar

    2016-01-01

    SUMMARY Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet. PMID:26935135

  10. Safety and Utility of Quantitative Sensory Testing among Adults with Sickle Cell Disease: Indicators of Neuropathic Pain?

    PubMed Central

    Ezenwa, Miriam O.; Molokie, Robert E.; Wang, Zaijie Jim; Yao, Yingwei; Suarez, Marie L.; Pullum, Cherese; Schlaeger, Judith M.; Fillingim, Roger B.; Wilkie, Diana J.

    2014-01-01

    Objectives Pain is the hallmark symptom of sickle cell disease (SCD), yet the types of pain that these patients experience, and the underlying mechanisms, have not been well characterized. The study purpose was to determine the safety and utility of a mechanical and thermal quantitative sensory testing (QST) protocol and the feasibility of utilizing neuropathic pain questionnaires among adults with SCD. Methods A convenience sample (N=25, 18 women, mean age 38.5 ± 12.5 [20–58 years]) completed self-report pain and quality-of-life tools. Subjects also underwent testing with the TSA-II NeuroSensory Analyzer and calibrated von Frey microfilaments. Results We found that the QST protocol was safe and did not stimulate a SCD pain crisis. There was evidence of central sensitization (n=15), peripheral sensitization (n=1), a mix of central and peripheral sensitization (n=8), or no sensitization (n=1). The neuropathic pain self-report tools were feasible with evidence of construct validity; 40% of the subjects reported S-LANSS scores that were indicative of neuropathic pain and had evidence of central, peripheral or mixed sensitization. Discussion The QST protocol can be safely conducted in adults with SCD and provides evidence of central or peripheral sensitization, which is consistent with a neuropathic component to SCD pain. These findings are novel, warrant a larger confirmatory study, and indicate the need for normative QST data from African American adults and older adults. PMID:25581383

  11. Theoretical modeling of mechanical homeostasis of a mammalian cell under gravity-directed vector.

    PubMed

    Zhou, Lüwen; Zhang, Chen; Zhang, Fan; Lü, Shouqin; Sun, Shujin; Lü, Dongyuan; Long, Mian

    2017-08-17

    Translocation of dense nucleus along gravity vector initiates mechanical remodeling of a eukaryotic cell. In our previous experiments, we quantified the impact of gravity vector on cell remodeling by placing an MC3T3-E1 cell onto upward (U)-, downward (D)-, or edge-on (E)- orientated substrate. Our experimental data demonstrate that orientation dependence of nucleus longitudinal translocation is positively correlated with cytoskeletal (CSK) remodeling of their expressions and structures and also is associated with rearrangement of focal adhesion complex (FAC). However, the underlying mechanism how CSK network and FACs are reorganized in a mammalian cell remains unclear. In this paper, we developed a theoretical biomechanical model to integrate the mechanosensing of nucleus translocation with CSK remodeling and FAC reorganization induced by a gravity vector. The cell was simplified as a nucleated tensegrity structure in the model. The cell and CSK filaments were considered to be symmetrical. All elements of CSK filaments and cytomembrane that support the nucleus were simplified as springs. FACs were simplified as an adhesion cluster of parallel bonds with shared force. Our model proposed that gravity vector-directed translocation of the cell nucleus is mechanically balanced by CSK remodeling and FAC reorganization induced by a gravitational force. Under gravity, dense nucleus tends to translocate and exert additional compressive or stretching force on the cytoskeleton. Finally, changes of the tension force acting on talin by microfilament alter the size of FACs. Results from our model are in qualitative agreement with those from experiments.

  12. Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma

    PubMed Central

    Alinari, Lapo; Yu, Bo; Christian, Beth A.; Yan, Fengting; Shin, Jungook; Lapalombella, Rosa; Hertlein, Erin; Lustberg, Mark E.; Quinion, Carl; Zhang, Xiaoli; Lozanski, Gerard; Muthusamy, Natarajan; Prætorius-Ibba, Mette; O'Connor, Owen A.; Goldenberg, David M.; Byrd, John C.; Blum, Kristie A.

    2011-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti–CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab–mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL. PMID:21228331

  13. Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma.

    PubMed

    Alinari, Lapo; Yu, Bo; Christian, Beth A; Yan, Fengting; Shin, Jungook; Lapalombella, Rosa; Hertlein, Erin; Lustberg, Mark E; Quinion, Carl; Zhang, Xiaoli; Lozanski, Gerard; Muthusamy, Natarajan; Prætorius-Ibba, Mette; O'Connor, Owen A; Goldenberg, David M; Byrd, John C; Blum, Kristie A; Baiocchi, Robert A

    2011-04-28

    Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.

  14. Non-genomic actions of thyroid hormone in brain development.

    PubMed

    Leonard, Jack L

    2008-10-01

    Thyroid hormone (TH) is essential for neuronal migration and synaptogenesis in the developing brain. Assembly of neuronal circuits depends on guidance cues provided by the extracellular matrix. These cues are interpreted by the migrating neuron and its growing neurites through transmembrane signaling proteins anchored in place by the actin cytoskeleton. One of the best examples of a non-genomic action of thyroid hormone is its dynamic regulation of the number and quantity of actin fibers in astrocytes. Thyroxine (T4) and its transcriptionally inactive metabolite, 3',5',3-triiodothyronine (reverse T3) are responsible for modulating microfilament organization, while the transcriptional activator, 3',3,5-triiodothyronine (T3) is inert. The biological consequence of the loss of the actin filaments in astrocytes is the inability of the cell to anchor laminin, to its cell surface, and the loss of this key guidance molecule interrupts neurite pathfinding and neuronal migration. These data provide the essentials to construct a physiological pathway where TH-dependent regulation of the polymerization state of actin in the astrocyte and the developing neuron modulates the production and recognition of guidance cues--cues that if disrupted lead to abnormal neuronal migration and neuronal process formation--and lead to the morphological deficits observed in the cretinous brain.

  15. Immunocytochemical localization of gelsolin in fibroblasts, myogenic cells, and isolated myofibrils.

    PubMed

    Dissmann, E; Hinssen, H

    1994-04-01

    Gelsolin was localized by immunofluorescence in fibroblasts and skeletal muscle cells using antibodies which eliminated the risk of detecting xenogenic plasma gelsolin. Gelsolin was consistently found to be closely associated with the elements of the microfilament system: In fibroblasts, a preferential labeling of the stress fibers was observed, whereas with myogenic cells and myofibrils isolated from skeletal muscle, a specific staining of the I-Z-I region in the sarcomeres was found. From double labeling of gelsolin and actin it became evident that the staining patterns for both proteins were practically coincident: The width and location of the fluorescent bands varied with the degree of contraction of the myofibrils. The region of cross-bridges in the A-zone, where thick and thin filaments overlap, remained unstained. The gelsolin staining of myofibrils was EGTA-resistant; it persisted after glycerol extraction and extensive washing. The presence of gelsolin in myofibrils after this treatment was also confirmed by immunoblotting. From these observations it was concluded that a significant part of the total gelsolin in skeletal muscle cells is tightly associated with the thin filaments, and is an integral part of the myofibrils even at low Ca(++)-concentrations. From the coincidence of actin and gelsolin staining in myofibrils it was concluded that gelsolin is localized along the whole length of the thin filaments in the sarcomere.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. PHBV/PAM Scaffolds with Local Oriented Structure through UV Polymerization for Tissue Engineering

    PubMed Central

    Wang, Yingjun

    2014-01-01

    Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100 μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance. PMID:24579074

  17. Effects of the in vitro administered ethanol and lipopolysaccharide toxin on membrane properties, intracellular free calcium and phagocytic function of isolated rat kupffer cells

    SciTech Connect

    Victorov, A.; Smith, T.; Abril, E.; Hamlin, E.; Earnest, D. )

    1991-03-11

    Low concentrations of ethanol slightly stimulated phagocytosis of cultured Kupffer cells (KC), producing practically no effect on membrane microviscosity and cytosolic free (Ca{sup 2+}){sub i}. On the contrary, high concentrations of ethanol significantly suppressed phagocytic function, increased fluidity of membrane lipids and caused a sustained rise in (Ca{sup 2}){sub i}; above the resting level of 41-85 nM. Treatment of KC with colchicine and cytochalasin B dramatically destructurized the plasma membrane lipids. Short term preincubation of KC with high doses of alcohol stimulated the disordering effects of both drugs, suggesting direct interaction of ethanol with microtubule and microfilament structures. The authors hypothesize that ethanol impairs phagocytosis of KC by concerted actions on membrane lipid fluidity, cytosolic free Ca{sup 2+} and functioning of cytoskeleton. On the other hand, incubation of KC with low concentrations of lipopolysaccharide (LPS) produced no changes in (Ca{sup 2+}){sub i}; or plasma membrane fluidity but reduced by several fold the fluidizing effect of subsequently added ethanol. They suggested that low doses of LPS, by activating second messengers other than Ca{sup 2+}, alter the functioning of the cytoskeleton and cause reorganization of the plasma membrane thus making KC membranes more resistent to the fluidizing action of ethanol and partially restoring the phagocytic function.

  18. Enhanced bovine serum albumin absorption on the N-hydroxysuccinimide activated graphene oxide and its corresponding cell affinity.

    PubMed

    Xiong, Kun; Fan, Qingbo; Wu, Tingting; Shi, Haishan; Chen, Lin; Yan, Minhao

    2017-12-01

    By successively reacting with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the carboxyl on the graphene oxide (GO) surface was successfully activated into NHS active ester. In this study, bovine serum albumin (BSA) was selected as a model protein, used for studying the protein absorption capacity of the NHS activated GO (GO-EDC-NHS). Approximately 12.75mg of BSA could be covalent bonded onto the GO-EDC-NHS surface (BSA-CB-GO), whereas only 6.83mg of BSA physical absorbed onto the GO surface (BSA-NB-GO). With a 168h of phosphate buffer saline (PBS) soaking, the BSA accumulative desorption ratio, which was accordingly assigned to the BSA-NB-GO and the BSA-CB-GO, was separately 29.91wt% and 2.95wt%. Consequently, it proved GO-EDC-NHS exhibited more stable and stronger BSA absorption capacity. As compared to the mouse bone marrow mesenchymal stem cells (mBMSCs) cultivated on the BSA-NB-GO surface, the immunofluorescence staining images showed that more vinculins and integrin α5 were visible in the mBMSCs cultivated on the BSA-CB-GO surface, they also produced more distinct stress fibers and actin-containing microfilaments. In summary, BSA-CB-GO possesses an excellent cell affinity, which can be considered as a promising functional material used for promoting the bone remodeling. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein

    PubMed Central

    1991-01-01

    The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27- kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C,